Hemoglobin Disorders: Molecular Methods and Protocols (Methods in Molecular Medicine)

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M E T H O D S I N M O L E C U L A R M E D I C I N E TM

Hemoglobin Disorders Molecular Methods and Protocols Edited by

Ronald L. Nagel, MD

Humana Press

Hemoglobin Disorders

METHODS IN MOLECULAR MEDICINE

TM

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METHODS IN MOLECULAR MEDICINE

TM

Hemoglobin Disorders Molecular Methods and Protocols Edited by

Ronald L. Nagel, MD Albert Einstein College of Medicine, Bronx, NY

Humana Press

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© 2003 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 www.humanapress.com All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Methods in Molecular Medicine™ is a trademark of The Humana Press Inc. For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact Humana at the above address or at any of the following numbers: Tel.: 973-256-1699; Fax: 973-256-8341; E-mail: [email protected]; Website: http://humanapress.com The content and opinions expressed in this book are the sole work of the authors and editors, who have warranted due diligence in the creation and issuance of their work. The publisher, editors, and authors are not responsible for errors or omissions or for any conequences arising fom the information or opinions presented in this book and make no warranty, express or implied, with respect to its contents.

Cover illustration: Fig. 4A from Chapter 10, “Nucleation and Crystal Growth of Hemoglobins: The Case of HbC,” by Peter G. Vekilov, Angela Feeling-Taylor, and Rhoda Elison Hirsch. Production Editor: Tracy Catanese Cover design by Patricia F. Cleary. Due diligence has been taken by the publishers, editors, and authors of this book to assure the accuracy of the information published and to describe generally accepted practices. The contributors herein have carefully checked to ensure that the drug selections and dosages set forth in this text are accurate and in accord with the standards accepted at the time of publication. Notwithstanding, as new research, changes in government regulations, and knowledge from clinical experience relating to drug therapy and drug reactions constantly occurs, the reader is advised to check the product information provided by the manufacturer of each drug for any change in dosages or for additional warnings and contraindications. This is of utmost importance when the recommended drug herein is a new or infrequently used drug. It is the responsibility of the treating physician to determine dosages and treatment strategies for individual patients. Further it is the responsibility of the health care provider to ascertain the Food and Drug Administration status of each drug or device used in their clinical practice. The publisher, editors, and authors are not responsible for errors or omissions or for any consequences from the application of the information presented in this book and make no warranty, express or implied, with respect to the contents in this publication.

This publication is printed on acid-free paper. ∞ ANSI Z39.48-1984 (American Standards Institute) Permanence of Paper for Printed Library Materials. Photocopy Authorization Policy: Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Humana Press Inc., provided that the base fee of US $20.00 is paid directly to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license from the CCC, a separate system of payment has been arranged and is acceptable to Humana Press Inc. The fee code for users of the Transactional Reporting Service is [0-89603-962-5/03 $20.00]. Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1 Library of Congress Cataloging in Publication Data Main entry under title: Methods in molecular medicine™. Hemoglobin disorders : methods and protocols / edited by Ronald L. Nagel. p. ; cm. -- (Methods in molecular medicine ; 82) Includes bibliographical references and index. ISBN 0-89603-962-5 (alk. paper); 1-59259-373-9 (e-book) 1. Hemoglobinopathy--Laboratory manuals. I. Nagel, R. L. (Ronald L.) II. Series. [DNLM: 1. Hemoglobinopathies--Laboratory Manuals. 2. Laboratory Techniques and Procedures--Laboratory Manuals. WH 25 H489 2003] RC641.7.H35H444 2003 616.1'51--dc21 2003040674

Preface Hemoglobin and Hemoglobinologists This volume, Hemoglobin Disorders: Molecular Methods and Protocols, will be introduced with a review of the great milestones in the field, and the scientists responsible for those achievements. The history of hemoglobin can be divided into three periods: the Classical period, the Modern period, and the Post-Modern period. I am inclined to include as the four major members of the classical period Francis Roughton, Quentin Gibson, Jeffries Wyman, and Linus Pauling, not only because of their achievements, but also because of the superb scientists they trained and/or influenced. Francis John Worsely Roughton (1899–1972) (Fig. 1), in his laboratory at Trinity College in Cambridge, England, made the first measurements of the rapid reaction of oxygen with hemoglobin at the millisecond scale, at first by flow-mixing methods and later by flash photolysis. He not only opened an era of molecular research of hemoglobin, but also invented the methodology for fast reactions through the use of laser technology, which was later improved by others so that even faster reactions could be detected. Another contribution of Roughton was the education of Quentin H. Gibson (Fig. 2), his favorite student, who, in his laboratory in Sheffield, continued to expand the horizon of ligand binding to hemoglobin, defining the oxygen binding constants for each of the hemes of hemoglobin. Though this did not, as expected, solve the underlying mechanism of ligand cooperativity as discussed below, it was nonetheless an important milestone. Roughton would later have a surprising influence in the Italian hemoglobin group because he trained Luigi Rossi-Bernardi, and because Quentin Gibson introduced Jeffries Wyman to Eraldo Antonini, the hemoglobin man in Rome in that period (1). In a meeting in Bellagio, Lake Como, Luigi regaled us with stories about this highly talented and very eccentric investigator. It was fortunate to science that eccentricity was perfectly acceptable in England, unlike in other places in the world. Finally, Quentin continued his highly productive career after emigrating to the United States in the early 1960s, working independently first in Britton Chance’s lab in Philadelphia and then at Cornell, where he trained John Olson, who brilliantly carried on the torch and is an author in this book. I consider Quentin my mentor, along with Helen Ranney. It was most exciting to solve the molecular basis of the hemoglobin/haptoglobin reaction together. v

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Fig. 1. Francis John Worsely Roughton

Quentin Gibson, who is an MD, was famous for having a lathe in the middle of the lab, useful for tinkering with homemade instrumentation. This wonderful “tinkering” habit of British scientists came in handy during World War II, to the benefit of the world. Gibson is still scientifically active, and has contributed widely to the hemoglobin ligand binding field (see Chapter 5). He also wrote his recollections of the life and work of Francis J. Roughton in 1973, after Roughton’s death at the age of 73 years (2). Fig. 2. Quentin H. Gibson Jeffries Wyman (1901–1995) (Fig. 3) was a Boston Brahmin and a remarkable American biophysicist whose grandfather was one of the founders of the National Academy of Science. A Harvard man, he developed an interest in proteins and in 1937 wrote his first hemoglobin paper on the pH titration curves, or oxydeoxy-hemoglobin (3). He exhibited a unique understanding of thermodynamics in his analysis of linked function reciprocal relations (1948). He later came back to this subject with a landmark book, Binding and Linkage: Functional

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Chemistry of Biological Macromolecules, coauthored with Stanley J. Gill, who derived great comfort from this enterprise in the last years of his life. According to Edsall (4), Wyman, while visiting colleagues in Japan in 1950, had an insight based on the work of Felix Haurowitz, a New York scientist who used to walk about with a vial of growing hemoglobin crystals in his vest pocket, so as to maintain the solution close to 37°C. Haurowitz did a remarkable and simple experiment: he reduced a crystal of oxyhemoglobin with dithionite and observed its breakage Fig. 3. Jeffries Wyman and dissolution. He concluded that these two ligand states of hemoglobin had different crystal habits. Wyman, in turn, concluded that the result was the consequence of hemoglobin in two different conformational states: in oxy (met in reality) and deoxy, a remarkable anticipation of Perutz’s work. Wyman’s wanderlust took him to the four corners of the world. After the death of his first wife, he left Harvard and the United States for Paris, where he was the first Cultural Attache to the American Embassy. After that, it was an International Organization job in Egypt, and then escapes to the Congo, Alaska, Papua, New Guinea, and so forth. But his most important visit by far was to Rome, where he became part of the hemoglobin team lead by Eraldo Antonini (1). Italy became his home for most of his life, in spite of the fact that he never obtained a permanent status, and needed to go to Switzerland every year to renew his visa. He never learned to speak Italian. The Rome group, integrated by Maurizio Brunori, Emilia Chiancone, and others, became a strong presence in the field, concentrating on the biophysical and biochemical aspects of hemoglobin. Another participant in this interactive hemoglobin world was Quentin Gibson, who collaborated with Eraldo early on and had to carry instrumentation and glass artifacts through the corridors and yards of the University of Rome, because it was unseemly for an Italian professor to do so. Maurizio, of course, became the leader of this highly productive group after the untimely death of Eraldo at the age of 52, keeping the high standards set by its founder. During my first visit to Rome, Maurizio introduced me to Wyman, and like everybody else, I was in awe of the magnetic field of his mind and his ability to contribute brilliantly to any problem that might be presented to him. Finally, the emergence of the Jacob-Monod-Changeux allosteric model fit Wyman’s insight into the workings of hemoglobin and rapidly adopted its

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Fig. 4. Linus Pauling

nomenclature. On the other the hand, Eraldo Antonini resisted this concept, postulating an alternative dimer-based model. The demonstration that haptoglobin binds exclusively hemoglobin dimers—and does not bind deoxyhemoglobin (5,6) because it does not dimerize—made this proposal untenable. The final member of the Classical period was Linus Pauling (1901–1994) (Fig. 4), a double Nobel Prize winner, and another eccentric and brilliant scientist. He should be considered a luminary in the field of hemoglobin for two reasons. First, he proposed (and demonstrated) that a hemoglobin abnormality had to be the reason for the sickling of red cells in sickle cell anemia. This disease had been discovered by Dr. James B. Herrick, a cardiologist, in Chicago in 1910 (7). The concept of sickle cell anemia as a “molecular disease” opened a new chapter in medicine (8). Second, Pauling discovered the differential magnetic susceptibility of oxy- and deoxy-hemoglobin, which is the basis of advanced methods of nuclear magnetic resonance imaging, allowing detection of deoxy-hemoglobin in tissues (9) and recently applied to the study of sickle transgenic mice (10).

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The Modern period was inaugurated with the discovery by Max Ferdinand Perutz (1914–2002) (11) (Fig. 5) that the isomorphic replacement method was applicable to large molecules, and that binding of mercury to the Cys93 did not distort the molecule. This solved the phase problem and aided in the description of the tridimensional structure of the hemoglobin molecule. This effort was stimulated by conversations with Felix Haurowitz and realized with the help of a small grant from the Rockefeller Foundation, obtained through the good offices of Sir Lawrence Bragg, inventor of crystallography. As a young investigator, I met Max Perutz in Cambridge shortly after he had published Fig. 5. Max Ferdinand Perutz his milestone work, and true to his modesty and bonhomie, he told me that he was happy to have finally published because doing so guaranteed him lab space at the Cavendish, which he was previously at risk of losing for lack of publications. This publication’s followup, on deoxyhemoglobin, allowed us to understand the molecular basis of cooperativity and was recognized with the Nobel Prize, which Perutz shared with Kendrew, who had worked on the less complex problem of the crystallography of myoglobin. After the Nobel Prize, he worked even harder. I saw him in Cambridge in the last few months before his death, and we discussed the need to understand why HbC has a highly increased tendency to crystallize. It was a fruitful exchange. We also talked about his leaving Vienna in 1936 to work with J. D. Bernal in crystallography. At the beginning of the war, Britain interned all immigrants born in enemy countries in mild detention camps, even if they were of Jewish origin. Fortunately, the authorities put him to work on plans to construct gigantic ice surfaces that could serve as airplane landing sites in the North Sea. For this plan they needed a crystallographer’s knowledge to help them strengthen the crystallized water, at which Max eventually succeeded through the use of wood pulp, although too late to be useful. Vernon M. Ingram clearly deserves a place in the Modern period. Also in Cambridge, using a sickle cell anemia patient's blood samples left behind by a colleague, he purified the hemoglobin, and ran a trypsin digestion and a combination of electrophoresis and paper chromatography (to be known as “fingerprinting”) on the sample, revealing that the mutation in sickle hemoglobin was limited to a single amino acid change: glutamic acid replaced by valine. This proved a momentous finding and the launching of a technique that was used

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widely for decades in the analysis of proteins. Vernon wrote a recollection of this discovery later (12). Another feature of the Modern period is the highly compatible, yet unlikely pairing of Reinhold and Ruth Benesh, called by many R2B2. Reinhold was a Polish immigrant who went to England to study chemistry, and survived by performing stand-up comedy in English vaudeville theaters. He eventually emigrated to the United States, met Ruth, and formed a powerful scientific team. Preparing for a lecture to students of medicine, he realized that 2,3-DPG existed in the red cell in almost identical quantities as hemoglobin. The next day they mixed 2,3-DPG and hemoglobin and observed a right shift of the oxygen equilibrium curve. A new allosteric effector had been found. This finding had tremendous scientific and medical impact. To date, a PubMed search for 2,3-DPG yields 1793 results. Reinhold also contributed to, among other things, the definition of the contact sites in α-chains that contribute to the stabilization of the sickle polymer. The Post-Modern period, being contemporary, cannot be judged in the same way as the two previous periods. But important accomplishments need to be recognized in the field of hemoglobins. First, George Stamatoyannopoulos merits special mention. Not only has he and his laboratory contributed enormously to the field and trained a slew of young scientists, but he has become the “cheerleader” of research in hemoglobin molecular biology. His “Switching Meetings,” at first in collaboration with Art Nienhuis and George Dover, have become, with time, a classic “George’s show.” Everybody waits for George’s phone call: “What have you done lately?” Stamatoyannopoulos has also been a constant and successful lobbyist to NIH for more money for globin research and for greater opportunities for young investigators to join the field. The explosion of molecular biology is one of the most important events characterizing this period. Too many important participants are worth mentioning, so I will limit the list to a few that contributed to the field up to 1990 (recent work is outlined in “late-breaking news”): A. W. Nienhuis F. G. Grosveld T. J. Ley L. I. Zon T. M. Towns (see Chapter 13) J. B. Ligrel G. Felsenfeld D. R. Higgs S. A. Liebhaber T. Papayannoupolous

S. H. Orkin Y. W. Kan S. L. Thein J. M. Old (see Chapters 7 and 8) T. M. Ryan (see Chapter 13) A. N. Schechter E. J. Benz J. B. Clegg S. M. Weissman B. G. Forget

Preface H. H. Kazazian R. Krishnamoorthy (see Chapter 12) A. Bank N. P. Anagnou J. L. Sleighton K. Adachi (see Chapter 14) S. A. Acharya (see Chapter 11) and many others.

xi N. J. Proudfoot D. Labie (see Chapter 12) J. D. Engel C. Driscoll W. G. Wood R. C. Hardison

Sir David Weatherall also deserves special mention. He is responsible for major developments in the understanding of thalasssemia in the last 40 years, including some all-encompassing and very readable textbooks on the subject (13). David Nathan is also a major figure in this field in America, contributing to both the scientific and clinical sides (14). Clinical advances can be credited to Sergio Piomelli in the general management of this difficult disease (14) and to G. Lucarelli (16) for his contribution on bone marrow transplantation of thalassemic patients in Italy and the world. A major and groundbreaking contribution to sickle cell anemia was the discovery by William Eaton and his group that the polymerization of HbS was a nucleation-driven reaction in its two forms: homogeneous and heterogeneous (17). In addition, they discovered that the delay time of polymerization was dependent exclusively on the initial concentration of Hb with the potential of modifying the extent of the phenotype (18). The next important discovery in the field, credited to Robert Hebbel and associates (19), was the capacity of young sickle cells to adhere to cultured endothelial cells. This finding was confirmed by Dhananjay K. Kaul in ex vivo and in vivo microcirculatory beds, and was followed by the demonstration that sickle vasocclusion occurred, not predominately in the capillaries as previously thought, but in the small venules, in which the adhesion of young sickle cells preceded obstruction by rigid sickle cells (20). The structure of the sickle polymer was resolved by a combination of the following discoveries: (1) the crystallography of sickle hemoglobin (21), (2) the study of the polymerization tendency of binary mixtures of sickle and other hemoglobin mixtures to define residues in the area of contact of the polymer (22), and (3) electron microscopy of the polymer and modeling (23,24). Another surprise was the linkage of the sickle mutation with several haplotypes of polymorphic sites in the globin gene cluster. This effort arose based on early work by Y. W. Kan and Stuart Orkin, which was followed by genetic epidemiological studies in Jamaica (25), Africa (26), and India (27,28). Besides demonstrating the multicentric origin of the sickle mutation, this effort revealed the linkage between severity and certain haplotypes and the

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role of -158 Xmn I polymorphism in the expression of HbF (29,30), in addition to their power as instruments in anthropological and gene flow studies. Alpha thalassemia was, after the ameliorating effect of HbF, the first modifier of sickle cell anemia found and most of the credit for this finding belongs to Steve Embury (31). The discovery of Locus Control Region (LCR), 5' to the β-like gene cluster, by Dorothy Tuan and Irving London (32) had unexpected consequences. In addition to the involvement of LCR in the development of appropriate expression of the β-like globins, it made possible the high and tissue-specific expression of transgenes in mouse models as well as in vectors containing antihemoglobinopathies for gene therapy. Although much progress has been made owing to the efforts of George Stamatoyannopoulos, Marc Groudine, and F. G. Grosveld, a definitive picture has not yet emerged. The development of transgenic sickle and thalassemic mice, very useful in the field despite unfriendly NIH committee reviews for many years, is a complicated history with many players, so I refer the interested reader to a recent review (33) and Chapter 13. The successful clinical trial, beyond any rational expectation, of hydroxyurea as a specific treatment for sickle cell anemia (34) is a great landmark in the history of sickle cell in America because it is the only drug approved by the FDA for the treatment of this disease. Investigations leading to this breakthrough involved Paul Heller, Joe DeSimone, George Stamatoyannopoulos, George Dover, and others. The leader of the clinical trial was Sam Charache, after years of frustrating rejections by unsympathetic and misguided reviewers, with the competent help of Martin Steinberg (35). The pioneering work of Chien Ho (see Chapter 15) on NMR of hemoglobin and hemoglobin variants was highly successful and contributed to, among other things, the molecular localization of the Bohr protons. Other less glamorous but equally important clinical advances can be credited to Helen Ranney, who contributed all of her scientific life to hemoglobin research, with her pioneering work on HbA1c as a noted example. She also contributed by organizing what I believe to be the first hemoglobinopathiesdedicated clinic in America, at Jacobi Hospital, Bronx, NY. The NIH Natural History initiative, under the leadership of Marilyn Gaston (36), saved many lives by demonstrating the effectiveness of penicillin prophylaxis in decreasing infections and modality in infants with sickle cell disease. The Herculean effort of Graham Serjeant, who headed an MRC unit in Jamaica dedicated to the care and study of sickle cell anemia patients, must be recognized. He produced considerable and reliable natural history clinical data on sickle cell anemia with much less funding than the NIH effort (37).

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The discovery of desferrioxamine and the use of chelation therapy to increase the life expectancy of patients with thalassemia major and thalassemia intermedia is also a major accomplishment. The compound is a natural product extracted for actinomycetes, and was reported to be an iron chelator useful in the treatment of hemochromatosis by P. Imhof of Ciba Geigy at the joint annual meeting of the 1962 Swiss Medical in Lugano. An annotation in Lancet (38) concludes that “it is unfortunate that in secondary hemochromatosis, usually the result of repeated transfusions in patients with aplastic anemia and other anemias when repeated blood letting is not possible, the drug is apparently less efficacious than in the idiopathic type.” Fortunately, this prediction did not come to pass, and the drug is now the mainstay of the treatment of severe thalassemia. The quest for a clearly effective oral form seems to be close at hand. Another aspect of research in hemoglobinopathies is the effort to characterize hemoglobin mutants, useful in many of the studies referred to above. In this realm, three investigators have been particularly successful. The first is Herman Lehmann (39,40), who emigrated to Britain early in life, worked in Cambridge, and spent World War II in the British Army in India, in which his training as a hematologist was welcome. He discovered HbS among the “tribals” of India, and contributed profusely to the works on hemoglobin , particularly in the identification and characterization of Hb mutants. He also predicted the duplication of the α-globin loci. The second great figure in this realm was Titus Huisman (41), who published 661 papers in his life, almost all on hemoglobin. He was a refined analytical biochemist and a highly focused and productive researcher. Finally, the successor in this field today is Henri Wajcman, editor of Hemoglobin, who runs a highly efficient reference laboratory in Paris for abnormal hemoglobins that has been enormously useful to all of us. Dr. Wajcman is an expert on unstable hemoglobins. Finally, in “late-breaking news,” the very recent correction of sickle cell anemia (42) and thalassemia (43) by transplantation of stem cells transducted with a lentivirus construct containing human globin genes in mice transgenic models is an encouraging event, and bodes well for the future of gene therapy in hemoglobinopathies. The remarkably successful adventures that have characterized research and clinical endeavors in hemoglobinopathies have been the product of the efforts of an army of highly qualified and imaginative investigators and clinicians, interested in diseases that affect not only Europe and North America, but most of the third world. In conclusion, the last century has been good to hemoglobin. Maybe because hemoglobin is red, which helped in its isolation, maybe because it is abundant, or maybe because, as the third book of the Torah (and the Old Testa-

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ment) says, “the soul of the flesh is the blood,” hemoglobin has been an active participant in the development of biochemistry, protein chemistry, molecular biology, human genetics, and molecular medicine. It is also apparent that behind it all there was a real network of investigators, sometimes interacting competitively, some times cooperatively, but always in contact. The network has indeed produced a cascade of findings and valuable and unforgettable human interactions. Maybe the lure of this unique and beautiful molecule attracted brilliant, eccentric, imaginative, and one-of-a-kind investigators who blazed a brilliant trail of successes.

Ronald L. Nagel, MD References 1. Brunori, M. (1999) Hemoglobin is an honorary enzyme. Trends Biochem. Sci. 24, 158–161. 2. Gibson, Q. H. (1973) Francis John Worsely Roughton, 1899–1972. Biogr. Mem. Fellows R Soc. 19, 563–582. 3. Wyman, J. and Allen, D. (1958) The problem of the heme interactions in hemoglobin and the nature of the Bohr effect. J. Polymer Sci. 7, 499–518. 4. Edsall, J. T. (1995) Jeffries Wyman, (1901–95) Nature 378, 556. 5. Nagel, R. L. and Gibson, Q. H. (1966) Kinetics of the reaction of carbon monoxide with the hemoglobin-haptoglobin complex. J. Mol. Biol. 22, 249–255. 6. Nagel, R. L., Rothman, M. C., Bradley, T. B., Jr., and Ranney, H. M. (1965) Comparative haptoglobin binding properties of oxyhemoglobin and deoxyhemoglobin. J. Biol. Chem. 240, 4543–2545. 7. Herrick, J. B. (1910) Peculiar elongated and sickle-shaped red blood corpuscles in a case of severe anemia. Arch. Intern. Med. 6, 517–521. 8. Pauling, L. (1977) Magnetic properties and structure of oxyhemoglobin. Proc. Natl. Acad. Sci. USA 74, 2612–2613. 9. Ogawa, S., Lee, T. M., Nayak, A. S., and Glynn, P. (1990) Oxygenation-sensitive contrast in magnetic resonance image of rodent brain at high magnetic fields. Magn. Reson. Med. 124, 68–78. 10. Fabry, M. E., Kennan, R. P., Paszty, C., et al. (1996) Magnetic resonance evidence of hypoxia in a homozygous α-knockout of a transgenic mouse model for sickle cell disease. J. Clin. Invest. 98, 2450–2455. 11. King, A. (2002) Restrospective: structural biology and biochemistry. Max Perutz (1914–2002). Science 295, 2382–2383. 12. Ingram, V. M. (1989) Abnormal human haemoglobins. I. The comparison of normal human and sickle-cell haemoglobins by “fingerprinting.” 1958. Biochim. Biophys. Acta. 1000, 151–157.

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13. Weatherall, D. and Clegg, J., eds. (2001) The Thalassemia Syndromes, Fourth Edition. Blackwell Science, Boston, MA. 14. Nathan, D. G. (1998) Genes, Blood and Courage: A Boy Named Immortal Sword. Harvard University Press, Cambridge, MA. 15. Piomelli, S. (1989) Cooley’s Anemia Management: 25 years of progress. Prog. Clin. Biol. Res. 309, 23–26. 16. Lucarelli, G., Andreani, M., and Angelucci, E. (2002) The cure of thalassemia by bone marrow transplantation. Blood Rev. 16, 81–85. 17. Eaton, W. A. and Hofrichter, J. (1990) Sickle cell hemoglobin polymerization. Adv. Protein Chem. 40, 63–279. 18. Eaton, W. A., Hofrichter, J., and Ross, P. D. (1976) Editorial: Delay time of gelation: a possible determinant of clinical severity in sickle cell disease. Blood 47, 621–627. 19. Hebbel, R. P., Yamada, O., Moldow, C. F., et al. (1980) Abnormal adherence of sickle erythrocytes to cultured vascular endothelium: possible mechanism for microvascular occlusion in sickle cell disease. J. Clin. Invest. 65, 154–160. 20. Kaul, D. K., Fabry, M. E., and Nagel, R. L. (1989) Microvascular sites and characteristics of sickle cell adhesion to vascular endothelium in shear flow conditions: pathophysiological implications. Proc. Natl. Acad. Sci. USA 86, 3356–3360. 21. Wishner, B. C., Ward, K. B., Lattman, E. E., and Love, W. E. (1975) Crystal structure of sickle-cell deoxyhemoglobin at 5 Å resolution. J. Mol. Biol. 98, 179–194. 22. Nagel, R. L., Johnson, J., Bookchin, R. M., et al. (1980) Beta-chain contact sites in the haemoglobin S polymer. Nature 283, 832–834. 23. Edelstein, S. J. (1981) Structure of the fibers of hemoglobin S. Tex. Rep. Biol. Med. 40, 221–232. 24. Watowich, S. J., Gross, L. J., and Josephs, R. (1989) Intermolecular contacts within sickle hemoglobin fibers. J. Mol. Biol. 209, 821–828. 25. Wainscoat, J. S., Bell, J. I., Thein, S. L., et al. (1983) Multiple origins of the sickle mutation: evidence from beta S globin gene cluster polymorphisms. Mol. Biol. Med. 1, 191–197. 26. Pagnier, J., Mears, J. G., Dunda-Belkhodja, O., et al. (1984) Evidence for the multicentric origin of the sickle cell hemoglobin gene in Africa. Proc. Natl. Acad. Sci. USA 8, 1771–1773. 27. Kulozik, A. E., Thein, S. L., Kar, B. C., et al. (1987) Raised Hb F levels in sickle cell disease are caused by a determinant linked to the beta globin gene cluster. Prog. Clin. Biol. Res. 251, 427–439. 28. Labie, D., Srinivas, R., Dunda, O., et al. (1989) Haplotypes in tribal Indians bearing the sickle gene: evidence for the unicentric origin of the beta S mutation and the unicentric origin of the tribal populations of India. Hum. Biol. 61, 479–491. 29. Labie, D., Dunda-Belkhodja, O., Rouabhi, F., et al. (1985) The -158 site 5' to the G gamma gene and G gamma expression. Blood 66, 1463–1465.

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30. Gilman, J. G. and Huisman, T. H. (1985) DNA sequence variation associated with elevated fetal G gamma globin production. Blood 66, 783–787. 31. Embury, S. H. (1989) Alpha thalassemia. A modifier of sickle cell disease. Ann. NY Acad. Sci. 565, 213–221. 32. Tuan, D. Y., Solomon, W. B., London, I. M., and Lee, D. P. (1989) An erythroidspecific, developmental-stage-independent enhancer far upstream of the human “beta-like globin” genes. Proc. Natl. Acad. Sci. USA 86, 2554–2558. 33 Nagel, R. L. and Fabry, M. E. (2001) The panoply of animal models for sickle cell anaemia. Br. J. Haematol. 112, 19–25, 34. Charache, S., Terrin, M. L., Moore, R. D., et al. (1995) Effect of hydroxyurea on the frequency of painful crises in sickle cell anemia. Investigators of the Multicenter Study of Hydroxyurea in Sickle Cell Anemia. N. Engl. J. Med. 332, 1317–1322. 35. Steinberg, M. H., Lu, Z. H., Barton, F. B., et al. (1997) Fetal hemoglobin in sickle cell anemia: determinants of response to hydroxyurea. Multicenter Study of Hydroxyurea. Blood 89, 1078–1088. 36. Gaston, M. H., Verterm, J. I., Woods, G., et al. (1986) Prophylaxis with oral penicillin in children with sickle cell anemia. A randomized trial. N. Engl. J. Med. 314, 1593–1599. 37. Serjeant, G. R. (2001) The emerging understanding of sickle cell disease. Br. J. Haematol. 112, 3–18. 38. Annotation (1962) A new treatment for haemochromatosis? Lancet i, 1172. 39. Lehmann, H. (1984) Sickle cell anemia 35 years ago: reminiscence of early African studies. Am. J. Pediatr. Hematol. Oncol. 6, 72–76. 40. Lehmann, H. (1984) The gradual understanding of thalassemia. Prog. Clin. Biol. Res. 165, 121–136. 41. Proceedings of the Titus H. J. Huisman Memorial Symposium (2001) Augusta, Georgia, USA. June 9, 2000. Hemoglobin 25, 117–258. 42. Pawliuk, R., Westerman, K. A., Fabry, M. E., et al. (2001) Correction of sickle cell disease in transgenic mouse models by gene therapy. Science 294, 2368–2371. 43. Imren, S., Payen, E., Westerman, K. A., et al. (2002) Permanent and panerythroid correction of murine β-thalassemia by multiple lentivirus integration in hematopoietic stem cells. Proc. Natl. Acad. Sci. USA 99, 14380–14385.

Contents Preface ............................................................................................................. v Contributors ................................................................................................... xix 1 X-ray Crystallography of Hemoglobins Martin K. Safo and Donald J. Abraham .............................................. 1 2 Analysis of Hemoglobins and Globin Chains by High-Performance Liquid Chromatography Henri Wajcman ..................................................................................... 21 3 Purification and Molecular Analysis of Hemoglobin by High-Performance Liquid Chromatograpy Belur N. Manjula and Seetharama A. Acharya ................................. 31 4 Oxygen Equilibrium Measurements of Human Red Blood Cells Jean Kister and Henri Wajcman ........................................................ 49 5 Measurement of Rate Constants for Reactions of O2, CO, and NO with Hemoglobin John S. Olson, Erin W. Foley, David H. Maillett, and Eden V. Paster .......................................................................... 65 6 Electrophoretic Methods for Study of Hemoglobins Henri Wajcman ..................................................................................... 93 7 DNA Diagnosis of Hemoglobin Mutations John M. Old ........................................................................................ 101 8 Methods for Analysis of Prenatal Diagnosis John M. Old ........................................................................................ 117 9 Hemoglobin Fluorescence Rhoda Elison Hirsch ......................................................................... 133 10 Nucleation and Crystal Growth of Hemoglobins: The Case of HbC Peter G. Vekilov, Angela Feeling-Taylor, and Rhoda Elison Hirsch .............................................................. 155 11 Semisynthesis of Hemoglobin Seetharama A. Acharya and Sonati Srinivasulu ........................... 177 12 β-Globin-like Gene Cluster Haplotypes in Hemoglobinopathies Shanmugakonar Muralitharan, Rajagopal Krishnamoorthy, and Ronald L. Nagel ...................................................................... 195

xvii

xviii

Contents

13 Transgenic Mice and Hemoglobinopathies Mary E. Fabry, Eric E. Bouhassira, Sandra M. Suzuka, and Ronald L. Nagel ...................................................................... 14 Recombinant Single Globin-Chain Expression and Purification Kazuhiko Adachi ................................................................................ 15 Nuclear Magnetic Resonance of Hemoglobins Jonathan A. Lukin and Chien Ho ..................................................... 16 Solubility Measurement of the Sickle Polymer Mary E. Fabry, Seetharama A. Acharya, Sandra M. Suzuka, and Ronald L. Nagel ...................................................................... Index ............................................................................................................

213 243 251

271 289

Contributors DONALD J. ABRAHAM • Department of Medicinal Chemistry, School of Pharmacy, and the Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, VA SEETHARAMA A. ACHARYA • Department of Medicine and Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY KAZUHIKO ADACHI • Division of Hematology, Children’s Hospital of Philadelphia, Philadelphia, PA ERIC E. BOUHASSIRA • Department of Medicine, Albert Einstein College of Medicine, Bronx, NY MARY E. FABRY • Department of Medicine, Albert Einstein College of Medicine, Bronx, NY ANGELA FEELING-TAYLOR • Sue Golding Graduate Division, Department of Anatomy and Structural Biology, and Medical Scientists Training Program, Albert Einstein College of Medicine, Bronx, NY ERIN W. FOLEY • Department of Biochemistry and Cell Biology, and the W. M. Keck Center for Computational Biology, Rice University, Houston, TX RHODA ELISON HIRSCH • Division of Hematology, Department of Medicine, and Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY CHIEN HO • Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA JEAN KISTER • INSERM U473, Le Kremlin Bicetre, France RAJAGOPAL KRISHNAMOORTHY • INSERM U468, Hôpital Robert Debré, Paris, France JONATHAN A. LUKIN • Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA DAVID H. MAILLETT • Department of Biochemistry and Cell Biology, and the W. M. Keck Center for Computational Biology, Rice University, Houston, TX BELUR N. MANJULA • Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY SHANMUGAKONAR MURALITHARAN • Department of Medicine, Albert Einstein College of Medicine, Bronx, NY RONALD L. NAGEL • Division of Hematology, Department of Medicine, and Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY xix

Contributors

xx

JOHN M. OLD • John Radcliffe Hospital, Institute of Molecular Medicine, Oxford, UK JOHN S. OLSON • Department of Biochemistry and Cell Biology, and the W. M. Keck Center for Computational Biology, Rice University, Houston, TX EDEN V. PASTER • Department of Biochemistry and Cell Biology, and the W. M. Keck Center for Computational Biology, Rice University, Houston, TX MARTIN K. SAFO • Department of Medicinal Chemistry, School of Pharmacy, and the Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, VA SONATI SRINIVASULU • Department of Medicine, Albert Einstein College of Medicine, Bronx, NY SANDRA M. SUZUKA • Department of Medicine, Albert Einstein College of Medicine, Bronx, NY PETER G. VEKILOV • Department of Chemical Engineering, University of Houston, Houston, TX HENRI WAJCMAN • INSERM U468, Hôpital Henri Mondor, Creteil, France

X-ray Crystallography of Hemoglobins

1

1 X-ray Crystallography of Hemoglobins Martin K. Safo and Donald J. Abraham 1. Introduction X-ray crystallography has played a key role in understanding the relationship between protein structure and physiological function. In particular, X-ray analysis of hemoglobin (Hb) crystals has been pivotal in the formulation of basic theories concerning the behavior of allosteric proteins. Methemoglobin (MetHb) from horse was the first three-dimensional (3D) structure of liganded Hb to be solved (1–4). It was followed by crystallographic determination of the unliganded (deoxygenated) form nearly a decade later (5). The X-ray analyses provided 3D atomic resolution structures and confirmed that Hb was tetrameric, containing two subunit types (α and β), and one oxygen-binding heme group per subunit. John Kendrew (myoglobin) and Max Perutz (Hb) received the Nobel Prize for their pioneering work, being the first to determine the 3D structures of proteins, using X-ray crystallography. Since the crystallographic determination of these structures, there has been an almost exponential increase in the use of X-ray crystallography to determine the 3D structures of proteins, i.e., as evidenced by the history of structures deposited in the protein data bank. Comparison of the quaternary structures of liganded and deoxygenated horse Hb clearly showed significantly different conformational states. The Hb X-ray structures were the first to confirm the two-state allosteric theory put forward by Monod et al. (6), which is referred to as the MWC model. The liganded Hb conformation conformed to the MWC relaxed (R) state, while unliganded Hb conformation conformed to the MWC tense (T) state. The source of the tension in the T state was attributed to crosslinking salt bridges and hydrogen bonds between the subunits. The relaxed (R) state has only a few intersubunit hydrogen bonds and salt bridges. From: Methods in Molecular Medicine, vol. 82: Hemoglobin Disorders: Molecular Methods and Protocols Edited by: Ronald L. Nagel © Humana Press Inc., Totowa, NJ

1

2

Safo and Abraham

Muirhead and Greer (7) published the first structure of human adult deoxygenated hemoglobin (deoxyHbA). Several years later, Baldwin and Chothia (8,9) and Baldwin (9) published the structure of human adult carbonmonoxyhemoglobin (HbCOA), and Shaanan (10) published the structure of human adult oxyhemoglobin (oxyHbA). Interestingly, the structure of oxyHbA was delayed because of complications resulting from heme iron autoxidation. Subsequently, a new quaternary ligand-bound Hb structure known as R2 (11) or Y (12,13) provided another relaxed structure. R2 was proposed to be a low-energy intermediate in the T-to-R allosteric transition. However, further analysis has revealed that R2 is not an intermediate but, rather, another relaxed end-state structure (14). Quite recently, our laboratory discovered two more novel HbCO A relaxed structures (R3 and RR2); RR2 has a structural conformation between that of R and R2 (unpublished results). The quaternary structural difference between T and R3 is as large as that of T and R2. However, R2 and R3 have very different conformations. The quaternary difference is determined by superimposing the α1β1 subunit interfaces and calculating the rotation angle between the nonsuperimposed α2β2 dimers (8,9). The first 3D structures of horse Hb were solved using isomorphous replacement techniques (1–3,5). A number of published Hb structures also crystallize isomorphously, thus making it possible to use phases from the known isomorphous Hb structure for further structural analysis. The development of molecular replacement methods (15,16) for the solution of protein structures enabled routine structure solutions for nonisomorphous Hb crystals. When the structure horse Hb was determined, no computer refinement programs existed. Therefore, the atomic positions were refined visually against the electron density map. With isomorphous mutant crystals (17) or isomorphous crystals with bound allosteric effectors (18), simple electron density difference map calculations have been shown to be powerful tools in analyzing structural differences. Currently, all new protein structures are refined using modern, faster computing methods, such as CNS (19) and REFMAC (20). The crystal structures of more than 250 Hbs have been solved and published, including mutants and Hb cocrystalized with allosteric effector molecules. Selected examples of native and mutant Hbs including quaternary states, crystallization conditions, and unit cell descriptions are given in Tables 1–3. The structures of mutant Hbs provided the first concrete correlation between structural changes and disease states, while Hb cocrystallized with small effector molecules has advanced our understanding of the fundamental atomic-level interactions that regulate allosteric function of an important protein. The general methodologies for isolating, purifying, crystallizing and crystal mounting for data collection follow. The X-ray structure solution of Hb and variants is routine and employs the techniques discussed above: isomorphous

Name

Quater- Chemical nary state form

3

Crystallization condition

Unit cell characteristicsa a = 63.2, b = 83.5, c = 53.8 Å, β = 99.3°, SG = P21, AU = 1 tetramer a = 97.1, b = 99.3, c = 66.1 Å, SG = P21212, AU = 1 tetramer a = 63.2, b = 83.6, c = 53.9 Å, β = 99.2°, SG = P21, AU = 1 tetramer SG = P21, AU = 1 tetramer a = 53.7, b = 53.7, c = 193.0 Å, SG = P41212, AU = 1 dimer a = 53.7, b = 53.7, c = 193.8 Å, SG = P41212, AU = 1 dimer a = 97.5, b = 101.7, c = 61.1 Å, SG = P212121, AU = 1 tetramer a = 62.8, b = 62.8, c = 320.9 Å, SG = P43212, AU = 1 tetramer a = 61.5, b = 61.5, c = 176.3 Å, SG = P4122, AU = 1 dimer a = 65.5, b = 154.6, c = 55.3 Å, SG = P21212, AU = 1 tetramer a = 106.1, b = 86.2, c = 64.3 Å, SG = P212121, AU = 1 tetramer

DeoxyHbA

T

Normal

DeoxyHbA

T

Normal

RSR13-deoxy T HbA complexb T DeoxyHbFc OxyHbA R

Normal

2.2–2.8 M NH4 phosph/sulfate, pH 6.5 10–10.5% PEG 6000, 100 mM KCl, 10 mM K phosph, pH 7.0 2.5–2.9 M NH4 phosph/sulfate, pH 6.5

Fetal Normal

2.2–2.8 M NH4 phosph/sulfate, pH 6.5 2.25–2.75 M Na/K phosph, pH 6.7

HbCO A

R

Normal

2.25–2.75 M Na/K phosph, pH 6.7

HbCO A

R2

Normal

CO Gower II (α2ε2)d HbCO Ae

R2 R3

16% PEG 6000, 100 mM, Na cacodylate, pH 5.8 Embryonic 21% MME PEG 5000, 0.2 M TAPS-KOH, pH 8.5 Normal 2.34–2.66 M Na/K phosph, pH 6.4–6.7

HbCO Ae

RR2

Normal

2.34–2.66 M Na/K phosph, pH 6.4–6.7

CNMetHbAf

Y

Normal

16–17% PEG 8000, 0.1 M Tris, 0.12% BOG


Table 1 Crystallization Conditions and Structural Properties of Selected Human Hbs Resolution (Å)

Reference

1.7

25

2.15

26

1.85

27

2.5 2.1

28 10

2.7

8

1.7

11

2.9

29

2.65

Unpublished data Unpublished data 13

2.18 2.09

a SG

, space group; AU, and asymmetric unit. is an allosteric effector. c The authors of deoxyHbF did not provide the cell constants, however, the crystal is isomorphous to the high-salt deoxyHbA crystal (25). d The quaternary structure of carbonmonoxy embryonic Gower II Hb lies between that of R and R2 states, though closer to the R2 state. e Relaxed end-state structures (see text). f The quaternary structures of Y and R2 state Hbs are similar. b RSR13

3

4

Table 2 Crystallization Conditions and Structural Properties of Selected Natural Mutant Human Hbs Name


4

Unit cell characteristicsa

a = 52.9, b = 185.7, c = 63.3 Å, β = 92.6°, SG = P21, AU = 2 tetramers a = 63.2, b = 83.6, c = 53.8 Å, β = 99.4°, SG = P21, AU = 1 tetramer a = 97.1, b = 99.3, c = 66.1 Å SG = P21212, AU = 1 tetramer a = 63.2, b = 83.6, c = 53.8 Å, β = 99.4°, SG = P21, AU = 1 tetramer a = 93.1, b = 93.1, c = 144.6 Å SG = P3221, AU = 1 tetramer a = 54.38, b = 54.38, c = 195.53 Å, SG = P41212, AU = 1 dimer SG = P21, AU = 1 tetramer SG = P21, AU = 1 tetramer

DeoxyHbA Sickle cell

T

Glu6βVal

33% PEG 8000, 5.5 mM citrate, pH 4.0–5.0

Catonsville

T

2.2–2.8 M NH4 Phosph/sulfate pH 6.5

Rothschild

T

Pro37α-GluThr38α Trp37βArg

Thionville

T

10–10.5% PEG 6000, 100 mM KCl, 10 mM K phosph, pH 7.0 2.2–2.8 M NH4 phosph/sulfate, pH 6.5

Cowtown

R

His146βLeu

2.25–2.75 M Na/K phosph, pH 6.7

Knossosb GrangeBlancheb Brocktonb Suresnesb Kansas

T T

Ala27βSer Ala27βVal

2.2–2.8 M NH4 phosph/sulfate, pH 6.5 2.2–2.8 M NH4 phosph/sulfate, pH 6.5

T T T

Ala138βPro Arg141αHis Asn102βThr

2.2–2.8 M NH4 phosph/sulfate, pH 6.8 2.2–2.8 M NH4 phosph/sulfate, pH 6.5 2.2–2.8 M NH4 phosph/sulfate, pH 6.5

SG = P21, AU = 1 tetramer SG = P21, AU = 1 tetramer a = 63.4, b = 83.6, c = 53.9 Å, β = 99.3 o, SG = P21, AU = 1 tetramer

2.05

24

1.7

30

2.0

26

1.5

31

3.0

12

2.3

32

2.5 2.5

33 33

3.0 3.5 3.4

34 35 36

Safo and Abraham

COYpsilanti Y

Val1αGlu AcetMet(-1)1α Asp99βTyr 2.25–2.30 M Na/K phosph, pH 6.7

a SG,

Resolution (Å) Reference


space group and; AU, asymmetric unit. authors of Hb Knossos, Grange-Blanche, Brockton, and Suresnes did not provide the cell constants, however, the crystals are isomorphous to the high-salt deoxyHbA crystal (25). b The


Name Yα42H

5


Unit cell characteristicsa a = 62.4, b = 81.2, c = 53.3 Å, β = 99.65°, SG = P21, AU = 1 tetramer a = 63.3, b = 83.4, c = 53.8 Å, β = 99.5°, SG = P21, AU = 1 tetramer a = 54.3, b = 54.3, c = 194.1 Å SG = P41212, AU = 1 dimer a = 62.9, b = 81.3, c = 111.4 Å SG = P212121, AU = 1 tetramer a = 62.9, b = 82.0, c = 53.9 Å, β = 99.0°, SG = P21, AU = 1 tetramer a = 102.5, b = 115.2, c = 56.7 Å SG = P212121, AU = 1 tetramer a = 63.5, b = 83.2, c = 54.0 Å, β = 99.15°, SG = P21, AU = 1 tetramer a = 96.7, b = 98.7, c = 66.0 Å, SG = P21212, AU = 1 tetramer a = 63.2, b = 83.4, c = 53.8 Å, β = 99.4°, SG = P21, AU = 1 tetramer a = 63.2, b = 83.7, c = 53.8 Å, β = 99.4°, SG = P21, AU = 1 tetramer

T

Tyr42αHis

2.2–2.8 M NH4 phosph/sulfate, pH 6.5

rHb(α96Val→Trp) T

Val96αTrp


rHb(α96Val→Trp) R

Val96αTrp

2.25–2.75 M Na/K phosph, pH 6.7

Deoxy-Hbβ6W

T

Glu6βTrp

Deoxy-rHb1.1

T

CNmet-rHb1.1

B

Deoxy-βV67T

T

Des-Arg141αHbA T

4–7 uL of 33 % PEG 8000, 5 uL of Na citrate, pH 4.8 Asn108βLys 2.2–2.8 M NH4 phosph/sulfate, pH 6.5 α1-Gly-α2 Asn108βLys 13 % PEG 3350, 10 mM KCN, α1-Gly-α2 150 mM NH4 acetate, pH 5.0 Val67βThr 2.2–2.8 M NH4 phosph/sulfate, pH 6.5

Bulltown

T

des-Arg141α 10–10.5 % PEG 6000, 100 mM KCl, 10 mM K phosph, pH 7.0 His146βGln 2.2–2.8 M NH4 phosph/sulfate, pH 6.5

Deoxy-βV1M

T

Val1βMet

a SG,



Table 3 Crystallization Conditions and Structural Properties of Selected Artificial Mutant Human Hbs

Resolution (Å) Reference 1.8

38

1.9

39

2.5

39

2.0

40

2.0

41

2.6

41

2.2

42

2.1

43

2.6

44

1.8

45

space group; AU, asymmetric unit.

5

6

Safo and Abraham

replacement, difference electron density calculations, molecular replacement, and structure refinement (for details, see references). 2. Materials 2.1. Purification of Human Hb for Crystallization 1. HbA is purified from outdated human red blood cells (RBCs) unsuitable for transfusion (~500 mL). Sickle cell Hb (HbS) is purified from sickle cell blood, normally obtained from homozygote sickle cell patients who receive blood-exchange transfusions. To avoid clotting, blood samples are normally stored with about 1/10 vol of an anticoagulant agent, such as EDTA, heparin, or potassium citrate. 2. Buffer stock solution (5–10 L) containing 50 mM Tris buffer (pH 8.6) with EDTA: The solution is made by mixing 50 vol of 0.1 M Trizma base, 12.4 vol of 0.1 N Trizma hydrochloride, adjusting the volume to 100 mL with deionized water containing 4 g of EDTA (see Note 1). 3. Stock saline solutions (3 and 1 L) of 0.9% (9 g/L) and 1.0% NaCl (10 g/L), respectively. 4. DEAE sephacel and chromatography column equipment. 5. Cellulose dialysis tubes (Fisher Pittsburgh, PA). 6. Carbon monoxide gas cylinder (Matheson, Joliet, IL) (see Note 2). 7. NaCl, Na dithionite, and K2HPO4. 8. Three Erlenmeyers or side arm flasks (1 L).

2.2. Crystallization of Human Hb 1. Cyrstallization procedures will be described for deoxyHbA, deoxyHbS, and COHb A. These methods are also applicable to other HBs. HbA and HbS isolated and purified as described in Subheading 3.1.2. are used for all crystallization setups.

2.2.1. High-Salt Crystallization of T-State deoxyHbA 1. HbA solution (12 mL) (60 mg/mL or 6g%): Dilute the protein with deionized water if necessary to obtain the above concentration. 2. 3.6 M precipitant solution (50 mL) (pH 6.5): This is made by mixing 8 vol of 4 M (NH4)2SO4, 1.5 vol of 2 M (NH4)2HPO4, and 0.5 vol of 2 M (NH4)H2PO4. 3. Deionized water (100 mL). 4. Ten 8-mL sterile interior vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ). 5. Stoppered glass jar (Aldrich, St. Louis, MO). 6. Parafilm. 7. Pipets and pipet tips (100 and 1000 mL). 8. Three 15- to 25-mL beakers or volumetric flasks. 9. Graduated cylinders (10- and 50-mL). 10. Mixture of FeSO4 (2 g) and Na citrate (1.5 g). 11. A few grains of Na dithionite. 12. Test tube rack.


7

2.2.2. High-Salt Crystallization of R-State HbCO A 1. HbA solution (12 mL) (40 mg/mL or 4g%) in a 50-mL round-bottomed flask equipped with a stir bar and a greased stopcock adapter. 2. 3.4 M precipitant solution (40 mL) (pH 6.4): This is made by mixing 7 vol of 3.4 M NaH2PO4 and 5 vol of 3.4 M K2HPO4 (see Note 3). 3. Deionized water (100 mL). 4. Toluene (50 µL). 5. Ten 8-mL sterile interior vacutainer tubes (Becton Dickinson). 6. Stoppered glass jar (Aldrich). 7. Pipets and pipet tips (100 and 1000 mL). 8. A few grains of Na dithionite. 9. Carbon monoxide gas cylinder (Matheson) (see Note 2) and nitrogen gas cylinder. 10. Test tube rack. 11. Vacuum pump and rubber tubing.

2.2.3. Low-Salt Crystallization of T-State deoxyHbS 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

HbS solution (1.2 mL) (120 mg/mL or 12g%). 50% (w/v) polyethylene glycol (PEG) 6000 (12 mL). 0.2 M citrate buffer (1 mL), pH 4.0–5.0 (Hampton Research, Laguna Hills, CA). Deionized water (10 mL). Ten 3-mL sterile interior vacutainer tubes (Becton Dickinson). Parafilm. Stoppered glass jar (Aldrich). Pipets and pipet tips (100 and 1000 mL). Two 15- to 25-mL beakers. A few grains of Na dithionite.

2.3. Crystal Preparation and Mounting The methods described here are for deoxyHbA and COHb A, and are also applicable to other Hb cystals.

2.3.1. Room Temperature Data Collection 1. Vacutainer tube containing T- or R-state crystals. 2. Capillary sealant, such as epoxy or paraffin wax or any wax with a low melting point. 3. Disposable pipets and pipet rubber bulb. 4. Stainless steel blunt-end needles (Fisher). 5. Disposable syringes (3–5 mL) (Fisher Scientific). 6. Sterilized paper wicks (Hampton Research). 7. Thin-walled quartz or borosilicate capillaries (Charles Supper, Natick, MA), ranging in size from 0.1 to 1.2 mm. 8. Soldering iron. 9. Sharp tweezer.

8

Safo and Abraham

2.3.2. Cryogenic Temperature Data Collection 2.3.2.1. T-STATE DEOXYHBA CRYSTAL 1. 2. 3. 4. 5. 6. 7. 8.

Vacutainer tube containing T-state crystals. Glycerol (100 µL). Small Dewar flask with liquid nitrogen. Thin fiber loop with diameter slightly larger than longest crystal dimension (Hampton Research). Cryovial and cryovial tong (Hampton Research). Disposable pipets and pipet rubber bulb. Glass slides. A few grains of Na Dithionite.

2.3.2.2. R-STATE COHB CRYSTAL 1. Vacutainer tube containing R-state crystals. 2. Cryoprotectant solution made by mixing 60 µL of mother liquor and 5–8 µL of glycerol. 3. Thin fiber loop with diameter slightly larger than longest crystal dimension. 4. Disposable pipets and pipet rubber bulb. 5. Glass slides.

3. Methods 3.1. Purification of Human Hb for Crystallization About 90% of RBC content is made up of Hb, and in healthy human adults, HbA accounts for more than 90% of the human Hb protein, while other minor components, such as fetal HbF (~1%) and hemoglobin HbA2 (2 to 3%), make up the remainder. The method described here for isolating HbA and HbS from blood or RBCs, and further purification by ion-exchange chromatography, is a modified version of Perutz’s (21) protocol. This procedure, using appropriate buffer eluents, has also been used to separate other variant forms of human Hb and Hb from other species.

3.1.1. Purification of HbA 1. Place three Erlenmeyer or side-arm flasks in a walk-in refrigerator and chill to 4°C. 2. Centrifuge the RBCs at 600g for 20 min at 4°C. 3. Gently aspirate the supernatant solution (debris, plasma, and excess serum) from the centrifuge bottles and discard. 4. Wash the RBCs three times with an excess volume of 0.9% NaCl, and then once with 1.0% NaCl, each time centrifuging and discarding the supernatant solution. 5. Pool the RBCs into a chilled flask and lyse the cells by adding 1 to 2 vol of 50 mM Tris buffer, pH 8.6 (containing EDTA) (see Note 4). 6. Allow the mixture to stand on ice for 30 min with occasional gentle stirring. 7. Centrifuge the Hb solution at 10,000g for 2 h at 4°C.


9

8. Pool the supernatant Hb solution, which is free of cell debris, into a chilled flask, and slowly add NaCl (40–60 mg/mL of Hb solution) while stirring the solution. 9. Centrifuge the Hb solution at 10,000g for 1 to 2 h at 4°C to remove any remaining cell stroma. 10. Pool the clear supernatant Hb solution into a chilled flask and discard the “syrupy” pellet. 11. Dialyze the Hb solution against 50 mM Tris buffer, pH 8.6 (containing EDTA), at 4°C to remove NaCl or other low molecular weight impurities (see Note 5). 12. Further purify the dialyzed Hb by ion-exchange chromatography using DEAE sephacel to separate the HbA from other Hb components (see Note 6): a. Equilibrate the resin with 50 mM Tris buffer, pH 8.6. b. Run the Hb solution through the column with 50 mM Tris buffer, pH 8.6 (containing EDTA), to allow the various Hb bands to separate. HbA2 (light band color) elutes first, followed by HbA (dark band color). The HbA fractions can be examined for purity by electrophoresis and only pure fractions (dark band) pooled together. 13. Concentrate the pooled fractions (40–100 mg/mL) with an Amicon stirred cell (Model 402) to a final HbA concentration of about 80–120 mg/mL (see Note 7). 14. Store the concentrated HbA, which is essentially the oxygenated form, at –80°C or freeze in liquid nitrogen. Hb stored at this temperature can remain suitable for crystal growth experiments for several years.

3.1.2. Purification of HbS HbS from homozygous sickle cell blood is isolated and dialyzed as described for HbA in Subheading 3.1.1. (steps 1–11). The HbS solution is further purified on a DEAE sephacel ion-exchange column using a buffer gradient of 50 mM Tris buffer, pH 8.6 (containing EDTA), and 50 mM Tris buffer, pH 8.4 (containing EDTA) (see Note 1). 1. Elute first HbA2 Tris buffer at pH 8.6, then HbS at pH 8.4. 2. Concentrate the pure HbS, identified by electrophoresis and store as indicated for HbA in Subheading 3.1.1. (steps 13 and 14).

3.2. Crystallization of Human Hb DeoxyHbA crystallizes from either high-salt or low-salt precipitants (7,21). The ligand-bound R-state Hbs, such as oxyHbA, HbCO A, and MetHbA; generally crystallize under high-salt conditions (8–10,21), while the ligand-bound R2- or Y-state HbAs also crystallize mainly under low-salt conditions (11,13). The most common approach to crystallizing Hb is the Perutz’s (21) batch method. Alternatively, the vapor diffusion method of hanging or sitting drop (22) is used, especially when only a small amount of protein is available. Here, detailed crystallization is described for both T- and R-state human HbA and

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Table 4 High-Salt Crystallization of deoxyHbA Tube

3.6 M NH4 phosph/ sulfate (mL)

Deionized H2O (mL)

0.5 M Fe citrate (mL)

1 2 3 4 5 6 7 8 9 10

4.90 4.80 4.70 4.60 4.50 4.40 4.30 4.20 4.10 4.00

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90

0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

6g% deoxy- Final salt HbA (mL) conc. (M) 1 1 1 1 1 1 1 1 1 1

2.94 2.88 2.82 2.76 2.70 2.64 2.58 2.52 2.46 2.40

includes the high-salt crystallization of deoxyHbA and HbCO A and the lowsalt crystallization of deoxyHbS. The crystallization methods described are modified batch methods by Perutz (21) and Wishner et al. (23) and can also be applied to Hb mutants and Hb from other species. See Notes 8 and 9 for important precautions regarding setting up T- and R-state crystals, respectively.

3.2.1. High-Salt Crystallization of T-State deoxyHbA 1. The materials in Subheading 2.2.1., with the exception of the stoppered glass jar and the parafilm, are put in an antechamber of a glove box. The vacutainer tubes should be unstoppered, labeled as shown in Table 4, and arranged on a test tube rack. All containers, including those with solvents, should be left open. 2. Alternately evacuate and fill the antechamber with nitrogen while stirring the HbA solution for 10–20 min to obtain completely deoxyHbA, water, and precipitant solutions (see Note 10). 3. Purge the anaerobic chamber of the glove box with nitrogen to ensure a complete anaerobic condition. 4. Transfer all materials from the antechamber to the anaerobic chamber. 5. Add 25 mL of deionized water to the FeSO4 and Na citrate mixture and shake for about 30 s. 6. Allow the solution to settle and decant. Use the supernatant (Fe citrate) solution for all experiments (see Note 11). 7. Measure the volume of precipitant solution with a graduated cylinder, and add water to restore to the original volume of 50 mL (3.6 M), if necessary. 8. Measure the volume of deoxyHbA solution with a graduated cylinder, and add water to restore to the original volume of 12 mL (60 mg/mL), if necessary. 9. Add a few grains of Na dithionite (or ~2 mM) to the deoxyHbA solution to reduce any ferric heme that may be present.


11

Table 5 High-Salt Crystallization of HbCO Aa Tube

3.4 M Na/K phosph (mL)

4g% HbCO A (mL)

Final salt conc. (M)

1 2 3 4 5 6 7 8 9 10

3.80 3.60 3.40 3.20 3.00 2.80 2.60 2.40 2.20 2.00

1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

2.69 2.66 2.63 2.59 2.55 2.51 2.46 2.40 2.34 2.27

aA

drop or two of toluene is added to each tube.

10. Measure the precipitant solution and water and add to the vacutainer tubes as indicated in Table 4. 11. Measure 1- and 0.1-mL aliquots of deoxyHbA and Fe citrate, respectively, and add to each vacutainer tube. 12. Stopper each vacutainer tube, and tilt at least twice to mix the solution. 13. Remove all the materials from the glove box and wrap parafilm around the stopper of each vacutainer tube. 14. Store the sealed vacutainer tubes in greased, stoppered glass jars filled with nitrogen. Crystals normally appear within 3–10 d and vary in size from microscopic to as large as 8 mm in any direction. The crystals belong to space group P21 with approximate unit cell constants of a = 63 Å, b = 83 Å, c = 53 Å, and β = 99°.

3.2.2. High-Salt Crystallization of R-State HbCO A 1. Add a few grains of Na dithionite to 12 mL of HbA (40 mg/mL) in a roundbottomed flask (three to five times the size of the volume of the HbA solution) fitted with a stopcock adapter and connected to both a vacuum pump and a nitrogen gas source with rubber tubing. 2. Alternately evacuate and flush with nitrogen for about 10 min. 3. Connect a CO source to a disposable pipet with rubber tubing (see Note 2). 4. Open the flask containing the deoxyHbA solution, and quickly bubble CO through the solution to make the HbCO A derivative. 5. Reconstitute the volume to 12 mL (40 mg/mL) with CO-purged deionized water. 6. Bubble CO through the precipitant solution. 7. Measure the precipitant solution and add to the vacutainer tubes as indicated in Table 5. 8. Measure 1-mL aliquots of HbCO A and add to each vacutainer tube.

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Table 6 Low-Salt Crystallization of deoxyHbS Tube

50% PEG 6000 (mL)

Deionize water (mL)

0.2 M Citrate (mL)

1 2 3 4 5 6 7 8 9 10

1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 0.6

0.15 0.25 0.35 0.45 0.55 0.65 0.75 0.85 0.95 1.05

0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05

12g% deoxyHbS (mL) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

9. Add a drop or two of toluene to each vacutainer tube (see Note 12). 10. Slowly bubble CO through each vacutainer tube, stopper, and tilt at least twice to mix the solution. 11. Seal the vacutainer tubes with rubber stoppers and store in greased, stoppered glass jars filled with nitrogen to minimize formation of MetHbA. Crystals normally appear within 3–10 d. The crystals are octahedral and belong to space group P41212, with approximate unit cell constants of a = 53 Å, b = 53 Å, and c = 193 Å. The method described to crystallize HbCO A is applicable to both oxyHbA and MetHbA (see Note 13).

3.2.3. Low-Salt Crystallization of T-State deoxyHbS 1. Place all materials (except stoppered glass jar and parafilm) in the antechamber of the glove box. The vacutainers should be unstoppered and labeled as shown in Table 6. All containers, including those of solvents, should be left opened (see Note 14). 2. Deoxygenate the HbS and other solutions (5–10 min) in the antechamber of the glove box. 3. Purge the anaerobic chamber and transfer all materials from the antechamber into the anaerobic chamber. 4. Add deionized water to restore the volume of the HbS to 1.2 mL, if necessary. 5. Add a few grains of Na dithionite (or ~2 mM) to the HbS solution. 6. Add deionized water to restore the volume of the precipitant solution to 12 mL, if necessary. 7. Measure the precipitant solution and deionized water and add to the vacutainer tubes as shown in Table 6. 8. Measure 0.1 mL-aliquots of deoxyHbS and add to each vacutainer tube. 9. Stopper each vacutainer tube and tilt at least twice to mix the solution.


13

10. Store the vacutainer tubes and contents as described in Subheading 3.2.1. (steps 13 and 14). Crystals grown by this method are twinned (23,24) and must be separated before X-ray data can be obtained. Final crystals have the symmetry of the monoclinic space group P21, with approximate cell constants of a = 53 Å, b = 184 Å, c = 63 Å, and β = 93° (see Note 15).

3.3. Crystal Preparation and Mounting Hb crystals, like most other protein crystals, are fragile because of their high solvent content and should be handled with care. For room temperature data collection, Hb crystals are mounted and sealed in a thin-walled glass capillary about twice the size of the crystal. For cryogenic data collection, crystals are mounted in a thin fiber loop with a layer of suitable cryoprotectant around the crystal.

3.3.1. Room Temperature Data Collection T-state crystals are prepared and mounted in the glove box, while R-state crystals are mounted outside the glove box. However, to minimize autoxidation, mount R-state oxyHbA crystals as described for T-state crystals. 1. Select at least two 8-cm-long capillaries, and, using a soldering iron, melt a ring of wax close to the middle of the capillary. 2. Use a sharp tweezer to cut the bottom part of the capillary, just below the ring of wax. The top part of the capillary with the wide mouth is retained. Seal the cut bottom (with the ring of wax) with melted wax or epoxy (see Note 16). 3. Using a microscope, select a few good crystals by marking outside the vacutainer tube where those crystals are. 4. For R-state crystals, proceed to step 9. 5. For T-state crystals, place the materials in Subheading 2.3.1., in addition to the prepared capillaries, in the antechamber of the glove box. 6. Alternately evacuate and fill the antechamber with nitrogen for 5–10 min. 7. Transfer all the materials to a nitrogen-purged anaerobic chamber (see Note 17). 8. With a blunt-end needle, introduce a small amount of mother liquor from the vacutainer tube into the upper third of the capillary (all the way to the top). 9. Using a disposable pipet with a rubber bulb, suck a suitable marked crystal up onto the solution in the capillary. Allow the crystal to flow down to the air space. If the crystal is less dense than the mother liquor, invert the capillary to allow the crystal to flow to the air space. 10. Carefully push the crystal with a thin fiber or the blunt end of the needle into the air space. 11. Remove the solution from the capillary with a syringe and needle. 12. Carefully dry excess liquid from the crystal with a filter paper strip, a smaller cut capillary, or even the tip of the blunt-end needle. Leave a thin film of mother liquor between the crystal and the capillary wall (see Note 18).

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13. Reintroduce a small amount of mother liquor into the capillary, about 5 mm from the crystal (~5-mm-long liquid). Do not fill all the way to the top (see Note 19). 14. Close the capillary with melted wax or epoxy.

3.3.2. Cryogenic Temperature Data Collection T-state crystals are prepared and mounted in the glove box, and R-state crystals are mounted outside the glove box. Slightly different procedures are used, so a protocol for each is given next. 3.3.2.1. T-STATE DEOXYHBA CRYSTAL 1. Place the materials in Subheading 2.3.2.1. in the anaerobic glove box as already described in Subheading 3.3.1. (steps 5–7). 2. Submerge the cryovial in the Dewar liquid nitrogen using the cryovial tong. 3. Prepare cryoprotectant solution by mixing 50 µL of mother liquor, 10–16 µL of glycerol, and a few grains of Na dithionite (see Note 20). 4. Pick up a crystal with the disposable pipet, and place it into 5 µL of cryoprotectant solution on a glass slide for about 30 s. 5. Transfer the crystal to another 5 µL of cryoprotectant solution for another 30 s. 6. Use a fiber loop to scoop the crystal. 7. Plunge the loop containing the crystal and the drop of cryoprotectant directly into the cryovial which is submerged in the liquid nitrogen. 8. Take the closed cryovial out of the glove box and mount the crystal on the goniometer head in the cold nitrogen gas stream (see Note 21).

3.3.2.2. R-STATE COHB A CRYSTAL 1. Pick up a crystal with a disposable pipet, and introduce it into 5 µL of cryoprotectant solution on a glass slide for about 30 s. 2. Transfer the crystal to another 5 µL of cryoprotectant solution for another 30 s. 3. Scoop up the crystal, which has a protective cover of cryoprotectant liquid, with a fiber loop. 4. Place the fiber loop on the goniometer head in the cold nitrogen gas stream.

4. Notes 1. For HbS purification, prepare an additional 3–5 L of buffer stock solution containing 50 mM Tris buffer (pH 8.4) with EDTA. The solution is made by mixing 50 vol of 0.1 M Trizma base and 17.2 vol of 0.1 N Trizma hydrochloride, and adjusting the volume to 100 mL with deionized water containing 4 g of EDTA. 2. CO should be handled with great care; it is extremely toxic. All experiments involving CO should be done in a fume hood in a well-ventilated room. 3. Alternatively, a precipitant solution consisting of equal volumes of 3.4 M NaH2PO4 and 3.4 M K2HPO4 (pH 6.7) may be used and 0.2 mL of distilled water added to each tube. 4. EDTA helps prevent oxidation of ferrous heme to ferric heme by chelating any heavy metals that may act as catalysts for the autoxidation process. The final


5.

6.

7.

8.

9.

10.

15

concentration of the purified HbA will depend on the amount of buffer added to lyse the cell. Strips of standard cellulose dialysis tubing that have been washed three or four times and boiled for 10 min in deionized water are used for the dialysis. This is done to remove traces of impure compounds that may contaminate the HbA. The dialyzing buffer should be 50- to 200-fold of the HbA volume and should be continuously stirred overnight. If possible, the buffer should be changed every 2 to 3 h. Alternatively, HbA is dialyzed with 10 mM phosphate buffer, pH 7.0. The same type of buffer is then used to purify the HbA, as described in the text, using G25 Sephadex (fine) column. Alternatively, HbA is concentrated by ultrafiltration through an Mr 10,000 pellicon cassette. The concentration of HbA can be determined using the Perutz (21) procedure. The concentration is measured by taking 1 mL of HbA solution and diluting it with 19 mL of deionized water and 80 mL of 0.07 M K2HPO4. Na dithionite powder (0.2 g) is then added to the solution to generate the fully reduced deoxyhemoglobin derivative. CO is then bubbled through the solution to produce the COHb A derivative. The extinction coefficient is measured at 540 nm, and the concentration of HbA is calculated by dividing the optical density by 8.03. All crystallization steps for deoxyHbA are performed under rigorous anaerobic conditions in a nitrogen atmosphere glove box. It is critical that all crystallization solvents be purged of oxygen and stored under nitrogen. These precautions are necessary to prevent formation of oxyHbA or MetHbA. Human R-state COHb A , oxyHbA, and MetHbA crystallize isomorphously, and the corresponding structures are very similar. OxyHbA is very susceptible to autoxidation, which leads to formation of MetHbA during crystallization and data collection. To slow autoxidation, EDTA (1 mM) is added to the precipitating agents to chelate traces of heavy metals that catalyze the autoxidation process. Autoxidation of oxyHbA proceeds very rapidly when deoxyHbA is present in the solution; therefore, oxygen should be bubbled through the HbA solution to completely oxygenate all the HbA. In addition, crystallization should be performed at a low temperature, preferably 4°C, to slow down autoxidation. Even though HbCO A is fairly stable for a long period, the presence of oxygen leads to gradual oxidation of the ferrous heme. Therefore, crystallization of HbCO A should be under a CO atmosphere to avoid possible oxidation of the heme. All solutions for HbCO A crystallization should be purged with CO before use. A simple glove bag or Plexiglas box with gloves can be substituted for a more expensive glove box. If a glove bag or Plexiglas box is used, the HbA solution has to be deoxygenated outside the glove box. The HbA solution is put in a roundbottomed flask (three to five times the size of the volume of the HbA solution) and then connected by rubber tubing to both a vacuum pump and a nitrogen gas source with a glass stopcock adapter. The HbA is alternately evacuated and flushed with nitrogen for 30–60 min to obtain a deoxyHbA solution. (For smaller volume, the deoxygenation time is decreased.) A larger flask prevents boiling

16

11.

12.

13.

14.

15.

16. 17.

18.

Safo and Abraham HbA solution from getting into the vacuum line during the evacuation cycle. Additionally, to avoid undue boiling and splashing of the HbA, the flask containing the HbA solution may be cooled briefly in an ice bath before evacuation. Next, all materials are put into the glove bag or Plexiglas box. With the exception of the deoxyHbA solution, all other solution-containing flasks (precipitant, water, and buffer) should be left open. Once all the materials are put in the glove bag or Plexiglas box, it is then purged continuously with nitrogen for at least 40 min before the flask containing the HbA solution is opened. If the chamber is not airtight, it should be purged continuously with nitrogen during the crystallization experiments (Subheading 3.2.1., steps 5–14). Fe Citrate solution is prepared in situ from FeSO4 and Na citrate in the glove box and used fresh because the compound is unstable and easily oxidizes to ferric citrate. Fe citrate is a mild reducing agent and helps prevent oxidation of the iron; it also acts as an antimicrobial agent to prevent growth of bacteria and fungi. Toluene, like similar organic solvents, reduces the effective electrostatic shielding between the macromolecules by decreasing the electrostatic properties of the precipitating solutions. This facilitates increased contact between the macromolecules and serves to induce crystallization. The presence of toluene is also effective in preventing microbial growth. Recently, we have discovered two new crystal forms of HbCO A (R3 and RR2; see Table 1) that grows under the same crystallization conditions. One crystal form is rectangular and needle-like and belongs to the space group P4122. The other crystal form, which is also needle-like, belongs to the space group P212121. Alternatively, the HbA is deoxygenated outside the glove box as indicated above. For a small quantity of solution, the deoxygenation time is reduced accordingly (see Note 10, and continue from Subheading 3.2.3., steps 4–10). Crystals must be transferred to a stabilizing solution made of glutaraldehyde, which strengthens the crystals before cutting. Glutaraldehyde stabilizes the crystals by crosslinking the subunits. Soak the crystals for 1 d in a mixture of 35% (v/v) PEG stock solution, 20% (v/v) 0.2 M citrate buffer (pH 5.6), 45% (v/v) of 2% Drabkin’s buffer, and 10 mM of Na dithionite. The temperature of the solution is subsequently lowered to 3°C, and glutaraldehyde solution (50% [w/v]) is then added. The mixture is allowed to stand overnight at 3°C. Without the wax, the capillary may shatter when cut. If a glove bag or Plexiglas box is used, make sure that all necessary materials are put in the chamber and then purged continuously with nitrogen for at least 40 min before the vacutainer tube containing the crystals is opened. If the chamber is not airtight, it should be purged continuously with nitrogen during the experiments. A large amount of mother liquor around the crystal may decrease the resolution and increase mosaicity and background noise. The crystal can also move freely or slip. While making sure that as much liquid as possible is removed, do not completely dry the crystal. Excess drying will dehydrate the crystal, which may result in cracking, increased mosaicity, poor diffraction, disorder, and a large reduction in cell volume.


17

19. Mother liquor in the capillary ensures that the crystal is kept in the saturated vapor of the mother liquor during room temperature data collection to prevent drying. 20. Paraffin oil (Hampton Research) can also be used as a cryoprotectant. After putting the crystal in the paraffin oil, make sure that all excess mother liquor in the paraffin oil drop is removed by passing the crystal back and forth in the paraffin oil. The drop should form a perfectly clear glass under the cold stream. White patches may lead to reduction in resolution and increase mosaicity. 21. Simple freezing of the crystal will result in the formation of ice in the interior of the crystal and will render it useless. The cryoprotectant forms a noncrystalline glass, which protects the crystal from freeze shock.

References 1. Perutz, M. F., Rossmann, M. G., Cullis, A. F., Muirhead, H., Will, G., and North, A. C. T. (1960) Structure of haemoglobin. A three-dimensional Fourier synthesis at 5.5Å resolution obtained by x-ray analysis. Nature 185, 416–422. 2. Perutz, M. F., Muirhead, H., Cox, J. M., Goaman, L. C., Mathews, F. S., McGandy, E. L., and Webb, L. E. (1968) Three-dimensional Fourier synthesis of horse oxyhaemoglobin at 2.8 Å resolution: (1) x-ray analysis. Nature 219, 29–32. 3. Perutz, M. F., Muirhead, H., Cox, J. M., and Goaman, L. C. (1968) Three-dimensional Fourier synthesis of horse oxyhaemoglobin at 2.8 Å resolution: the atomic model. Nature 219, 131–139. 4. Ladner, R. C., Heidner, E. J., and Perutz, M. F. (1977) The structure of horse methaemoglobin at 2.0 Å resolution. J. Mol. Biol. 114, 385–414. 5. Bolton, W. and Perutz, M. F. (1970) The three dimensional Fourier synthesis of horse deoxyhaemoglobin at 2.8 Å resolution. Nature 228, 551, 552. 6. Monod, J., Wyman J., and Changeux J.-P. (1965) On the nature of allosteric transitions: a plausible model. J. Mol. Biol. 12, 88–118. 7. Muirhead, H. and Greer, J. (1970) Three-dimensional Fourier synthesis of human deoxyhaemoglobin at 3.5 Angstrom units. Nature 228, 516–519. 8. Baldwin, J. and Chothia, C. (1979) Haemoglobin: the structural changes related to ligand binding and its allosteric mechanism. J. Mol. Biol. 129, 175–220. 9. Baldwin, J. (1980) The structure of human carbonmonoxy haemoglobin at 2.7 Å resolution. J. Mol. Biol. 136, 103–128. 10. Shaanan, B. (1993) Structure of oxyhaemoglobin at 2.1 Å resolution. J. Mol. Biol. 171, 31–59. 11. Silva, M. M., Rogers, P. H., and Arnone, A. (1992) A third quaternary structure of human Hb at 1.7 Å resolution. J. Biol. Chem. 267, 17248–17256. 12. Smith, F. R., Lattman, E. E., and Carter, C. W. Jr. (1991) The mutation β99 AspTyr stabilizes a new composite quaternary state of human Hb. Proteins 10, 81–91. 13. Smith, F. R. and Simmons, K. C. (1994) Cyanomet human Hb crystallized under physiological condition exhibits the Y quaternary structure. Proteins 18, 295–300. 14. Janin, J.,and Wodak, S. J. (1993) The quaternary structure of carbonmonoxy Hb Ypsilanti. Proteins 15, 1–4. 15. Rossmann, M. G. and Hodgkin, D. C. (1972) in The Molecular Replacement Method (Rossmann, M. G., ed.), Gordon & Breach, New York, pp. 36–38.

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16. Navaza J. (1994) AMoRe: an automated package for molecular replacement. Acta Crystallogr. D50, 157–163. 17. Luisi, B. F. and Nagai, K. (1986) Crystallographic analysis of mutant human haemoglobins made in Escherichia coli. Nature 320, 555, 556. 18. Wireko, F. C., Kellogg, G. E., and Abraham, D. J. (1992) Allosteric modifiers of hemoglobin. 2. Crystallographic determined binding sites and hydrophobic binding/interaction analysis of novel hemoglobin oxygen effectors. J. Med. Chem. 34, 758–767. 19. Brunger, A. T., Adams, P. D., Clore, G. M., et al. (1998) Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D54, 905–921. 20. Murshudov, G., Vagin, A., and Dodson, E. (1997) Application of maximum likelihood methods for macromolecular refinement. Acta Crystallogr. D53, 240–255. 21. Perutz, M. F. (1968) Preparation of haemoglobin crystals. J. Crystal Growth 2, 54–56. 22. McPherson, A. (1982) Preparation and Analysis of Protein Crystals (McPherson, A., ed.), John Wiley & Sons, New York. 23. Wishner, B. C., Ward, K. B., Lattman, E. E., and Love, W. E. (1975) Crystal structure of sickle-cell deoxyHb at 5 Å resolution. J. Mol. Biol. 98, 179–194. 24. Harrington, D. J., Adachi, K., and Royer, W. E. Jr. (1997) The high resolution crystal structure of DeoxyHb S. J. Mol. Biol. 272, 398–407. 25. Fermi, G., Perutz, M. F., Shaanan, B., and Fourme, R. (1984) The crystal structure of human deoxyHb at 1.7 Å resolution. J. Mol. Biol. 175, 159–174. 26. Kavanaugh, J. S., Rogers, P. H., Case, D. A., and Arnone, A. (1992) High-resolution X-ray study of deoxyhemoglobin Rothschild 37β Trp ∏ Arg: a mutation that creates an intersubunit chloride-binding site. Biochemistry 31, 4111–4121. 27. Safo, M. K., Moure, C. M., Burnett, J., Joshi, G. S., and Abraham, D. J. (2001) High resolution crystal structure of deoxy T-state hemoglobin complexed with a potent allosteric effector. Protein Science 10, 951–957. 28. Frier, J. A., and Perutz, M. F. (1977) Structure of human foetal deoxyhaemoglobin. J. Mol. Biol. 112, 97–112. 29. Sutherland-Smith, A. J., Baker, H. M., Hofmann, O. M., Brittain, T., and Baker, E. D. (1998) Crystal structure of a human embryonic haemoglobin: the carbonmonoxy form of Gower II (α2ε2) haemoglobin at 2.9 Å resolution. J. Mol. Biol. 280, 475–484. 30. Kavanaugh, J. S., Moo-Penn, W. F., and Arnone, A. (1993) Accommodation of insertions in helices: the mutation in hemoglobin Catonsville (Pro 37α-Glu-Thr 38α) generates a 3(10) → α bulge. Biochemistry 32, 2509–2513. 31. Vasseur, C., Blouquit, Y., Kister, J., Prome, D., Kavanaugh, J. S., Rogers, P. H., Guillemin, C., Arnone, A., Galacterose, F., Poyart, C., Rosa, J., and Wajcman, H. (1992) Hemoglobin Thionville: An alpha-chain variant with a substitution of a glutamate for valine at NA-1 and having an acetylated methionine NH2 terminus. J. Biol. Chem. 267, 12,682–12,691.


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32. Derewenda, Z., Dodson, G., Emsley, P., Harris, D., Nagai, K., Perutz, M., and Reynaud, J.-P. (1990) Stereochemistry of carbon monoxide binding to normal and Cowtown haemoglobins. J. Mol. Biol. 211, 515–519. 33. Huang, Y., Pagnier, J., Magne, P., Bakloute, F., Kister, J., Delaunay, J., Poyart, C., Fermi, G., and Perutz, M. F. (1990) Structure and function of hemoglobin variants at an internal hydrophobic site: consequences of mutation at the beta 27 (B9) position. Biochemistry 29, 7020–7023. 34. Moo-Penn, W. F., Jue, D. L., Johnson, M. H., Olsen, K. W., Shih, D., Jones, R. T., Lux, S. E., Rodgers, P., and Arnone, A. (1988) Hemoglobin Brockton [β138(H16) Ala → Pro]: an unstable variant near the C-terminus of the b-subunits with normal oxygen-binding properties. Biochemistry 27, 7614–7619. 35. Poyart, C., Bursaux, E., Arnone, A., Bonaventura, J., and Bonaventura, C. (1980) Structural and functional studies of hemoglobin Suresnes (Arg 141α2 → His α2): consequences of disrupting an oxygen-linked anion-binding site. J. Biol. Chem. 255, 9465–9473. 36. Anderson, N. L. (1975) Structures of deoxy and carbonmonoxy haemoglobin Kansas in the deoxy quaternary conformation. J. Mol. Biol. 94, 33–49. 37. Tame, J. R. H. and Vallone, B. (2000) The structures of deoxy human haemoglobin and the mutant Hb Tyra42His at 120 K. Acta Crystallogr. D56, 805–811. 38. Puius, Y. A., Zou, M., Ho, N. T., Ho, C., and Almo, S. C. (1998) Novel watermediated hydrogen bonds as the structural basis for the low oxygen affinity of the blood substitute candidate rHb(α96Val → Trp). Biochemistry 37, 9258–9265. 39. Harrington, D. J., Adachi, K., and Royer, W. E. Jr. (1997) Crystal structure of deoxy-human hemoglobin Gluβ6 → Trp. Implication for the structure and formation of the sickle cell fiber. J. Biol. Chem. 273, 32,690–32,696. 40. Kroeger, K. S. and Kundrot, C. E. (1997) Structures of Hb-based blood substitute: Insights into the function of allosteric proteins. Structure 5, 227–237. 41. Pechik, I., Ji, C., Fidelis, K., Karavitis, M., Moult, J., Brinigar, W. S., Fronticelli, C., and Gilliland, G. L. (1996) Crystallographic, molecular modeling, and biophysical characterization of the Valineβ67 (E11) → Threonine variant of hemoglobin. Biochemistry 35, 1935–1945. 42. Kavanaugh, J. S., Chafin, D. R., Arnone, A., Mozzarelli, A., Rivetti, C., Rossi, G. L., Kwiatkowski, L. D., and Noble, R. W. (1995) Structure and oxygen affinity of crystalline desArg141 alpha human hemoglobin A in the T state. J. Mol. Biol. 248, 1136–1150. 43. Shih, D. T. B., Luisi, B. F., Miyazaki, G., Perutz, M. F., and Nagai, K. (1993). A mutagenic study of the allosteric linkage of His(HC3)146β in haemoglobin. J. Mol. Biol. 230, 1291–1296. 44. Kavanaugh, J. S., Rogers, P. H., and Arnone, A. (1992) High-resolution X-ray study of deoxy recombinant human hemoglobins synthesized from β-globins having mutated amino termini. Biochemistry 31, 8640–8647.

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Analysis of Hbs by HPLC

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2 Analysis of Hemoglobins and Globin Chains by High-Performance Liquid Chromatography Henri Wajcman 1. Introduction In recent years, high-performance liquid chromatography (HPLC) has become a reference method for the study of hemoglobin (Hb) abnormalities. This technique is used in two distinct approaches. The first is quantitative analysis of the various Hb fractions by ion-exchange HPLC, which is now done in routine hospital laboratories mostly by using fully automated systems. The second is reverse-phase (RP)-HPLC, which is of interest for more specialized studies (see Note 1). 2. Materials and Methods 2.1. Ion-Exchange HPLC Separation of Hbs Cation-exchange HPLC is the method of choice to quantify normal and abnormal Hb fractions (1–4). This is the method of reference for measuring glycated Hb for monitoring diabetes mellitus. It is also generally used for measuring of the levels of HbA2, HbF, and several abnormal Hbs. According to some researchers, this method could even replace electrophoretic techniques for primary screening of Hbs of clinical significance (3,5–7) or, at least, should be an additional tool for the identification of Hb variants (8). Automated apparatuses have been developed for large series measurement. I describe the Bio-Rad Variant Hemoglobin Testing System (Bio-Rad, Hercules, CA), using the β Thalassemia Short program as an example of this type of equipment.

From: Methods in Molecular Medicine, vol. 82: Hemoglobin Disorders: Molecular Methods and Protocols Edited by: Ronald L. Nagel © Humana Press Inc., Totowa, NJ

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2.1.1. Bio-Rad Variant Hb Testing System The Bio-Rad apparatus is a fully automated HPLC system, using double wavelength detection (415 and 690 nm). The β Thalassemia Short program is the most widely used system for HbA2 and HbF measurements, but other elution methods, including specific columns, buffers, and software, are available from the manufacturer according to the test to perform. This program has been designed to separate and determine, in 5 to 6 min, area percentage for HbA2 and HbF and to provide qualitative determinations of a few abnormal Hbs. Windows of retention time have been established for presumptive identification of the most commonly occurring Hb variants. The β Thalassemia Short program uses a 3.0 × 0.46 cm nonporous cation-exchange column that is eluted at 32 ± 1°C, with a flow rate of 2 mL/min, by a gradient of pH and an ionic strength made of two phosphate buffers provided by the manufacturer. This material and procedure have been used worldwide in many laboratories over the last several years. Since recommendations for experimental procedure are fully detailed by the manufacturer, I describe only a few additional notes of practical import. 1. Blood is collected on adenine citrate dextrose (ACD). 2. Samples for analysis (about 0.2% Hb) are obtained by hemolysis of 20 µL of blood in 1 mL of a buffer containing 5 g/L of potassium hydrogenophthalate, 0.5 g/L of potassium cyanide, 2 mL of a 1% solution of saponine, and distilled water. This procedure for sample preparation, which is currently used for HPLC determination of HbA1c, avoids some of the Hb components present in low amounts (about 1%) eluted together with HbF in the HbF retention time window (8). 3. Twenty microliters of hemolysate is applied onto the column for analysis.

Under these experimental conditions an excellent agreement is found between chromatographic measurement of HbF, down to 0.2%, and resistance to alkali denaturation, up to 15% (9). Presumptive identification of the most commonly occurring variants (Hb S, HbC, HbE, and HbD Punjab) is made using the retention time windows named S-Window, D-Window, A2-Window, and C-Window, which have been specified by the manufacturer. Aged Hb specimens display some degraded products that are eluted in the P2 and P3 windows (e.g., glutathione-Hb) (Table 1). Slight differences in the elution time of the various Hb components are observed from column to column and from one reagent batch to another, which should be taken into account by a program supplied by the manufacturer. The elution time of an Hb component varies also slightly according to its concentration in the sample. For a given column, a more accurate calibration than that proposed by the manufacturer could be obtained using HbA2 as reference. The concentration of this Hb, which varies between narrow limits, prevents significant modification of its elution time.


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Table 1 Analyte Identification Windowa Analyte name F P2 P3 A0 A2 D-window S-window C-window a Example

Retention time (min)

Band (min)

Window (min)

1.15 1.45 1.75 2.60 3.83 4.05 4.27 5.03

0.15 0.15 0.15 0.40 0.15 0.07 0.15 0.15

1.00 -1.30 1.30-1.60 1.50-1.90 2.20-3.30 3.68-3.98 3.98-4.12 4.12-4.42 4.88-5.18

provided by manufacturer.

Two methods are available for comparing data when the elution time of HbA2 differs between two runs done with a different column or reagent batch. The first consists of slightly modifying the experimental procedure (temperature or pH) to reproduce exactly the elution times of the previous runs. The second method consists of establishing a normalized retention scale taking as references two Hbs eluted within a linear part of the gradient. The elution patterns of more than 100 variants have been published, but, in my opinion, these data should be used as a confirmatory test for characterization of a variant after a careful multiparameter electrophoretic study (8) rather than as a primary identification method.

2.1.2. Alternative Methods When a dedicated machine is not available for Hb analysis, or when the chromatographic separation is done for “preparative” purposes, alternative techniques have to be used. These procedures are suitable for conventional HPLC equipment. Several anion-exchange and cation-exchange HPLC columns may be used for Hb separation; some are silica based and others are synthetic polymers. These methods have been well standardized for several years (10,11). PolyCat A (Poly LC, Columbia, MD) is one of the more popular phases for Hb separations (6). It consists of 5-µm porous (100-nm) spherical particles of silica coated with polyaspartic acid. For analytical purposes, a 5.0 × 0.40 cm column is used; elution is obtained at 25°C with a flow rate of 1 mL/min, by developing in 20 min at pH 6.58 a linear gradient of ionic strength from 0.03 to 0.06 M NaCl in a 50 mM Bis-Tris, 5 mM KCN buffer. The presence of KCN is necessary to convert methemoglobin into cyanmethemoglobin, which displays ion-exchange chromatographic properties similar to those of oxyhemoglobin (see Note 2).

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2.2. HPLC Analysis of Globin Chains 2.2.1. Analysis of HbF Composition (see Note 3) The solvent system, acetonitrile–trifluororoacetic acid (TFA), which is used for RP-HPLC, dissociates the Hb molecule into its subunits and removes the heme group. This method is therefore used to analyze or separate the globin chains. This kind of study may be useful in the investigation of many human Hb disorders. For instance, the determination of HbF composition (Gγ:Aγ ratio) is of interest in several genetic and acquired disorders. A good separation is obtained between the Gγ and AγI, with most of the RP columns by using a very flat acetonitrile gradient. By contrast, it is often much more difficult to separate Gγ from AγT, a frequent allele of AγI. Among the procedures that have been successfully proposed for this analysis, one of the most popular is the RP-HPLC method described by Shelton et al. (12). They used a Vydac C4 column (The Separation Group, Hesperia, CA) eluted at a flow rate of 1 mL/min by developing in 1 h a linear gradient from 38 to 42% acetonitrile in 0.1% TFA with detection at 214 nm. Under these conditions, the chains were eluted in the following order: β, α, AγT, Gγ, and AγI. In recent years, a modification introduced in the manufacturing process of this type of column (13) made necessary the use the higher acetonitrile concentrations to elute the γ-chains. Unfortunately, it also resulted in the low resolution of AγT. 2.2.1.1. RP PERFUSION CHROMATOGRAPHY

Perfusion chromatography involves a high-velocity flow of the mobile phase through a porous chromatographic particle (14–16). The Poros R1® media (Applied Biosystems, Foster City, CA) used in this technique consists of 10-µm-diameter particles. These particles are made by interadhering under a fractal geometry poly(styrene-divinylbenzene) leading to throughpores of 6000- to 8000-Å-diameter microspheres with short, diffusive 500- to 1000-Ådiameter pores connected to them. As a result, relatively low pressures are obtained under high flow rates. The Poros R1® beads may be considered a fimbriated stationary phase having retention properties somewhat similar to those of a classic C4 support (15). The column (10 × 0.46 cm) is packed on a conventional HPLC machine at a flow rate of 8 mL/min using the Poros selfpack technology® according to the manufacturer’s protocol. More than a thousand runs may be performed without alteration of the resolution. 2.2.1.1.1. Sample Preparation 1. Samples containing about 0.1 mg of Hb/mL are obtained by lysis, in 1 mL water (or 5 mM KCN), of 2–5 µL of washed red blood cells (RBCs). 2. Membranes are removed by centrifuging at 6000g for 10 min.


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3. According to the HbF level, 20–100 µL of these hemolysates are applied onto the column. To avoid additional chromatographic peaks owing to glutathione adducts, 10 µL of a 50 mM solution of dithiothreitol in water is added per 100 µL of sample. An in-line stainless steel filter (0.5-µm porosity) needs to be used to protect the column.

2.2.1.1.2. Equipment. Any conventional HPLC machine can be used. In the method described here, the analyses were performed on a Shimadzu LC-6 HPLC machine equipped with an SCL-6B system controller, an SIL-6B autoinjector, and a C-R5A integrator (Shimadzu, Kyoto, Japan). A flow rate of 3.0–4.5 mL/min was convenient for synchronization of injection, integration, and column equilibration. 2.2.1.1.3. Experimental Procedure (see Note 4). Using a flow rate of 3 mL/min, the various γ-chains are isolated by developing in 9 min a linear gradient from 37 to 42% acetonitrile in a 0.1% solution of TFA in water. In practice, this is done by using two solvents (A: 35% acetonitrile, 0.1% TFA in water; B: 50% acetonitrile, 0.1% TFA in water) and a linear gradient from 15 to 45% B. Before injection, the column is equilibrated by a 10 column volume wash with the starting solvent, thus allowing completion of a cycle of analysis every 14 min. Elution is followed at 214 nm (wavelength at which double bonds absorb), and the recorder is set to 0.08 AUFS. Higher flow rates may be used, but the slope of the gradient will need to be increased in proportion. Keeping the same initial and final acetonitrile concentrations as above, elution is achieved in 6 min at a flow rate of 4.5 mL/min and in 4 min at a flow rate of 6.0 mL/min.

2.2.2. RP-HPLC Analysis of Globin Chains (see Note 5) Globin chain analysis is also important as an additional test that allows discrimination between Hb variants for the identification of structural abnormalities. Several RP-HPLC procedures have been proposed (10,14,17,18). On a conventional HPLC apparatus, a 20 × 0.46 cm column packed with Lichrospher 100 RP8 (Merck, Darmstadt, Germany) is used. Samples are prepared as described in Subheading 2.2.1.1.1. Elution is obtained at 45°C with a flow rate of 0.7 mL/min using a 90-minute linear gradient of acetonitrile, methanol, and NaCl made by a mixture of two solvents (18). Solvent A contains acetonitrile, methanol, and 0.143 M NaCl, pH 2.7 (adjusted by a few drops of 1 N HCl), in the proportion of 24, 38, and 36 L/L, respectively. Solvent B is made from the same reagents but in the proportion of 55, 6, and 39 L/L, respectively. The gradient starts with 10% B and ends with 70% B. The design of the gradient may be modified according to the machine, the geometry of the column, and the separation to be achieved. Elution can be followed at 214 or 280 nm. Globin chains are eluted in the same order as on the Vydac C4 column.

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A kit for globin chain analysis with similar performance is also commercially available from Bio-Rad (ref. 270.0301).

2.2.3. Scaled Up Methods for Chain Separation For biosynthetic or structural studies, milligram amounts of globin chains need to be separated. This can be achieved either by scaling up the RP-HPLC procedure using semipreparative size columns or by cation-exchange -HPLC done in the presence of dissociating concentrations of urea. 2.2.3.1. SEMIPREPARATIVE SIZE RP-HPLC COLUMNS

2.2.3.1.1. Samples. Globin solution rather than Hb solution is used. Globin is prepared from a 1% Hb solution obtained by hemolysing washed RBCs in distilled water. Stromas are removed by centrifuging at 6000g for 30 min, and the globin is precipitated by the acid acetone method. Usually, the sample is made from 1 to 2 mg of globin dissolved in 250 µL of 0.1% TFA, which requires the use of a 500-µL injection loop. 2.2.3.1.2. Chromotographic Procedure. A 240 × 10 mm Vydac C4 column (ref. 214TP510) is used. Elution is obtained by a gradient of acetonitrile in 0.1% TFA made by two solvents (solvent A contains 35% acetonitrile and solvent B 45%). A typical elution program, using a flow rate of 1.2 mL/min, consists of a 10-min equilibration at 35% B, 70 min of a linear gradient from 35 to 55% B, 30 min of a linear gradient from 55 to 90% B, and 5 min of an isocratic step at 90% B for cleaning the column. Elution of the column is followed at 280 nm with a full scale of 0.16 absorbance units (AU). 2.2.3.2. CATION-EXCHANGE HPLC IN PRESENCE OF 6 M UREA USING A POLYCAT COLUMN

Procedures that are modified from the classic CM cellulose chromatography described by Clegg et al. (19) may be transposed to the HPLC technology (20). The retention capacity of this type of column is higher than that of RP supports, allowing the handling of larger samples. I describe here a method using a PolyCat 300-Å, 10-µm particle column (150 × 4 mm). 2.2.3.2.1. Reagents and Buffers. Two buffers are used. Buffer A consists of 6 M urea, 0.1 M sodium acetate, and 0.4% β-mercaptoethanol, with the pH adjusted to 5.8 by acetic acid. Buffer B consists of 6 M urea, 0.25 M sodium acetate, and 0.35% β-mercaptoethanol, with the pH adjusted to 5.8 by acetic acid. Both buffers need to be filtered through a membrane with 0.45-µm porosity before being used. In addition, an in-line stainless steel filter (0.5-µm porosity) is needed to protect the column. 2.2.3.2.2. Samples. Up to 5–10 mg of globin, prepared by the acid acetone method, is dissolved in 200–600 mL of buffer A.


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2.2.3.2.3. Chromatographic Procedure. Elution is obtained by a gradient of ionic strength developed with the two buffers. A typical elution program, using a flow rate of 1.0 mL/min, consists of a 10-min equilibration at 0% B, 5 min of a linear gradient from 0 to 25% B, 50 min of a linear gradient from 25 to 100% B, and 5 min of an isocratic step at 100% B for cleaning the column. Elution of the column is followed at 280 nm with a full scale of 0.32 UA. 3. Notes 1. Why should one method be preferred over another? The choice of a separation method between RP or ion-exchange chromatography depends on the purpose of the separation. Ion-exchange is the only chromatographic method that allows preparation of native Hb fractions. The presence of cyanide ions in the buffers (or during sample preparation) will nevertheless hinder any further oxygenbinding study. If the aim of the separation is to obtain Hbs suitable for functional studies, the technique will have to be modified accordingly by removing cyanide from all the steps. It may be of interest in some cases to work with carbonmonoxyhemoglobin, since Hb is very stable under this form and procedures are available to return to the oxyform. For several applications, salts in excess also need to be removed. RP separation methods always lead to denatured proteins that cannot be used for functional studies. Techniques involving an ionic strength gradient can only be used for analytical purposes. By contrast, using fully volatile buffers, such as the acetonitrile-TFA system, the isolated globin fractions can be vacuum dried and readily used for further structural studies such as mass spectrometry measurements. 2. To isolate amounts of Hb in the milligram range, larger columns (15.0 × 0.46 cm) may be used. According to the separation to be achieved, the dimensions of the column, and the apparatus used, slightly different experimental conditions may have to be designed. Elution is followed at 415 nm for analytical purposes or at 540 nm in preparative runs. This buffer system is not suitable for ultraviolet (UV) detection. The use of an in-line stainless steel filter (0.5-µm porosity) is recommended to increase the column life expectancy. Reproducibility requires careful preparation of the buffers and temperature control. Since in these chromatographic methods the elution is recorded at one of the wavelengths of absorption of the heme, any factor modifying the absorption spectrum of the Hb molecule will hinder accurate quantitative measurement. For instance, unstable Hb variants, which lose their heme groups or lead to hemichrome formation, will be underestimated. HbMs, which are hardly converted into cyanmethemoglobin, display a much higher extinction coefficient than oxyhemoglobin at 415 nm and a lower one at 540 nm. As a consequence, HbMs will be overestimated when measured at the first wavelength, and underestimated at the second one. A modified experimental procedure allowing for a simultaneous measurement of HbF, glycated Hb, and several other Hb adducts has been proposed by using a combination of pH and ionic gradients (11).

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3. In my laboratory, for routine determination of the γ-chain composition, we replaced this procedure with an RP perfusion chromatography using a Poros R1® column (Applied Biosystems) (14). 4. To obtain good reproducibility, we recommend using the same glassware for preparing the solvents. Solvents may be kept refrigerated at 4°C for a few days. Accurate balance of the TFA between both solvents is important to avoid baseline drift. Acetonitrile must be of HPLC grade with low UV absorbancy in the 210-nm region. With this Poros R1 column, the α-chain is eluted before the β-chain. Resolution may be improved by modifying the geometry of the column or the design of the gradient. A 10 × 0.2 cm column may be used to improve separation between the various γ- or adult chains. In this case, with a flow rate of 1 mL/min, after 5 min of equilibration at 5% B, the column is eluted using a 15-min linear gradient between 5 and 25% B of the described solvents. This is followed by a 2-min isocratic elution at 25% B. 5. Several columns may be used, but I have found that a method adapted from that described in ref. 17 leads to a good resolution. Other columns or techniques may nevertheless be more appropriate for some specific separations. When chromatographic methods are used for globin chain quantification, it is important to consider the absorption coefficient of the various chains at the wavelength of detection. In some cases, it may be identical, such as when comparing the various γ-chains. In other cases, the absorption may differ considerably; for example, at 280 nm, γ-chains, because of their 3 Trp residues, have a higher ε coefficient than β-chains (2Trp) and α-chains (1 Trp). Abnormal Hbs containing a number of aromatic residues different from the normal may also display modified absorption coefficient.

References 1. Wilson, J. B., Headlee, M. E., and Huisman, T. H. J. (1983) A new high-performance liquid chromatographic procedure for the separation and quantitation of various hemoglobin variants in adults and newborn babies. J. Lab. Clin. Med. 102, 174–185. 2. Kutlar, A., Kutlar, F., Wilson, J. B., Headlee, M. E., and Huisman, T. H. J. (1984) Quantitation of hemoglobin components by high-performance cation-exchange liquid chromatography: its use in diagnosis and in the assessment of cellular distribution of hemoglobin variants. Am. J. Hematol. 17, 39–53. 3. Rogers, B. B., Wessels, R. A., Ou, C. N., and Buffone, G. J. (1985) High-performance liquid chromatography in the diagnosis of hemoglobinopathies and thalassemias. Am. J. Clin. Pathol. 84, 671–674. 4. Samperi, P., Mancuso, G. R., Dibenedetto, S. P., Di Cataldo, A., Ragusa, R., and Schiliro, G. (1990) High performance liquid chromatography (HPLC): a simple method to quantify HbC, O-Arab, Agenogi and F. Clin. Lab. Haematol. 13, 169–175. 5. Shapira, E., Miller, V. L., Miller, J. B., and Qu, Y. (1989) Sickle cell screening using a rapid automated HPLC system. Clin. Chim. Acta 182, 301–308.


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6. Ou, C. N. and Rognerud, C. L. (1993) Rapid analysis of hemoglobin variants by cation-exchange HPLC. Clin. Chem. 39, 820–824. 7. Papadea, C. and Cate, J. C. (1996) Identification and quantification of hemoglobins A, F, S, and C by automated chromatography. Clin. Chem. 42, 57–63. 8. Riou, J., Godart, C., Hurtrel, D., Mathis, M., Bimet, C., Bardakdjian-Michau, J., Préhu, C., Wajcman, H., and Galactéros, F. (1997) Evaluation of cation-exchange high-performance liquid-chromatography for presumptive identification of hemoglobin variants. J. Clin. Chem. 43, 34–39. 9. Préhu,C., Ducrocq, R., Godart, C., Riou, J., and Galactéros, F. (1998) Determination of HbF levels: the routine methods. Hemoglobin 22, 459–467. 10. Huisman, T. H. J. (1998) Separation of hemoglobins and hemoglobin chains by high performance liquid chromatography. J. Chromatogr. 418, 277–304. 11. Bisse, E. and Wieland, H. (1988) High-performance liquid chromatographic separation of human hemoglobins. Simultaneous quantitation of fetal and glycated hemoglobins. J. Chromatogr. 434, 95–110. 12. Shelton, J. B., Shelton, J. R., and Schroeder, W. A. (1984) High-performance liquid-chromatographic separation of globin chains on a large-pore C4 column. J. Liq. Chromatogr. 7, 1969–1977. 13. Vydac. (1994–1995) HPLC columns and separation materials, Technical Bulletin. 14. Wajcman, H., Ducrocq, R., Riou, J., Mathis, M., Godart, C., Préhu, C., and Galacteros, F. (1996) Perfusion chromatography on reversed-phase column allows fast analysis of human globin chains. Anal. Biochem. 237, 80–87. 15. Afeyan, N. B., Gordon, N. F., Mazsaroff, I., Varady, L., Fulton, S. P., Yang, Y. B., and Regnier, F. E. (1990) Flow-through particles for the high-performance liquid chromatographic separation of biomolecules: perfusion chromatography. J. Chromatogr. 519, 1–29. 16. Afeyan, N. B., Fulton, S. P., and Regnier, F. E. (1991) Perfusion chromatography material for proteins and peptides. J. Chromatogr. 544, 267–279. 17. Leone, L., Monteleone, M., Gabutti, V., and Amione, C. (1985) Reversed-phase high performance liquid chromatography of human hemoglobin chains. J. Chromatogr. 321, 407–419. 18. Wajcman, H., Riou, J., and Yapo, A. P. (2002) Globin Chains Analysis by RP-HPLC: recent developments. Hemoglobin 26, 271–284. 19. Clegg, J. B., Naughton, M. A., and Weatherall, D. J. (1966) Abnormal human hemoglobins: separation and characterization of the a and b chains by chromatography, and the detereminatioin of two new variants, Hb Chesapeake and Hb J (Bangkok) J. Mol. Biol. 19, 91–108. 20. Brennan, S. O. (1985) The separation of globin chains by high pressure cation exchange chromatography. Hemoglobin 9, 53–63.

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Purification and Molecular Analysis of Hb by HPLC

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3 Purification and Molecular Analysis of Hemoglobin by High-Performance Liquid Chromatography Belur N. Manjula and Seetharama A. Acharya 1. Introduction Hemoglobin (Hb) is a tetrameric protein (mol wt = 64,500) and is the major protein component of red blood cells (RBCs). In normal human erythrocytes, HbA composes about 90% of the total Hb. It is made up of two identical α-chains and two identical β-chains. Besides HbA, human erythrocytes contain small amounts of other forms of Hb as fetal hemoglobin (HbF, α2γ2) and HbA2 (α2δ2), and products of posttranslational modifications as HbA1c. HbS, the sickle cell Hb, is a genetic variant of HbA and is the most widely studied pathological form of Hb (1). Hb is a subject of active research not only for its molecular, genetic, and clinical aspects, but also as a prototype of allosteric proteins. Purification and characterization of Hbs has become easier and faster with the advent of highpressure and high-performance instrumentation, high-sensitivity detectors, and the availability of a wide variety of high-resolution column-packing materials. Methodological development using very small quantities of the protein is feasible, and the analytical methods are readily scalable. Here, we describe three different modes of high-performance liquid chromatography (HPLC) that are used in our laboratory for the purification and characterization of Hb, and modified or mutant Hb. Hb is purified by ion-exchange chromatography (IE-HPLC), its size is analyzed by size-exclusion chromatography (SEC-HPLC) (under native and dissociating conditions), its globin chain separation is accomplished by reverse phase HPLC (RP-HPLC), and tryptic peptide mapping of globin chains is also carried out by RP-HPLC. Preparative runs are generally carried out on an From: Methods in Molecular Medicine, vol. 82: Hemoglobin Disorders: Molecular Methods and Protocols Edited by: Ronald L. Nagel © Humana Press Inc., Totowa, NJ

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AKTA Protein Purification System (Amersham Pharmacia Biotech), which is also used for analytical runs. Other instrumentation used for the analytical runs includes a fast protein liquid chromatography (FPLC) system (Amersham Pharmacia Biotech) for SEC and ion-exchange chromatography, and a Shimadzu Liquid Chromatography System for RP-HPLC. Examples of ionexchange chromatographic purifications are given for analytical-scale runs (100 µg to 1 mg), small-scale preparative runs (up to 50 mg), and large-scale preparative runs (up to 3 g). The analytical-scale runs are useful not only for methodological development, but also for characterization purposes and for monitoring the progress of a chemical modification reaction. The SEC-HPLC runs are illustrated with analytical (1 mg) and semipreparative runs (100 mg). Examples of RP-HPLC are for analytical-scale runs (120 µg). 2. Materials 2.1. Purification of Human Hb by Ion-Exchange Chromatography (see Notes 1 and 2)

2.1.1. Anion-Exchange Chromatography 2.1.1.1. PURIFICATION OF HB ON DEAE-SEPHAROSE FAST FLOW: SMALL-SCALE PURIFICATION (SEE NOTES 1 AND 2) 1. 2. 3. 4. 5.

XK 16/10 chromatographic: column (Amersham Pharmacia Biotech). DEAE-Sepharose Fast Flow anion exchanger: (Amersham Pharmacia Biotech). Buffer A: 50 mM Tris-Ac, pH 8.5. Buffer B: 50 mM Tris-Ac, pH 7.0. Amersham Pharmacia Biotech AKTA Protein Purification System.

2.1.1.2. PREPARATIVE-SCALE PURIFICATION OF HB ON Q-SEPHAROSE HIGH PERFORMANCE CHROMATOGRAPHIC COLUMN 1. 2. 3. 4. 5.

XK26/70 column (Amersham Pharmacia Biotech) (see Notes 1 and 2). Q-Sepharose High Performance column-packing material (Amersham Pharmacia Biotech). Buffer A: 50 mM Tris-Ac, pH 8.5. Buffer B: 50 mM Tris-Ac, pH 7.0. Amersham Pharmacia Biotech AKTA Protein Purification System.

2.1.1.3. CATION-EXCHANGE CHROMATOGRAPHY: RECHROMATOGRAPHY OF Q-SEPHAROSE HIGH PERFORMANCE PURIFIED HBA ON CM-SEPHAROSE FAST FLOW 1. 2. 3. 4. 5.

XK26/70 chromatographic column (Amersham Pharmacia Biotech). CM-Sepharose Fast Flow cation exchanges (Amersham Pharmacia Biotech). Buffer A: 10 mM potassium phosphate, pH 6.35, 1 mM EDTA. Buffer B: 15 mM potassium phosphate, pH 8.5, 1 mM EDTA. Amersham Pharmacia Biotech AKTA Protein Purification System.


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2.2. Ion-Exchange Chromatography as an Analytical Tool 2.2.1. Characterization of Recombinant Hb by Cation-Exchange Chromatography on a Mono S Column 1. Mono S HR5/5 column (1 mL) (Amersham Pharmacia Biotech) (see Notes 3 and 4). 2. Buffer A: 10 mM potassium phosphate, pH 6.5. 3. Buffer B: 15 mM potassium phosphate, pH 8.5. 4. Pharmacia FPLC protein purification system. 5. Shimadzu UV-VIS detector at 540 nm. 6. Shimadzu Chromatopac CR7A plus data processor.

2.2.2. Monitoring Progress of a Chemical Modification Reaction Analysis of Amidated HbS by Analytical Anion-Exchange Chromatography on HiTrap Q Column 1. 2. 3. 4.

HiTrap Q, 1 mL column (Amersham Pharmacia Biotech) (see Note 3). Buffer A: 50 mM Tris-Ac, pH 8.5. Buffer B: 50 mM Tris-Ac, pH 7.0. Amersham Pharmacia Biotech AKTA Protein Purification System.

2.3. SEC of Hb 2.3.1. Analytical SEC 1. Pharmacia Superose 12 HR 10/30, two columns in series (column volume [CV], 47 mL) (see Note 4). 2. Buffer: 50 mM Bis-Tris (pH 7.4) or phosphate-buffered saline (PBS), pH 7.4, for analysis of tetrameric and size-enhanced Hbs; 50 mM Bis-Tris and 0.9 M MgCl2 (pH 7.4), for evaluating the stabilization of the tetrameric structure of Hb. 3. Pharmacia FPLC Protein Purification System. 4. Detector: Shimadzu UV-VIS detector at 540 nm. 5. Shimadzu CR7A plus data processor.

2.3.2. Semipreparative SEC 1. 2. 3. 4.

Pharmacia XK26/70 column. Superose 12 prep-grade packing material (Pharmacia). Buffer: PBS, pH 7.4. Pharmacia Biotech AKTA Protein Purification System.

2.4. Globin Chain Analysis of Hb by RP-HPLC Analysis 1. 2. 3. 4.

Column: Vydac Protein C4 column (4.6 × 250 mm). Solvent A: H2O, 0.1% trifluoroacetic acid (TFA). Solvent B: acetonitrile, 0.1% TFA. Shimadzu Liquid Chromatography System consisting of two LC-6A pumps, an SPD-6A UV detector, an SCL-6B System Controller, and Class VP chromatography software.

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3. Methods 3.1. Purification of Human HbA by Ion-Exchange Chromatography HbA is purified from erythrocytes obtained from adult human blood. The erythrocytes are gently washed with cold PBS, pH 7.4, and lysed with 4 volumes of water. The lysate containing the Hb is separated from the cell debris by centrifugation. The lysate is dialyzed extensively against PBS, pH 7.4, to strip the protein of 2,3-diphosphoglycerate. Because Hb can exist as an anion or a cation, depending on buffer conditions, it can be purified by either anion- or cation-exchange chromatography, or a combination of the two. Routinely, the erythrocyte lysate is first purified on a Q-Sepharose High Performance column or on a DEAE-Sepharose Fast Flow column followed by a second chromatography on a CM-Sepharose Fast Flow column. All purifications are carried out at 4°C.

3.1.1. Anion-Exchange Chromatography 3.1.1.1. PURIFICATION OF HB ON DEAE-SEPHAROSE FAST FLOW: SMALL-SCALE PURIFICATION

A typical elution profile of a human RBC lysate (25 mg of human RBC lysate injected in 500 µL of buffer A) is shown in Fig. 1. This represents an analytical-scale run of the same sample for which a preparative run is given in Subheading 3.1.1.2. on a Q-Sepharose High Performance column (Fig. 2). Runs like this are useful for the evaluation of the run conditions prior to preparative runs. The total run time is 5 h 6 min, the total volume is ~612 mL, and the gradient time/volume is 2 h/240 mL. 1. Pack the column (1.6 cm × 6 cm, CV = 12 mL) according to the manufacturer’s directions. 2. Wash the column with 1 CV each of water, buffer A, and buffer B. 3. Equilibrate the column with 10–25 CV of buffer A at a flow rate of 2 mL/min. 4. Inject the sample and wash the column with 1 CV of buffer A to elute unbound protein. 5. Elute the bound protein with a linear gradient of 0–100% buffer B in 20 CV. 6. Monitor the column effluent at 540, 600, and 630 nm (see Note 5). 7. Clean the column with 10 CV of buffer B. 8. Reequilibrate with 20 CV of buffer A.

3.1.1.2. PURIFICATION OF HB ON Q-SEPHAROSE HIGH PERFORMANCE: PREPARATIVE RUN

A typical chromatographic profile of a human erythrocyte lysate (load: ~40 mL containing ~3 g of Hb) is shown in Fig. 2. The protein eluting at 1500 mL (~65% buffer B) corresponds to HbA. The fractions corresponding to this peak


35

Fig. 1. Small-scale anion-exchange chromatography of human red cell lysate on a DEAE-Sepharose Fast Flow column (1.6 × 6 cm) at 4°C. Buffer A: 50 mM Tris-Ac, pH 8.5; buffer B: 50 mM Tris-Ac, pH 7.0. The column was equilibrated with buffer A, and a decreasing pH gradient of 0–100% buffer B over 20 CV was used for elution of the protein. Protein load: 25 mg.

are pooled, concentrated, and subjected to further purification by cationexchange chromatography on a CM-Sepharose Fast Flow column (Subheading 3.1.2.). The following run takes about 25 to 26 h. 1. Pack the Q-Sepharose High Performance ion-exchange column at 4°C in a Pharmacia XK26/70 column according to the manufacturer’s directions. Typically, a 2.6 × 58 cm column (~290-mL column volume) is used for the purification of 2.5–3 g of Hb. 2. Wash the column first with 1 CV of water, followed by 1 to 2 CV each of 20% buffer B and 100% buffer B. 3. Equilibrate the column with at least 10 CV of 20% buffer B, at a flow rate of 1.5 mL/min. 4. Dialyze the red cell lysate extensively against 20% buffer B, and filter through a 0.2-µm filter. 5. Load the lysate onto the column manually using line A18 of Pump A. 6. Elute the protein with a linear gradient of decreasing pH consisting of 20–100% buffer B in 8 column volumes (2320 mL). 7. Monitor the column effluent simultaneously at three wavelengths; 540, 600, and 630 nm (see Note 5).

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Manjula and Acharya

Fig. 2. Preparative-scale anion-exchange chromatography of human red cell lysate on Q-Sepharose High Performance column (2.6 × 58 cm) at 4°C. Buffer A: 50 mM Tris-Ac, pH 8.5; buffer B: 50 mM Tris-Ac, pH 7.0. The column was equilibrated with 20% buffer B, and a decreasing pH gradient of 20–100% buffer B over 8 CV was used for elution of the protein. Protein load: 3.0 g; fraction size: 20 mL.

3.1.2. Rechromatography of Q-Sepharose High Performance Purified HbA on a CM-Sepharose Fast Flow Column A typical chromatographic profile is shown in Fig. 3. The protein eluting at 1960 mL (~78% buffer B) corresponds to HbA. Pool the HbA-containing fractions, concentrate in an Amicon stirred cell to a concentration of 64–128 mg/mL, dialyze against the buffer of choice, and store either in liquid nitrogen or at –80°C. 1. Dialyze the HbA obtained from the Q-Sepharose High Performance column (~1.3 g in 60 mL) against 10 mM potassium phosphate buffer; 1 mM EDTA, pH 6.35 (see Note 6). 2. Load the dialyzed HbA on the CM-Sepharose Fast Flow column (2.6 cm × 59 cm), preequilibrated with the same buffer. The large sample volume is not a consideration, since the protein binds to the column at the initial conditions. Up to 3 g of Hb can be purified on a 2.6 × 59 cm column. 3. Elute the protein with a linear gradient of increasing pH, consisting of 0–100% buffer B over 8 column volumes (~2500 mL).


37

Fig. 3. Repurification of HbA purified on Q-Sepharose High Performance column (see Fig. 2) by cation-exchange chromatography on a CM-Sepharose Fast Flow column (2.6 × 59 cm) at 4°C. Buffer A: 10 mM potassium phosphate, 1 mM EDTA, pH 6.35; buffer B: 15 mM potassium phosphate, 1 mM EDTA, pH 8.5. The column was equilibrated with buffer A and an increasing pH gradient of 0–100% buffer B over 8 CV was used for elution of the protein. Protein load: ~1.3 g; fraction size: 12 mL. The effluent was monitored at 540, 600, and 630 nm. Elution profile for 540 nm is shown.

3.2. Ion-exchange Chromatography as an Analytical Tool 3.2.1. Characterization of HbA Expressed in Transgenic Swine by Cation-Exchange Chromatography on a Mono S Column The ion-exchange chromatographic procedures are also valuable as fast techniques for the analysis of Hb variants and recombinant hemoglobins. The elution positions are dependent on the surface topology of the Hb. The Mono S column (Amersham Pharmacia Biotech) distinguishes between the correctly folded and misfolded forms of recombinant HbA (rHbA) (2–4). Adachi et al. (2) have reported that the rHbA obtained from their yeast expression system contains a misfolded form of HbA in addition to the correctly folded form. The misfolded and the correctly folded forms of rHbA exhibit distinct elution positions on a Mono S column. Studies by Shen et al. (3,4) have shown that rHbA containing incorrectly inserted heme can be resolved from the species containing the correctly inserted heme on a Mono S column. In our studies, the chromatographic profile of the transgenic swine HbA on a Mono S column is identical to that of wild-type HbA (Fig. 4), which, in conjunction with NMR

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Fig. 4. Comparison of elution profiles of wild-type HbA and HbA expressed in transgenic swine, on a Mono S HR5/5 column. The flow rate was 1 mL/min. Protein load: 1 mg. Buffer A: 10 mM potassium phosphate, pH 6.5; buffer B: 15 mM potassium phosphate, pH 8.5. After injection of the protein, the column was washed with 2 CV of buffer A, and the bound protein was eluted with a linear gradient consisting of 0–100% buffer B over 45 CV. The effluent was monitored at 540 nm.

and functional studies (5), has established the absence of misfolded forms in this preparation. 1. Equilibrate the Mono S column with 10 mM potassium phosphate, pH 6.5 (buffer A), at a flow rate of 1 mL/min. 2. Inject 1 mg of the HbA or TgHbA in 25 µL of buffer A. 3. Wash the column with 2 CV of buffer A. 4. Elute the protein with a linear increasing pH gradient consisting of 0–100% buffer B over 45 CV. 5. Monitor the column effluent at 540 nm. 6. Regenerate the column in situ by washing first with ~5 CV of 100% buffer B followed by reequilibration with 25 CV of buffer A (0% buffer B).


39

Fig. 5. Chromatography of amidated HbS on a 1-mL HiTrap Q column at room temperature. Buffer A: 50 mM Tris-Ac, pH 8.5; buffer B: 50 mM Tris-Ac, pH 7.0. The column was equilibrated with 10% buffer B. The column was washed with 5 CV of 10% buffer B after injection of the sample, and a decreasing pH gradient of 0–100% buffer B over 20 CV was used for elution of the protein. Protein load: 1 mg. The effluent was monitored at 540 nm.

3.2.2. Monitoring Progress of a Chemical Modificattion Reaction: Analysis of Amidated HbS by Analytical Anion-Exchange Chromatography on HiTrap Q 3.2.2.1. PREPARATION OF AMIDATED HBS

HbS was amidated with ethanolamine, through a carbodimide and sulfo-Nhydroxy-succinimide-mediated reaction, according to the previously described procedures (6,7). 3.2.2.2. CHROMATOGRAPHY OF AMIDATED HBS ON HITRAP Q COLUMN

The chromatographic profile of an HbS preparation amidated with ethanolamine is illustrated in Fig. 5. As can be seen, the amidated HbS can be separated well from the unreacted HbS. Thus, this profile illustrates the feasibility of establishing conditions for the separation of modified and unmodified HbS using small amounts of the protein and within a short period of time. Protein loads as little as 100 µg are sufficient for such runs. Thus, these columns are highly useful for methodological development as well as for monitoring the time course of a protein modification reaction.

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Manjula and Acharya

Total run time is 60 min, total volume is ~60 mL, and sample run time/ volume is 25 min/25 mL. 1. Equilibrate the column with 10% buffer B, at a flow rate of 1 mL/min. 2. Inject a sample of 1 mg of HbS amidated with ethanolamine, and wash the column with 5 CV of 10% buffer B. 3. Elute the protein with a gradient of 10–100% buffer B in 20 CV. 4. Monitor the column effluent at 540 nm. 5. Regenerate the column in situ by washing first with 10 CV of 100% buffer B, followed by reequilibration with 25 CV of 10% buffer B.

3.3. SEC of Hb Three applications of SEC on Pharmacia Superose 12 are described; two applications are at an analytical level and the third is at a preparative level.

3.3.1. Analytical SEC 3.3.1.1. ESTABLISHING STABILIZATION OF TETRAMERIC STRUCTURE OF HB BY INTERDIMERIC (I.E., INTRATETRAMERIC) CROSSLINKING

The samples are generally injected in a volume of 25 µL. Since the SEC runs are under isocratic conditions, unlike the ion-exchange columns, no column regeneration step is necessary. Once the protein is eluted from the column and the baseline is stable, the column is ready for the analysis of the next sample. The run time for HbA is approx 70 min. Thus, it is possible to analyze several samples during the course of a working day. Under nondenaturing conditions, SEC analysis of Hb serves as a highly useful tool to establish the tetrameric structure and to analyze polymeric forms of Hb. In the presence of 0.9 M MgCl2, Hb dissociates into its constituent dimers (8). However, if the like chains are crosslinked, then the tetrameric structure is stabilized and the protein elutes as a tetramer. Thus, SEC analysis under dissociating conditions is a valuable tool to monitor the stabilization of the tetrameric structure of Hb by interdimeric (i.e., intratetrameric) crosslinking. These two modes of the SEC analyses are illustrated in Fig. 6A and 6B, from an analysis of HbA reacted with the bifunctional maleimide, bis-maleidophenyl PEG2000 (Bis-Mal-PEG2000) (9). SEC analysis of Bis-Mal-PEG2000-reacted HbA in 50 mM Bis-Tris-Ac, pH 7.4, a low-ionic-strength buffer, revealed that >95% of the protein elutes in the same position as the tetrameric HbA, and only a trace amount is present as an octameric species (Fig. 6A). By contrast, SEC analysis of the same BisMal-PEG2000-reacted HbA preparation on the same Superose 12 column but in the presence of 0.9 M MgCl2 revealed that nearly all of the Bis-MalPEG2000-reacted HbA still elutes at the 64,000-Dalton position (Fig. 6B, upper


41

Fig. 6. SEC of Bis-Mal-PEG2000-reacted HbA on Superose 12 at room temperature. Two Superose 12 HR 10/30 columns connected in series were used. (A) the buffer used was 50 mM Bis-Tris-Ac, pH 7.4; (B) the buffer used was 50 mM Bis-Tris-Ac, 0.9 M MgCl2, pH 7.4. For both (A) and (B) the flow rate was 0.5 mL/min, the protein load was 800 µg and the effluent was monitored at 540 nm (see Note 7).

panel), whereas the control HbA is completely dissociated into its constituent αβ dimers (Fig. 6B, lower panel). Thus, SEC analyses establish the stabilization of the tetrameric structure of HbA by intramolecular crosslinking with Bis-Mal-PEG2000. 3.3.1.1.1. SEC Analysis in 50 mM Bis-Tris-Ac, pH 7.4: Nondenaturing Conditions 1. Equilibrate the column with 50 mM Bis-Tris-Ac, pH 7.4, at a flow rate of 0.5 mL/min. 2. Inject the sample. 3. Elute the column with 50 mM Bis-Tris-Ac, pH 7.4.

42

Manjula and Acharya 3.3.1.1.2. SEC Analysis in 50 mM Bis-Tris-Ac; 0.9 M MgCl2, pH 7.4: Denaturing Conditions

1. Equilibrate the column with 2 CV of 50 mM Bis-Tris-Ac, 0.9 M MgCl2 (pH 7.4) at a flow rate of 0.5 mL/min. 2. Inject the sample. 3. Elute the column with 50 mM Bis-Tris-Ac, 0.9 M MgCl2, pH 7.4.

3.3.1.2. DETERMINING HYDRODYNAMIC VOLUME OF SIZE-ENHANCED HBS

Conjugation of large nonprotein molecules like PEG increases the hydrodynamic volume of the protein. SEC analysis serves as a useful tool to determine the increase in the hydrodynamic volume of Hb as a function of the chain length of the conjugated PEG molecule, and as a function of the number of PEG chains conjugated to the protein. 3.3.1.2.1. Preparation of PEGylated Hb

HbA in PBS, pH 7.4, is reacted with maleidophenyl derivatives of PEG 5000, PEG 10000, and PEG 20000 (unpublished). This results in the surface decoration of HbA at its two β93 cysteines. Homogeneous preparations of Hb carrying two copies of PEG 5K, 10K, and 20K were isolated by ion-exchange chromatography. 3.3.1.2.2. SEC Analysis

SEC analysis of the PEGylated Hbs is carried out on an analytical Superose 12 column, equilibrated and eluted with PBS, pH 7.4, as described in Subheading 3.3.1.1. The results are shown in Fig. 7. The PEGylated HbAs elute earlier than HbA on the Superose 12 column. The actual molecular mass of the three surface-decorated HbAs carrying two PEG chains of 5, 10, and 20 kDa is 74, 84, and 104 kDa, respectively. Thus, no resolution among three surface-decorated HbAs can be expected on the Superose 12 column based on the differences in their actual mass. Nevertheless, the three PEGylated HbAs are well resolved from each other, indicating an apparent increase in their size. The retention time of the PEGylated HbA decreases with the increase in the length of the attached PEG chain, indicating a progressive increase in the apparent size of HbA on surface decoration with PEG molecule of increasing chain length. Intertetrameric crosslinking of HbA with Bis-Mal-PEG600 results in the formation of defined oligomeric forms of HbA with molecular weights that are multiples of 64 kDa (see Subheading 3.3.2. for an example of a preparative run). Comparison of the retention times of surface-decorated HbAs with those of oligomerized HbAs indicated that the size enhancement is a linear function of the mass of the PEG chains attached and is approx 8 to 10 times that anticipated based on the actual molecular size of the attached PEG chain.


43

Fig. 7. Comparison of hydrodynamic volumes of PEGylated HbAs by SEC on Superose 12 at room temperature. Two Superose 12 HR 10/30 columns connected in series were used. Elution buffer: PBS, pH 7.4; flow rate: 0.5 mL/min; protein load: 800 µg in each case.

3.3.2. Semi-Preparative SEC: Purification of Oligomeric Forms of Hb SEC can also be used in a preparative mode for the purification of Hb based on its size. In the example shown here, HbA, stabilized against dissociation by intratetrameric crosslinking, was oligomerized by intertetrameric crosslinking with Bis-Mal-PEG600, and the products of the reaction were separated by SEC on a semipreparative Superose 12 column. A typical chromatographic profile is shown in Fig. 8. Reanalysis of the fractions from this run on an analytical column confirmed that the fractions eluting at the peak positions of 204, 182, and 169 mL represent tetrameric, octameric, and dodecameric Hb, respectively. Fractions containing the respective oligomeric forms of Hb are pooled, concentrated, and further purified by rechromatography on the same Superose column. 1. Equilibrate a semipreparative column of Superose 12 (2.6 × 65 cm, CV = 325 mL) with 2 to 3 CV of PBS, pH 7.4, at a flow rate of 1 mL/min. 2. Load ~100 mg of protein in 1 mL of PBS, pH 7.4. 3. Elute the column with PBS, pH 7.4.

3.4. Globin Chain Analysis of Hb by RP-HPLC Analysis The globin chains of Hb can be separated by RP-HPLC (10,11). Thus, RP-HPLC analysis is a useful technique for determining the purity of an HbA

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Fig. 8. Purification of intertetramerically crosslinked HbA on a semipreparative Superose 12 column (2.6 × 65 cm) at 4°C. Elution buffer: PBS, pH 7.4; flow rate: 1 mL/min; protein load: ~100 mg in 1 mL of PBS, pH 7.4; fraction size: 1.5 mL. The effluent was monitored at 540 nm.

preparation; identifying of the chain modified after a chemical reaction; and isolating of the globin chains for peptide mapping, mass analysis, amino acid analysis, and sequencing A comparison of the RP-HPLC profiles of HbA and Bis-Mal-PEG2000reacted HbA is shown in Fig. 9. This result shows that the β-chains of HbA are completely modified after reaction with Bis-Mal-PEG2000. The modified β-globin eluted as a distinct peak, after the α-chain. Thus, RP-HPLC analysis is a useful tool to identify the globin chain modified after a chemical modification reaction. The globin chains can be isolated for mass analysis and peptide mapping to identify the location of the modification on the polypeptide chain. For such applications, a semipreparative C4 column (10 × 250 cm) is used. One to two milligrams of Hb can be applied on the semipreparative column. A flow rate of 2 mL/min and the same gradient as for the analytical run can be employed. The fractions containing the separated globin chains are collected, and the solvent is removed either in a Speedvac or by lyophilization. Peptide


45

Fig. 9. RP-HPLC analysis of globin chains of Bis-Mal-PEG2000-reacted HbA on a Vydac C4 column (4.6 × 250 mm, 300 °A). Solvent A: water, 0.1% TFA; solvent B: acetonitrile, 0.1% TFA. The column was equilibrated with 35% solvent B, and a linear gradient of 35–50% solvent B in 100 min was employed for the elution. Flow rate: 1 mL/min. The effluent was monitored at 210 nm.

mapping of the globin chains is carried out by RP-HPLC on a Vydac C18 column (12). 1. Equilibrate a Vydac C4 column with 35% acetonitrile, 0.1% TFA at a flow rate of 1 mL/min. 2. Mix 10–100 µL of HbA (50–150 µg) with 1 mL of 0.3% TFA in a 1.5 mL microfuge tube, vortex, and centrifuge at 12,000 rpm for 4 min to clarify the sample. 3. Inject the supernatant onto the column. 4. Elute the globin chains with a linear gradient of 35–50% acetonitrile, 0.1% TFA in 100 min. 5. Monitor the effluent at 210 nm. 6. Regenerate the column in situ by washing with 100% acetonitrile, 0.1% TFA for 15 min, followed by reequilibration with 35% acetonitrile, 0.1% TFA (about 30 min).

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4. Notes 1. The DEAE-Sepharose Fast Flow, the Q-Sepharose High Performance, and the CM-Sepharose Fast Flow columns described here were run using an Amersham Pharmacia Biotech AKTA Protein Purification System. However, these runs can also be carried out on other chromatography systems such as the Pharmacia FPLC system or even by conventional techniques using a peristaltic pump and a twochamber gradient system. 2. The Fast Flow resins have greater mechanical strength than the cellulose-based resins and thus permit higher flow rates. Hence, the run can be completed within a much shorter duration than the Whatman cellulose ion exchangers. In the examples shown, the run time on the Q-Sepharose High Performance and the CM-Sepharose Fast Flow ion exchangers is approximately one-third that on corresponding cellulose-based ion-exchange columns. The High Performance and the Fast Flow ion exchangers can be regenerated and reequilibrated within the column after each run, and the columns can be reused for several runs. 3. Typically, for the analytical- and small-scale ion-exchange columns, the column cleaning and reequilibration steps are programmed as part of the method. Depending on the length of the gradient, the run time for a 1-mL column can vary from 30 to 60 min. Thus, these procedures permit evaluation of several run conditions within a short period of time, utilizing only small quantities of protein (as little as 100 µg of protein per run). 4. Care in the solvent and sample preparation is a crucial step especially for the high-pressure columns such as Mono S and Superose 12 HR 10/30. Routinely, all buffers are freshly prepared and filtered through a 0.2-µm filter. Larger samples are also filtered through a 0.2 µm-filter prior to loading on the column. Smallervolume samples for the Mono S, HiTrap, and analytical Superose 12 are clarified by centrifuging in a microfuge at 12,000 rpm for 4 min. 5. The AKTA Protein Purification System permits simultaneous monitoring of the effluent at three wavelengths. Routinely, all the Hb purifications are monitored at 540, 600, and 630 nm. Monitoring at 600 nm is useful in cases in which the absorbance at 540 nm is too high, and monitoring at 630 nm is useful for the determination of the relative amounts of met-Hb in the column fractions. 6. Since the starting pH for the CM-Sepharose Fast Flow column is > k'X[X]), the observed replacement rate becomes equal to kX. The same principle applies if a reagent is added to consume the dissociated ligand.

Rate Constant Measurement for Reactions with Hb

67

Fig. 1. Time course for CO binding to human deoxyHb in 0.1 M phosphate, pH 7.0, 20°C. In a Gibson-Dionex stopped-flow apparatus equipped with a 2-cm path length, 100 µM CO was mixed 1:1 with 10 µM deoxyHb. The reactant concentrations after mixing were [CO]total = 50 µM and [Hb]total = 5 µM, and the time course was followed at 436 nm. (䊊) Observed data points; solid line —— a fit to single exponential expression with an observed rate constant equal to 7.9 s–1. (Top) Plotted differences between observed data and fitted line (residuals). (Inset) Dependence of fitted pseudo firstorder observed rate constant on [CO]total after mixing.

In ligand consumption experiments, an excess of consuming agent is usually added so that the observed rate equals that for ligand dissociation. The rate of oxygen dissociation, kO2, is normally measured by reacting HbO2 (deoxygen complex with reduced hemoglobin) with either excess CO or high concentrations of dithionite to consume free O2 (2,5,11). The rate of CO dissociation, kCO, is measured by reacting carbon monoxide hemoglobin (HbCO) with excess NO or a protein that scavenges CO (2,5,11,12). The rate of NO dissociation, kNO,

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Table 1 Typical Rate Constants for CO, O2, and NO Binding to Human Hb at pH 7.0, 20–25°Ca T-state parameters Protein

k'T (µM –1s–1)

kT (s–1)

R-state parameters KT (µM –1)

Hb (tetramer) CO 0.12 0.16 0.75 5–10 500–1000 ~0.01 O2 NO 25 (~0.001) (~25,000) α-Subunit (tetramer) CO 0.14 0.15 0.9 O2 7.1 2000 0.004 β-Subunit (tetramer) CO 0.10 0.17 0.6 O2 6.4 1500 0.004

k'R kR (µM –1s–1) (s–1)

KR (µM –1)

6.0 66 60

0.008 20 0.00003

750 3.2 2,000,000

5.0 40

0.008 15

620 2.7

7.0 90

0.007 31

1000 2.9

a The T-state parameters were derived from measurements of the first step in ligand binding (i.e., k'1 = 4k'T and k1 = kT). The R-state parameters were derived from measurements of the last step in ligand binding (i.e., k'4 = k'R and k4 = 4kR). The values for O2 and CO in the first two rows were taken from analyses of the data in Figs. 1–5, Sawicki and Gibson (31), and Mathews and Olson (8). The values for NO were taken from Cassoly and Gibson (17), Moore and Gibson (13), and Eich et al. (41). The O2 and CO parameters for the individual α- and β-subunits in T- and Rstate Hb were taken from Unzai et al. (38) and the references therein. In the case of metal hybrid Hbs, the T state was often defined as in the presence of inositol hexaphosphate at pH 6.5, which can give abnormally high dissociation rate constants (see ref. 38).

can be measured by reacting HbNO with both excess CO to displace the bound ligand and excess dithionite to consume the newly released NO (13). The value of kNO can also be determined by mixing HbNO with excess molecular oxygen, which both displaces the NO and, when bound to the heme group, consumes free NO by dioxygenation (see Eq. 11; [14]).

2.2. Time Resolution: Rapid Mixing vs Flash Photolysis In general, the association of carbon monoxide with heme proteins is markedly slower than that of dioxygen or nitric oxide (NO) and is the easiest ligandbinding reaction to measure (Table 1). The rate-limiting step for CO binding is internal bond formation with the heme iron. CO enters and leaves the protein hundreds of times before it finally forms a bond with the iron atom, and, as a result, the overall bimolecular rate constant is normally small. By contrast, NO is so reactive that every ligand molecule that enters the protein combines with the iron atom before it has a chance to escape. Thus, NO binding is limited only by the rate of ligand entry into the protein. Dioxygen shows intermediate behavior,


69

being limited in part by the rate of movement into Hb and by the the rate of Fe-O2 bond formation (for a more complete discussion and references see ref. 15). The association rate constant for CO binding to Hb is usually small enough (0.1 to 5 µM–1s–1; Table 1) to allow measurement in simple rapid mixing, stopped-flow instruments with dead times of 2 to 3 ms. For example, at 100 µM free CO, the observed pseudo first rate for CO binding to low concentrations (≤20 µM) of human deoxyhemoglobin A (deoxyHbA) is 100 µM · ~0.1 µM–1s–1 = 10 s–1, which prescribes a half-time of ~70 ms. If the Hb is in the high-affinity, rapidly reacting conformation, the observed rate increases ~50-fold, yielding a half-time of ~1.5 ms. However, lower concentrations of CO can be used to increase the half-time of the reaction to ≥3 ms to allow measurements in rapid mixing devices. Alternatively, these more rapid CO reactions can be measured by the photolysis techniques described in Fig. 2. The association rate constants for NO and O2 binding to Hb are quite large (5–100 µM –1s–1) and normally can only be measured using laser photolysis techniques (7). For example, at 100 µM ligand, the observed rates of O2 and NO binding to high-affinity forms (R state) of human deoxyHbA are 100 µM · ~50 µM–1s–1 = 5000 s–1, yielding half-times of ~0.1 ms. These reactions are too rapid to be detected in rapid mixing experiments but are readily measured using laser photolysis techniques with excitation pulses ≤0.5 µs. In the case of O2 binding to the low-affinity, slowly reacting forms of Hb, the value of k'O2 is much smaller, ~5 µM–1s–1, which would lower the association rate to ~500 s–1 at 100 µM free ligand. Unfortunately, the dissociation rate constant for the low-affinity form of human Hb is on the order of 1000 s–1 (see Table 1). Since the observed pseudo first-order rate constant is the sum of the forward and backward rates (Eq. 1), the value of kobs is ≥1500 s–1, and t1/2 for the reaction is ≤0.5 ms, which precludes rapid mixing experiments. As result, there is no simple way of measuring O2 binding to deoxyHb by rapid mixing, and photolysis techniques with short excitation pulses are required. In the case of NO binding, the dissociation rate constants for either the lowor high-affinity forms of human hemoglobin are very small, 0.001–0.00001 s–1 (Table 1). Thus, NO binding to either the R or T states of deoxyHb can be measured by stopped flow, rapid mixing techniques. However, very low Hb (1–5 µM) and NO (1–10 µM) concentrations must be used to keep the observed rates in the range of 50–500 s–1 (16,17). 3. Results 3.1. CO Binding The association rate constant for CO binding can be measured by mixing a solution of deoxyHb with anaerobic buffer solutions containing various

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Fig. 2. Time courses for CO rebinding to human deoxyHb after photolysis with a 1-ms excitation pulse from a photographic flash lamp. The conditions are the same as those in Fig. 1, and the reactions were followed at 436 nm. Photolysis was carried out with two Sunpack 540 Strobes as described in Mathews and Olson (8). (A) Photolysis of 150 µM HbCO at four different excitation intensities. (Inset) A rapid, monophasic time course is observed when the extent of photolysis is ≤10%. (B) Complete photolysis at lower concentrations of HbCO.

amounts of dissolved ligand and measuring the disappearance of the deoxyHb absorption peak at 430 nm or appearance of the HbCO peak at 420 nm. In these experiments, small amounts of sodium dithionite are often added to ensure removal of all oxygen from both reactant solutions. A sample time course for CO binding to human deoxyHbA at pH 7.0 is shown in Fig. 1. The dependence of the apparent pseudo first-order rate constant on ligand concentration is shown in the inset. There is a linear dependence of the overall pseudo firstorder rate constant on [CO] after mixing. The slope of the curve is 0.15 µM–1s– 1 for HbA in 0.1 M phosphate buffer, pH 7.0, 20°C. The intercept of the k obs vs [CO] plot is effectively 0 since the values for CO dissociation are very small (from 0.1 to 0.005 s–1; Table 1). As noted by Gibson and Roughton 45 yr ago (reviewed in ref. 2), the time course for CO binding to deoxyHbA shows complex accelerating behavior. A fit to a single exponential decay expression shows systematic deviations from the observed data (Fig. 1, top). It is clear that the observed rate is increasing as


71

the reaction proceeds. A quantitative analysis of CO binding to human deoxyHb requires analysis in terms of the four-step Adair scheme: Hb4 + X

k'1 k1

Hb4X + X

k'2 k2

Hb4X2 + X

k'3 k3

Hb4X3 + X

k'4 k4

Hb4X4

(4)

Under most circumstances, the back reactions for CO binding can be neglected since the absolute value of the dissociation rate constants, k1 to k4, are very small compared to the rates of association (i.e., k'1[CO] >> k1; k'2[CO] >> k2; and so on). In effect, CO binding can be described by four consecutive irreversible reactions, whose analytical solutions under pseudo first-order conditions can be represented by sums of exponentials (see refs. 2 and 6). Assignment of rate constants to all four steps in CO binding is extremely difficult because the observed time course for CO binding is not very different from a simple exponential decay (Fig. 1). An independent experimental determination of the rate constant for the last step, k'4, is required. Gibson was the first to solve this problem by using partial photolysis techniques to measure this rate constant directly (reviewed in refs. 2 and 4). He constructed a Xe flash lamp (pulse length of ~10 µs) that could be used to flash photolyze the Fe-CO bond and drive the ligand out into the solvent. After the flash, CO rebinds to the newly produced Hb4(CO)3, Hb4(CO)2, Hb4(CO), and Hb4 intermediates, depending on the extent of photolysis. At 100% photolysis, fully deoxygenated Hb tetramers are generated; at ≤10% photolysis, the only reactive species is Hb4(CO)3, and the last step in ligand binding can be followed directly. Over the last 20 yr, these photolysis experiments have been extended to nanosecond and picosecond time regimes using ultrafast lasers. On these very short time scales, first-order, internal ligand rebinding is observed. These ultrafast processes are called geminate rebinding because the same iron/ligand pair is involved in bond reformation. Geminate recombination provides detailed information on the factors governing iron-ligand bond formation (15) and allows mapping of ligand movement into and out of the protein (18). In all of the following discussion, however, only long laser (~1-µs) or photographic flash (~1-ms) pulses are considered. In these experiments, only ligand rebinding from the solvent is being measured, all the processes are bimolecular, and the observed rates depend on the first power of the external ligand concentration. Sample time courses for CO rebinding to deoxyHbA after photolysis by a 1-ms pulse from photographic Xe flash lamps are shown in Fig. 2. When the total protein concentration is kept high ([Hb]total ≈ 150 µM; Fig. 2A), the time course for CO rebinding to human HbA after complete photolysis resembles that seen in rapid-mixing experiments (Fig. 1). Acceleration is seen in both cases. Decreasing the excitation light intensity with neutral-density filters leads to biphasic time courses (Fig. 2A, lower curves). The first phase represents rebinding to a rapidly

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reacting form of Hb, with an apparent bimolecular rate constant of ~6 µM–1s–1. At 5–10% photolysis, only the rapid phase is observed. This time course is assumed to represent the binding of the last CO molecule to hemoglobin (i.e. Hb4X3 + X → Hb4X4 in Eq. 4), and the observed bimolecular rate constant represents k'4 in the Adair scheme.

3.2. Analysis in Terms of Two States The deoxyheme group in the Hb4X3 intermediate was originally designated as Hb*, owing to its rapid reaction with CO (2). In the early 1970s, the twostate model of Monod, Wyman, and Changeux (19) was adopted as the standard for interpreting cooperative ligand binding. It is still the best first approximation for comparing mutant and naturally occurring Hbs. In this model, deoxyHb starts out in the low-affinity T state where the reactivity toward ligands is low. As ligands successively bind, the tetramer switches to the high-affinity R state so that the reactivities of the remaining deoxyheme group in Hb4X3 toward O2 and CO are ~300 to 1000 times greater, respectively, than any of the groups in Hb4 (Table 1). In the simple two-state model, the equilibrium constant for T-to-R isomerization in the completely unliganded Hb4 species is defined as L = [T]/[R] and is on the order of 1,000,000 for native HbA at pH 7.0 (20,21). The ratio of the ligand association equilibrium constants for binding to the T vs the R states is KT/KR ≈ 0.002–0.005. As the reaction proceeds, the tetramer isomerization constant decreases by L(KT/KR)n in which n is the number of ligands bound. For example, the binding of three ligands causes the isomerization constant to decrease from 106 to 10–2, and Hb4X3 is predominantly in the R state. Thus, under physiological conditions, measurement of the kinetics of the first step in the Adair scheme (Eq. 4) provides an estimate of the rate parameters for the T quaternary state, and time courses for the last step provide estimates of the corresponding R-state rate constants. Using these definitions, the Hb* species can be equated with the rapidly reacting R-state conformation (5,8,11).

3.3. Dimers and Monomers Are Rapidly Reacting In the late 1950s, Gibson (22) reported that the rapidly reacting form of Hb could also be seen after complete photolysis when the protein concentration was low (≤100 µM for human Hb, Fig. 2B). This apparent anomaly was resolved when Antonini and coworkers (11) were able to show that tetrameric, fully liganded Hb dissociates into dimers with an equilibrium dissociation constant K4,2 = 2–10 µM (for a review see ref. 11). In flash photolysis experiments with dilute HbCO (≤10 µM), a large fraction of the heme groups is present as dimers since the equilibrium dissociation constant for Hb4(CO)4 is ~1–5 µM under physiological conditions (11,21,23). By


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contrast, native deoxyHb has an equilibrium dissociation constant ≤10–10 µM at neutral pH and is completely tetrameric at micromoar heme concentrations (21). After photodissociation of Hb2(CO)2, the newly formed deoxy dimers remain in the rapidly reacting or Hb* conformation and have to reaggregate to tetramers before switching back to the slowly reacting, T-state form (23). Dimer aggregation (k'2,4 ≈ 0.1 µM–1s–1) is relatively slow compared to CO rebinding at high ligand concentrations (11,24,25). The mechanism for interpreting time courses at low Hb concentrations and after complete photolysis is as follows:

(5)

The fraction of rapidly reacting Hb after complete photolysis is a measure of the amount of Hb2(CO)2 dimers present in the original solution. The fraction of heme groups that is present as dimers is given by Eq. 6:

(6)

Edelstein et al. (23) have measured the fraction of rapidly reacting species as a function of total heme concentration and shown that the K4,2 value determined from these kinetic analyses is identical to that determined by molecular weight measurements using ultracentrifugation. At roughly the same time, Antonini, Brunori, and coworkers (11) showed that isolated α- and β-chains are also rapidly reacting, and that all three species—triliganded tetramers, dimers, and monomers—show roughly the same R-state ligand-binding parameters (for newer reviews, see refs. 5,8).

3.4. CO Dissociation Rate constants for CO dissociation from Hb are measured by mixing HbCO complexes with a high concentration of NO. In general, the rate constant for NO binding to Hb is ≥10 times that for CO binding. As a result, the observed replacement rate is a direct measure of kCO (see Eq. 3, where k'CO[CO]/ k'NO[NO] is ~0 as long as [CO] ≤ [NO]). For these types of replacement reac-

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tions, the intermediates containing unliganded heme groups are Hb4(CO)3, Hb4(CO)2(NO), Hb4(CO)(NO)2, and Hb4(NO)3. All these species contain three bound ligands. Consequently, the ligand replacement reaction measures only the first step in dissociation and, in general, shows simple behavior. Sharma et al. (12,26) have used microperoxidase (a small heme-containing degradation product of cytochrome-c) to scavenge CO from fully liganded Hb and attempted to determine all four rate constants for CO dissociation from Hb4(CO)4. Although it is difficult to analyze these data quantitatively owing to complex side reactions of microperoxidase, Sharma et al.’s (26) work has defined the value of k1 for CO dissociation from Hb4CO to be ~0.1 s–1.

3.5. O2 Binding As described previously, O2 reacts too rapidly with both the high- and lowaffinity states of Hb to allow direct measurement of the association rate constant by rapid-mixing methods. Instead, laser photolysis techniques must be employed. The quantum yield for complete O2 dissociation is ~0.05, compared with ~0.7 for photodissociation of CO (27,28). As result, complete photolysis cannot be achieved by conventional photographic flash lamps. The best choice is a flash lamp–driven dye laser with a pulse length on the order of 500 ns and an energy output of 1–3 J (see ref. 7). Although easier to use, YAG lasers have a pulse length of ≤10 ns, after which substantial internal geminate recombination can occur. In the case of HbO2, the extent of internal recombination is ≥50%. Consequently, complete photolysis of O2 cannot be achieved with a 9-ns pulse regardless of the energy output of the laser. By contrast, a dye laser pulse of ~0.5 µs is long enough to pump all the O2 out of the protein if sufficient energy is available in the excitation pulse. Sample time courses for O2 rebinding to human HbA at low and high free [O2] are shown in Figs. 3A and 3B, respectively. Even at high protein concentration, the observed time courses are biphasic at all levels of photolysis. The fastest process represents O2 association with rapidly reacting Hb* or R-state forms of Hb. The slower processes represent rebinding to low-affinity T-state forms. At low [O2], the rapid phase comprises about 30% of the absorbance change after complete photolysis. This result for O2 rebinding contrasts with that seen for CO rebinding after complete photolysis using a longer pulse. In the latter case, only one slow phase is observed at high protein concentration (100% photolysis; Fig. 3A). The persistence of rapid O2 rebinding occurs because the switch from the rapidly reacting R-state conformation to the slowly reacting T-state tetrameric form is not instantaneous (29–31). The rates for the R-to-T switch are between 1000 and 10,000 s–1 and on the same order as the apparent rates of rebinding at 100 µM O2 (~1000 s–1 to the T state and ~10,000 s–1 to the R state; see Table 1). This interpretation is shown schematically in Eq. 7:


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Fig. 3. Photolysis of human HbO2 at low and high [O2] in 0.1 M phosphate, pH 7.0, 20°C. A Phase-R 2100B dye laser was used to produce a 0.5-µs excitation pulse at 577 nm, and the extent of photolysis was attenuated with neutral-density filters (8). O2 rebinding was monitored at 436 nm. (A) Time courses at 100 µM free O2. Under these conditions, the majority of the rebinding reaction is slow at 100% photolyis. (B) Time courses at 1260 µM O2. Under these conditions, most of the reaction is fast. (Insets) At ≤10% photolysis, the reaction is monophasic and very rapid.

(7)

After the excitation pulse, there is competition between rapid O2 rebinding to the Hb* form of the newly formed deoxy tetramers and the conformational transition to the more slowly reacting T-state tetramer (Eq. 7). If the rate of O2 association is increased by raising its concentration, the percentage of slowly reacting form decreases because more of the O2 molecules rebind to the R-state form before it

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can switch to the slowly reacting T-state conformation. Thus, at 1250 µM free O2 (1 atm), the percentage of rapid rebinding increases to ~65% at full photolyis (Fig. 3B). Complete analyses of O2 rebinding time courses are very complex and require, in addition to the normal ligand-binding parameters, the assignment of multiple R-to-T conformational change rate constants for all the various Adair intermediates in Eq. 4 plus consideration of dimerization (see refs. 9 and 10). However, the results in Fig. 3 do allow a qualitative estimation of O2 association rate constants. The time courses can be fitted to two exponential expressions, and the observed fast and slow rate parameters can be assigned to the R and T forms. Plots of kobs (fast and slow) vs [O2] are roughly linear at high ligand concentrations, and the apparent rate constants are k'TO2 ≈ 9 µM–1s–1 and k'RO2 ≈ 60 µM–1s–1. The rate of the slower phase does show a complex dependence on [O2] at low levels of ligand and incomplete saturation, making it difficult to assign exact values for k'1 in the Adair scheme (10,30,31). By contrast, at high [O2] and ≤10% photolysis, the value of k'4 is readily obtained since only the rapid phase of rebinding is observed (Fig. 3B, inset).

3.6. O2 Dissociation Time courses for oxygen dissociation from Hb can be measured in stopped flow, rapid-mixing experiments using the ligand replacement and consumption reactions described in Eq. 2. When HbO2 is reacted with anaerobic buffer containing very high concentrations of sodium dithionite, a single phase is observed with an overall apparent rate constant of ~60–100 s–1 at pH 7.0, 20°C (Fig. 4, lower curve). If the reaction is carried out again with CO in the dithionite solution, the observed rate is two- to threefold smaller, ~30 s–1 (Fig. 4, upper curve). This difference is a reflection of the cooperative nature of O2 release in the absence of replacing ligands. This situation is shown schematically in Eq. 8.

(8)


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Fig. 4. Time courses for O2 dissociation from human Hb in 0.1 M phosphate, pH 7.0, 20°C. HbO2 (10 µM) in air-equilibrated buffer was mixed with anaerobic buffer containing excess sodium dithionite (lower curve) and with buffer equilibrated with 1 atm of CO containing excess dithionite (upper curve). The reaction was monitored by absorbance increases at 424 nm.

When O2 is consumed by dithionite in the absence of other ligands, the rate of dissociation increases as the protein switches from the R to the T conformation. This switch occurs after ~2 ligands are lost. As a result, the first O2 dissociates at the R-state rate, which is ~30 s–1. When the second ligand dissociates, the remaining sites lose O2 immediately because their rates of dissociation are 20- to 50-fold higher. Thus, the second rate is two to three times that of the first rate, and the time course shows acceleration. Consequently, the overall firstorder rate constant is two to three times greater than that for R-state Hb. When CO is present, the protein remains fully liganded, except for the transient formation of triliganded intermediate with one empty site. Under these conditions, only O2 dissociation from fully liganded tetramers is being measured (see Eq. 4). The reaction of dithionite with free O2 leads to the formation of hydrogen peroxide, which can cause secondary oxidative reactions. In addition, dithionite can also reduce methemoglobin (MetHb) impurities and degradation products. Both of these side reactions can cause large spectral changes that can interfere

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with measurement of the “true” O2 dissociation reaction. In our view, the best method for measuring the value of k4 for O2 dissociation is to mix HbO2 with buffer equilibrated with 1 atm of CO and then to vary the free [O2] in the Hb sample. The observed replacement rate should be given by the expression in Eq. 3, in which X = O2 and Y = CO. A sample time course for the reaction of human HbO2 with CO is shown in Fig. 5A. The dependence of robs on [CO]/[O2] is shown in the inset. The expression used to fit these data comes from rearranging Eq. 3:

(9)

kRO2 is defined as the intrinsic rate constant for O2 dissociation from a heme group in fully liganded Hb. The value of k4 in the Adair scheme (Eq. 4) would be 4kRO2 since there are four possible sites from which the O2 can be dissociated in Hb4(O2)4 (for more complete discussion of statistical factors see refs. 2, 5, and 6) The fitted value for the limiting rate in the inset to Fig. 5A is 29 s–1 in 0.1 M phosphate, pH 7.0, 20°C. This value of kRO2 is equal to the observed rate obtained when HbO2 was mixed with buffer containing both CO and high concentrations of sodium dithionite (Fig. 4, upper curve). This analysis also allows a quantitative determination of the ratio k'RO2/k'RCO, which can be used to confirm assignments made in partial photolysis measurements with the corresponding Hb4(O2)4 and Hb4(CO)4 complexes. Thus, analysis of the CO replacement reaction can also be used to obtain values for k'RO2 if the rate of CO binding to R-state Hb has already been measured.

3.7. Differences Between α- and β-Subunits Close examination of time courses for both O2 replacement and rebinding after ≤10% photolysis shows systematic deviations from simple exponential behavior (Fig. 5A, B, top). The pattern of residuals indicates two components, one reacting ~2 times faster than the other. This systematic deviation from simple monophasic behavior was first noted for the replacement reaction by Olson and Gibson in 1971 (32) and attributed to differences between the α- and β-subunits within tetrameric Hb. Their interpretation has been confirmed by four different sets of experiments over the past 30 yr. Large ligands such as alkyl isocyanides enhance subunit differences (33–35). Chemical modification of the β Cys93 with either mercurials or alkylating agents selectively increases the rate of O2 dissociation from β-subunits (32). Hybrid recombinant Hbs have


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Fig. 5. Time courses for O2 displacement by CO and O2 rebinding after partial photolysis (≤10%) of HbO2 at pH 7.0, 20°C. (A) HbO2 in air was mixed with buffer equilibrated with 1 atm of CO. The concentrations after mixing were [HbO2] = 5 µM, [O2] = 131 µM, and [CO] = 500 µM. (䊊) Observed data; solid line —— a fit to a single exponential expression with kobs = 9.5 s–1. (Top) Differences between observed data and fitted line (residuals). (Inset) Dependence of apparent pseudo first-order rate constant on [CO]/[O2]. The circles represent the observed rate constants and the line a fit to Eq. 9. (B) Time courses for O2 rebinding after 10% photolysis of 100 µM HbO2 taken from the inset in Fig. 3B. (Inset) The circles represent observed data and the line a fit to a single exponential expression with kobs = 76,000 s–1.

been constructed in which one type of subunit is mutated to be either more or less reactive toward ligands, and the other is kept as the wild-type chain (36,37). These mutant hybrids have been used to define the ligand-binding parameters of the “normal” α- and β-subunits (8). Metal hybrid Hbs have been constructed to examine the individual subunits in both the high- and low-affinity quaternary states. The most definitive hybrids contain Cr- and Ni-porphyrin substitutions in one type of subunit and Fe-porphyrin in the other (see ref. 38 and references therein). The Cr(III)-porphyrin groups are inert to ligands but remain 6-coordinated owing to covalently bound water that promotes the high-affinity R state. Ni(II)-porphyrin is also inert but remains 5- or 4-coordinated, biasing the tetramer toward the T

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Table 2 Simple Methods for Assigning Rate Constants to T (Initial Step) and R (Final Step) Forms of Human Hb Ligand

Rate parameter

Technique

CO

k'T (association) k'R (association)

Stopped flow Conventional flash Stopped flow Laser photolysis photolysis Laser photolysis Stopped flow Stopped flow

O2

kR (dissociaton) k'T (association)

NO

k'R (association) kR (dissociation) k'T (association) k'R (association) kR (dissociation)

Laser photolysis Conventional mixing

k'NO,ox

Stopped flow

Reaction Hb4 + CO – simple binding Hb4(CO)4 – partial (≤10%) photolysis Hb4(CO)4 + NO – ligand replacement Hb4(O2)4 – slow phase after 100% Hb4(O2)4 – partial (≤10%) photolysis Hb4(O2)4 + CO – ligand replacement Hb4 + NO – simple binding at very low [Hb],[NO] Hb4(NO)4 – partial (≤10%) photolysis Hb4(NO)4 + CO(excess DT) or Hb4(NO)4 + O2 – ligand replacement and NO scavenging (very slow) Hb4(O2)4 + NO – NO dioxygenation, Hb oxidation at very low [HbO2], [NO]

quaternary structure. Unzai et al. (38) have used (α(Fe)β(Cr))2 and (α(Cr)β(Fe))2 to assign rate constants to the individual subunits in the R conformation and (α(Fe)β(Ni))2 and (α(Ni)β(Fe))2 to assign T-state rate parameters. Quantitative analyses of time courses for ligand binding to Hb become difficult if subunit differences are taken into account. Twenty different intermediates must be considered if the two quaternary states and their rates of interconversion are considered explicitly. This situation is made even more difficult if low concentrations of heme are used and dissociation into dimers occurs. However, this complexity should not inhibit investigators from making the kinetic measurements shown in Figs. 1–5, particularly if the goal is to survey mutant or species differences. Simple experiments that are readily carried out are provided in Table 2. Rate constants for the ligand binding to the R state (defined here as the last step in the Adair scheme in Eq. 4) are readily determined from simple exponential analysis of ligand replacement time courses and rebinding time courses after ≤10% photolysis. The rate constant for CO association to T-state deoxyHb can be estimated by simple mixing experiments. The rate constant for O2 association to the T state can be obtained from analysis of the slow phases observed after total photolysis of HbO2 at high ligand concentrations. The dissociation rate constants for the first step in ligand binding to the T state are much more difficult to measure for native tetrameric Hb.


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In the case of O2, the T-state dissociation rate constant is often estimated by analyzing oxygen equilibrium curves, fixing the R-state parameters and the T-state association rate constants from direct kinetic measurements, and then fitting for the allosteric constant (L = [Hb4(T)]/[Hb4(R)]) and kT. In general, simple analyses that assume subunit equivalence and only two states are inadequate for understanding the detailed structural mechanisms underlying cooperative ligand binding. However, the results in Table 1 show that the differences between the subunit rate constants are less than a factor of 2 for native human HbA. Unzai et al. (38) have argued that even though the structural mechanism for the change in ligand affinity differs significantly between the α- and β-subunits, the two subunits have evolved similar rate and equilibrium constants for O2 binding in order to maximize the amount of observed cooperativity. Thus, the parameters for a simple two-step scheme do provide a useful framework for understanding O2 transport (first three rows in Table 1). The efficiency of O2 delivery depends primarily on the extent and rate of changes in fractional saturation of Hb at different oxygen tensions. As a result, the simple set of parameters, which assumes subunit equivalence, is primarily sufficient to simulate the O2 transport properties of native Hb in capillary experiments (39,40).

3.8. Reversible NO Binding The reactions of NO with Hb are important for detoxifying NO and preventing its transport. The two key reactions are the reversible binding of NO to the ferrous iron atom in deoxyHb and the oxidative reaction of NO with bound O2 to produce nitrate and metHb. In both cases, the rate-limiting step is the capture of NO in the distal pocket of Hb subunits (41). Once inside the protein, NO reacts extremely rapidly, presumably on picosecond time scales, with either the heme iron atom or bound O2 atoms. As a result, both reactions are very rapid: k'NO and k'NO,ox ≈ 60 µM–1s–1 at pH 7.0, 20°C (41). The reaction of NO with deoxyHb is very rapid even when the protein is fully unliganded and in the T quaternary state. In 1975, Cassoly and Gibson (17) reported that the bimolecular rate constant, k'NO, is ~40 µM–1s–1 for both the first and last steps in ligand binding. More recent measurements suggest that k'1 for NO may be twofold less than k'4 (unpublished data), and there may also be small subunit differences (see ref. 41). However, to first approximation, Cassoly and Gibson (17) were correct. The rate of NO binding to deoxyHb is governed exclusively by the rate of ligand entry into the protein and not the R-to-T transition, which affects primarily reactivity at the iron atom. Since the NO dissociation rate constants are very small, 0.001–0.00003 s–1 (13,42), it is possible to measure the rate of the overall binding reaction in rapid-

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Fig. 6. Time courses for the reactions of human HbO2 and sperm whale MbO2 with equimolar amount of NO in 0.1 M phosphate, pH 7.0, 20°C. The concentrations of reactants after mixing were 1 µM. If the reaction is second order and irreversible, plots of 1/[HbO2] vs time should be linear, and the slope determines the bimolecular rate of NO dioxygenation, k'NO,ox. (Inset) Plots for all three proteins.

mixing experiments. However, very low protein and ligand concentrations must be used, and only small, very rapid absorbance changes can be observed. The half-time of the reaction at 1 µM deoxyHb and 1 µM NO is 1/(~40 µM–1s–1 · 1 µM) ≈ 0.025 s. If the NO concentration is raised to 10 µM, the rate becomes ~400 s–1, the half time is ~1.6 ms, and >70% of the absorbance change occurs in the dead time of the apparatus (for equivalent time courses, see Figs. 6 and 7). In addition, great care must be taken to maintain anaerobic conditions in the absence of any added dithionite since both O2 and dithionite will consume the small amounts of NO required for the experiments. NO rebinding can be measured at high ligand concentrations using laser photolysis techniques. The problem in this case is that the quantum yield for complete photodissociation of NO into the solvent phase is ~0.001 at room


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Fig. 7. Reactions of HbO2 and MbO2 with excess NO. The conditions are the same as those in Fig. 6. (Top panel) Observed time courses for reaction of 10 µM NO with human HbO2 and sperm whale MbO2. The secondary phases represent binding of NO to newly formed ferric forms of the proteins. (Bottom panel) Dependence of observed pseudo first-order rate constants for the fast phases on [NO] after mixing. The reaction with the V68F MbO2 mutant is included to show that the linearity is observed over a wide range of NO concentrations (see ref. 41).

temperature. The practical consequence is that no more than 20–30% photolysis can be achieved, even with a 0.5-µs excitation pulse of ~2 to 3 J (28). Thus, only partial photolysis time courses can be measured. The observed rates under these R-state conditions are ~60 µM–1s–1 at pH 7.0, 20°C (41), which is about 1.5- to 2-fold greater than the association rate constant measured by mixing fully deoxygenated Hb with NO at very low heme concentrations (17).

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NO dissociation from fully liganded Hb can be measured in three ways. First, Moore and Gibson (13) showed that kRNO can be measured directly by mixing HbNO with a concentrated solution of dithionite containing 1000 µM (1 atm) CO. As shown in Eq. 10, the added CO will occupy the deoxy sites formed by NO dissociation long enough for the NO to be consumed by dithionite:

(10)

In the absence of CO, the reaction of released NO with the newly formed deoxyHb site will compete effectively with the reaction with dithionite, and the observed rate will be much smaller than the true value of kNO. Second, Sharma and Ranney (42) used excess deoxyMb to scavenge NO from nitrosylHb; however, this method is complex owing to the need for very high concentrations of deoxyMb, which makes simple absorbance measurements difficult. Third, an equally valid method is to expose HbNO to high concentrations of O2 and measure the rate of autoxidation to MetHb. The mechanism of this reaction is as follows:

(11)

In this case, the newly formed deoxyHb site reacts rapidly with O2 since the ratio k'O2[O2]/k'NO[NO] is always kept ≥100 (note that in Table 1, k'O2 ≈ k'NO for R-state Hb). The newly dissociated NO will react rapidly with bound O2 to form metHb and nitrate. NO can also react with free O2 to produce nitrite. The latter reaction is very slow at low NO concentrations (t1/2 ≈ minutes at 1 µM NO (43)) when compared with the reactions of NO with Hb and HbO2 (t1/2 ≈ 0.1–10 ms at 1–10 µM heme (41)). Foley et al. (unpublished observation) and Eich (14) have shown that there is a 1:1 correlation between the rates of autoxidation of ~30 different mutant nitrosylmyoglobins and the corresponding rate constants for NO dissociation


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measured using the CO/dithionite method of Moore and Gibson (13). The same correlation holds for nitrosylHb. NO binding at low levels of Hb saturation is complicated by the formation of pentacoordinate NO-heme complexes in α-subunits, particularly at low pH and in the presence of organic phosphates (44,45). The formation of the pentacoordinate complex is slow (t1/2 ≈ 1 s) compared to ligand binding (47). Its formation also causes ligand reequilibration from an equal mixture of αNO and βNO to predominately αNO complexes in the first Adair intermediate, Hb4NO (46). These complications are manifested as slow, small absorbance changes when deoxyHb is mixed with subsaturating levels of NO. Fortunately, these secondary changes have little effect on the measured rate constants as long as NO is in excess in the mixing or photolysis experiments.

3.9. NO Dioxygenation by HbO2 It has been known for a long time that the addition of NO to either HbO2 or oxymyoglobin causes a very rapid and stoichiometric oxidation of the heme group and formation of nitrate (see references in refs. 47 and 48). This reaction has been used for more than 20 yr as a simple assay for NO synthase activity (for a review, see ref. 49). However, the physiological importance of this process has become clear only within the last 10 yr. HbO2 in red cells and oxymyoglobin in muscle tissue detoxify NO by converting it to NO3–. This scavenging function prevents NO from being transported into actively respiring tissue, where it would inhibit both aconitase and cytocrome oxidase and shut down oxidative phosphorylation (50–53). Gardner (54–56) has called this activity NO dioxygenation and has shown that it is catalyzed efficiently by flavohemoglobins from various microorganisms, in which the expression of the gene is turned on by the addition of toxic levels of NO. Extracellular Hb scavenges NO much more rapidly than Hb packaged in red cells owing to its closer proximity to the endothelium and extravasation into the vessel walls (57,58). The net results are loss of NO signal molecules, little activation of guanylyl cyclase, sustained smooth muscle contraction, and elevated of blood pressure. Thus, we and others have looked for ways to reduce the reactivity of HbO2 with NO in order to design safer and more effective Hb-based blood substitutes (58–61). Time courses for the reaction of 1 µM NO with 1 µM HbO2, oxymyoglobin, and a slowly reacting mutant of myoglobin (V68F, Val68[E11] to Phe) are shown in Fig. 6. Plots of 1/[HbO2]remaining or 1/[MbO2]remaining vs time are linear, indicating simple, irreversible reactions. The bimolecular rate constants for HbO2, wild-type MbO2, and V68F MbO2 are 65, 35, and 10 µM–1s–1, respectively. Further proof that the reaction is bimolecular and depends directly on the first power of [NO] is shown in Fig. 7B. When the reactions are carried out

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under pseudo first-order conditions, [NO] ≥ 5 · [HbO2], there is a linear dependence of kobs on [NO], and the slopes of these plots give rate constants equal to those determined in the equimolar-mixing experiments. The problems associated with the NO dioxygenation reaction are shown in Fig. 7. First, the reactions are very fast and difficult to measure, and it is hard to get more than 2 or 3 points for kobs vs [NO] plots. At 10 µM NO, well over half the reaction with HbO2 is “lost” in the dead time of the apparatus (Fig. 7A). Second, excess free NO can react with newly formed MetHb and MetMb species, causing slow absorbance changes that often occur in the opposite direction of the oxidation reaction (Fig. 7A, right). Third, care must be taken to keep O2 out of the NO solutions and the plastic portions of the mixing device. Otherwise, NO will react slowly with free O2 to produce nitrite in a reaction that consumes 4 NO mol/O2. The resultant nitrite will eventually oxidize HbO2 but at a much slower rate. Fourth, at high pH (≥8.5), a spectral intermediate can be observed and has been assigned to an Fe3+-peroxynitrite complex by Herold and coworkers (63,64) (Fig. 8). Thus, the kinetic scheme for NO dioxygenation reactions needs at least two steps at alkaline pH:

(12)

The peroxynitrite intermediate, Hb(Fe3+OONO–), is formed by a bimolecular process with a rate constant similar to that observed for the overall reaction at pH 7.0, ~50–70 µM–1s–1 (62). This intermediate decays by a first-order process into MetHb and nitrate with no release of peroxynitrite or any ferryl heme formation (63). The rate of decay of the intermediate for both HbO2 and MbO2 increases with decreasing pH. At pH 7.0, it decays too rapidly to be observed. The overall reaction is limited by the bimolecular capture of NO, and simple monophasic kinetic behavior is observed (Fig. 6). In the case of Hb at high pH, the time course of the intermediate decay is biphasic, and Herold (63) has suggested that this is owing to subunit differences. Although difficult to measure, the physiological importance of the NO dioxygenation reaction requires that it be examined routinely in studies of both native and recombinant Hbs. The simplest approach is that shown in Figs. 6 and 7. For example, Eich et al. (41) used these types of experiments to show


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Fig. 8. Reaction of HbO2 with NO in 0.1 M borate, pH 9.0, 20°C. HbO2 (50 µM after mixing) was reacted with an equimolar amount of NO in an OLIS RSM stoppedflow apparatus, and spectra were collected as rapidly as possible (one spectrum every 1 millisecond) in the visible wavelength region (470–670 nm). As shown originally by Herold et al. (64), a spectral species (– – –) resembling the complex of nitrite with MetHb is formed in the dead time of the apparatus. This intermediate decays in a firstorder process to the final hydroxymethemoglobin product (· · ·) with an overall halftime of ~0.020 s.

that Val(E11) to Phe or Trp substitutions in β-subunits and Leu(B10) to Phe or Trp substitutions in α-subunits reduce the rate of NO dioxygenation by HbO2 markedly, up to 10- to 30-fold. Herold et al. (63) used similar methods to characterize the chemistry of the NO dioxygenation reaction with both oxymyoglobin and HbO2. Acknowledgments This research was supported by United States Public Health Service grants GM 35649 and HL 47020 and grant C-612 from the Robert A. Welch Foundation. DHM and EWF were recipients of predoctoral fellowships from NIH Biotechnology Training Grant GM 08362.

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References 1. Hartridge, H. and Roughton, F. J. W. (1923) A method of measuring the velocity of very rapid chemical reactions. Proc. Roy. Soc. A 104, 376–394. 2. Gibson, Q. H. (1959) The kinetics of reactions between haemoglobins and gases, in Progress in Biophysical Chemistry, vol. 9 (Butler, J. A. and Katz, B., eds.), Pergamon, New York, pp. 1–53. 3. Gibson, Q. H. and Milnes, L. (1964) Apparatus for rapid and sensitive spectrophotometry. Biochem. J. 91(1), 161–171. 4. Gibson, Q. (1978) Flash photolysis techniques. Methods Enzymol. 54, 93–101. 5. Olson, J. S. (1981) Stopped-flow, rapid mixing measurements of ligand binding to hemoglobin and red cells. Methods Enzymol. 76, 631–651. 6. Olson, J. S. (1981) Numerical analysis of kinetic ligand binding data. Methods Enzymol. 76, 652–667. 7. Sawicki, C. A. and Morris, R. J. (1981) Flash photolysis of hemoglobin. Methods Enzymol. 76, 667–681. 8. Mathews, A. J. and Olson, J. S. (1994) Assignment of rate constants for O2 and CO binding to alpha and beta subunits within R- and T-state human hemoglobin. Methods Enzymol. 232, 363–386. 9. Henry, E. R., Jones, C. M., Hofrichter, J., and Eaton, W. A. (1997) Can a twostate MWC allosteric model explain hemoglobin kinetics? Biochemistry 36(21), 6511–6528. 10. Gibson, Q. H. (1999) Kinetics of oxygen binding to hemoglobin A. Biochemistry 38(16), 5191–5199. 11. Antonini, E., and Brunori, M. (1971) Hemoglobin and myoglobin in their reactions with ligands, in Frontiers in Biology (Neuberger, A. and Tatum, E. L., eds.), North-Holland, Amsterdam. 12. Sharma, V. S., Ranney, H. M., Geibel, J. F., and Traylor, T. G. (1975) A new method for the determination of ligand dissociation rate constant of carboxyhemoglobin. Biochem. Biophys. Res. Commun. 66(4), 1301–1306. 13. Moore, E. and Gibson, Q. (1976) Cooperativity in the dissociation of nitric oxide from hemoglobin. J. Biol. Chem. 251, 2788–2794. 14. Eich, R. F. (1997), Reactions of nitric oxide with myoglobin, PhD thesis, Rice University, Houston, TX. 15. Olson, J. S., and Phillips, G. N. Jr. (1996) Kinetic pathways and barriers for ligand binding to myoglobin. J. Biol. Chem. 271(30), 17,593–17,596. 16. Gibson, Q. H. and Roughton, F. J. (1965) Further studies on the kinetics and equilibria of the reaction of nitric oxide with haemoproteins. Proc. R. Soc. Lond. B. Biol. Sci. 163(991), 197–205. 17. Cassoly, R. and Gibson, Q. (1975) Conformation, co-operativity and ligand binding in human hemoglobin. J. Mol. Biol. 91(3), 301–313. 18. Scott, E. E., Gibson, Q. H., and Olson, J. S. (2001) Mapping the pathways for O2 entry into and exit from myoglobin. J. Biol. Chem. 276(7), 5177–5188. 19. Monod, J., Wyman, J., and Changeux, J.-P. (1965) On the nature of allosteric transitions: a plausible model. J. Mol. Biol. 12, 88–118.


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20. Gibson, Q. H. and Edelstein, S. J. (1987) Oxygen binding and subunit interaction of hemoglobin in relation to the two-state model. J. Biol. Chem. 262(2), 516–519. 21. Ackers, G. K. (1998) Deciphering the molecular code of hemoglobin allostery. Adv. Protein Chem. 51, 185–253. 22. Gibson, Q. H. (1959) The Photochemical formation of a quickly reacting form of hemoglobin. Biochem. J. 71, 293–303. 23. Edelstein, S. J., Rehmar, M. J., Olson, J. S., and Gibson, Q. H. (1970) Functional aspects of the subunit association-dissociation equilibria of hemoglobin. J. Biol. Chem. 245(17), 4372–4381. 24. Andersen, M. E., Moffat, J. K., and Gibson, Q. H. (1971) The kinetics of ligand binding and of the association-dissociation reactions of human hemoglobin: properties of deoxyhemoglobin dimers. J. Biol. Chem. 246(9), 2796–807. 25. McGovern, P., Reisberg, P., and Olson, J. S. (1976) Aggregation of deoxyhemoglobin subunits. J. Biol. Chem. 251(24), 7871–7879. 26. Sharma, V. S., Schmidt, M. R., and Ranney, H. M. (1976) Dissociation of CO from carboxyhemoglobin. J. Biol. Chem. 251(14), 4267–4272. 27. Gibson, Q. H., Olson, J. S., McKinnie, R. E., and Rohlfs, R. J. (1986) A kinetic description of ligand binding to sperm whale myoglobin. J. Biol. Chem. 261(22), 10,228–10,239. 28. Olson, J. S., Rohlfs, R. J., and Gibson, Q. H. (1987) Ligand recombination to the alpha and beta subunits of human hemoglobin. J. Biol. Chem. 262(27), 12,930–12,938. 29. Sawicki, C. A. and Gibson, Q. H. (1976) Quaternary conformational changes in human hemoglobin studied by laser photolysis of carboxyhemoglobin. J. Biol. Chem. 251(6), 1533–1542. 30. Sawicki, C. A. and Gibson, Q. H. (1977) Properties of the T state of human oxyhemoglobin studies by laser photolysis. J. Biol. Chem. 252(21), 7538–7547. 31. Sawicki, C. A. and Gibson, Q. H. (1977) Quaternary conformational changes in human oxyhemoglobin studied by laser photolysis. J. Biol. Chem. 252(16), 5783–5788. 32. Olson, J. S., Andersen, M. E., and Gibson, Q. H. (1971) The dissociation of the first oxygen molecule from some mammalian oxyhemoglobins. J. Biol. Chem. 246(19), 5919–5923. 33. Reisberg, P. I. and Olson, J. S. (1980) Kinetic and cooperative mechanisms of ligand binding to hemoglobin. J. Biol. Chem. 255(9), 4159–4169. 34. Reisberg, P. I. and Olson, J. S. (1980) Rates of isonitrile binding to the isolated alpha and beta subunits of human hemoglobin. J. Biol. Chem. 255(9), 4151–4158. 35. Reisberg, P. I. and Olson, J. S. (1980) Equilibrium binding of alkyl isocyanides to human hemoglobin. J. Biol. Chem. 255(9), 4144–4150. 36. Mathews, A. J., Olson, J. S., Renaud, J. P., Tame, J., and Nagai, K. (1991) The assignment of carbon monoxide association rate constants to the alpha and beta subunits in native and mutant human deoxyhemoglobin tetramers. J. Biol. Chem. 266(32), 21,631–21,639. 37. Mathews, A. J., Rohlfs, R. J., Olson, J. S., Tame, J., Renaud, J. P., and Nagai, K. (1989) The effects of E7 and E11 mutations on the kinetics of ligand binding to R state human hemoglobin. J. Biol. Chem. 264(28), 16,573–16,583.

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38. Unzai, S., Eich, R., Shibayama, N., Olson, J. S., and Morimoto, H. (1998) Rate constants for O2 and CO binding to the alpha and beta subunits within the R and T states of human hemoglobin [in process citation]. J. Biol. Chem. 273(36), 23,150–23, 159. 39. Lemon, D. D., Nair, P. K., Boland, E. J., Olson, J. S., and Hellums, J. D. (1987) Physiological factors affecting O2 transport by hemoglobin in an in vitro capillary system. J. Appl. Physiol. 62(2), 798–806. 40. Page, T. C., Light, W. R., and Hellums, J. D. (1998) Prediction of microcirculatory oxygen transport by erythrocyte/hemoglobin solution mixtures. Microvasc. Res. 56(2), 113–126. 41. Eich, R. F., Li, T., Lemon, D. D., Doherty, D. H., Curry, S. R., Aitken, J. F., Mathews, A. J., Johnson, K. A., Smith, R. D., Phillips, G. N. Jr., and Olson, J. S. (1996) Mechanism of NO-induced oxidation of myoglobin and hemoglobin. Biochemistry 35(22), 6976–6983. 42. Sharma, V. S. and Ranney, H. M. (1978) The dissociation of NO from nitrosylhemoglobin. J. Biol. Chem. 253(18), 6467–6472. 43. Beckman, J. S. and Koppenol, W. H. (1996) Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am. J. Physiol. 271(5 Pt. 1), C1424–C1437. 44. Perutz, M. F., Kilmartin, J. V., Nagai, K., Szabo, A., and Simon, S. R. (1976) Influence of globin structures on the state of the heme. Ferrous low spin derivatives. Biochemistry 15(2), 378–387. 45. Hille, R., Olson, J. S., and Palmer, G. (1979) Spectral transitions of nitrosyl hemes during ligand binding to hemoglobin. J. Biol. Chem. 254(23), 12,110–12,120. 46. Hille, R., Palmer, G., and Olson, J. S. (1977) Chain equivalence in reaction of nitric oxide with hemoglobin. J. Biol. Chem. 252(1), 403–405. 47. Doyle, M. P., Pickering, R. A., DeWeert, T. M., Hoekstra, J. W., and Pater, D. (1981) Kinetics and mechanism of the oxidation of human deoxyhemoglobin by nitrites. J. Biol. Chem. 256(23), 12,393–12,398. 48. Wade, R. S. and Castro, C. E. (1996) Reactions of oxymyoglobin with NO, NO2, and NO2- under argon and in air. Chem. Res. Toxicol. 9(8), 1382–1390. 49. Stuehr, D. J. (1997) Structure-function aspects in the nitric oxide synthases. Annu. Rev. Pharmacol. Toxicol. 37, 339–359. 50. Gladwin, M. T., Ognibene, F. P., Pannell, L. K., Nichols, J. S., Pease-Fye, M. E., Shelhamer, J. H., and Schechter, A. N. (2000) Relative role of heme nitrosylation and beta-cysteine 93 nitrosation in the transport and metabolism of nitric oxide by hemoglobin in the human circulation. Proc. Natl. Acad. Sci. USA 97(18), 9943–9948. 51. Brunori, M. (2001) Nitric oxide moves myoglobin centre stage. Trends Biochem. Sci. 26(4), 209–210. 52. Brunori, M. (2001) Nitric oxide, cytochrome-c oxidase and myoglobin. Trends Biochem. Sci. 26(1), 21–23. 53. Thomas, D. D., Liu, X., Kantrow, S. P., and Lancaster, J. R. Jr. (2001) The biological lifetime of nitric oxide: implications for the perivascular dynamics of NO and O2. Proc. Natl. Acad. Sci. USA 98(1), 355–360.


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54. Gardner, P. R., Gardner, A. M., Martin, L. A., Dou, Y., Li, T., Olson, J. S., Zhu, H., and Riggs, A. F. (2000) Nitric-oxide dioxygenase activity and function of flavohemoglobins. sensitivity to nitric oxide and carbon monoxide inhibition. J. Biol. Chem. 275(41), 31,581–31,587. 55. Gardner, P. R., Gardner, A. M., Martin, L. A., and Salzman, A. L. (1998) Nitric oxide dioxygenase: an enzymic function for flavohemoglobin. Proc. Natl. Acad. Sci. USA 95(18), 10,378–10,383. 56. Gardner, A. M., Martin, L. A., Gardner, P. R., Dou, Y., and Olson, J. S. (2000) Steady-state and transient kinetics of Escherichia coli nitric-oxide dioxygenase (flavohemoglobin). The B10 tyrosine hydroxyl is essential for dioxygen binding and catalysis. J. Biol. Chem. 275(17), 12,581–12,589. 57. Liu, X., Miller, M. J., Joshi, M. S., Sadowska-Krowicka, H., Clark, D. A., and Lancaster, J. R. Jr. (1998) Diffusion-limited reaction of free nitric oxide with erythrocytes. J. Biol. Chem. 273(30), 18,709–18,713. 58. Doherty, D. H., Doyle, M. P., Curry, S. R., Vali, R. J., Fattor, T. J., Olson, J. S., and Lemon, D. D. (1998) Rate of reaction with nitric oxide determines the hypertensive effect of cell-free hemoglobin [see comments]. Nat. Biotechnol. 16(7), 672–676. 59. Olson, J. S. (1994) Genetic engineering of myoglobin as a simple prototype for hemoglobin- based blood substitutes. Artif Cells Blood Substit Immobil Biotechnol 22(3), 429–441. 60. Olson, J. S., Eich, R. F., Smith, L. P., Warren, J. J., and Knowles, B. C. (1997) Protein engineering strategies for designing more stable hemoglobin-based blood substitutes. Artif. Cells Blood Substit Immobil. Biotechnol. 25(1–2), 227–241. 61. Tsai, C. H., Fang, T. Y., Ho, N. T., and Ho, C. (2000) Novel recombinant hemoglobin, rHb (beta N108Q), with low oxygen affinity, high cooperativity, and stability against autoxidation. Biochemistry 39(45), 13,719–13,729. 62. Herold, S. (1999) Kinetic and spectroscopic characterization of an intermediate peroxynitrite complex in the nitrogen monoxide induced oxidation of oxyhemoglobin. FEBS Lett. 443(1), 81–84. 63. Herold, S., Exner, M., and Nauser, T. (2001) Kinetic and mechanistic studies of the NO*-mediated oxidation of oxymyoglobin and oxyhemoglobin. Biochemistry 40(11), 3385–3395. 64. Herold, S. (1999) Mechanistic studies of the oxidation of pyridoxalated hemoglobin polyoxyethylene conjugate by nitrogen monoxide. Arch. Biochem. Biophys. 372(2), 393–398.

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Electrophoretic Methods for Study of Hbs

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6 Electrophoretic Methods for Study of Hemoglobins Henri Wajcman 1. Introduction Electrophoresis, a technique consisting of the migration of electrically charged molecules under an applied electric field, occupies one of the most important places in the history of the study of hemoglobin (Hb). HbS, the first abnormal Hb described, was discovered in 1949 by Pauling et al. (1), using moving boundary electrophoresis. Later, Hb variants were detected by zone electrophoresis on paper, starch gel, or cellulose acetate (2,3). With the exception of cellulose acetate electrophoresis, which is still used in some laboratories, these procedures have been replaced by isoelectric focusing (IEF) (4). In IEF, a pH gradient is established by carrier ampholytes subjected to an electric current. The Hb molecule migrates across this gradient until it reaches the position where its net charge is zero (isoelectric point [pI]). It then concentrates into a sharp band. The most traditional and largely used methods of identifying and studying normal and mutant Hbs are the panoply of electrophoretic methods. This chapter describes the strengths and weakness of the most commonly used electrophoretic methods to separate Hbs. This electrophoretic approach needs to be assessed by other criteria, taking into accounts geographic and ethnic distribution as well as hematological and clinical presentation. In some cases additional tests such as biophysical or functional properties or mass spectrometry determinations may be required. For the last 20 yr in the Hemoglobin laboratory of Henri Mondor Hospital (Créteil, France) we have taken a multicriteria approach to the study of Hb, leading to a presumptive diagnosis for most of the known Hb variants. The electrophoretic mobility of more than 400 Hb variants is stored in a data bank under a format convenient for comparison based on an approach close to that From: Methods in Molecular Medicine, vol. 82: Hemoglobin Disorders: Molecular Methods and Protocols Edited by: Ronald L. Nagel © Humana Press Inc., Totowa, NJ

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proposed some 20 yr ago by Barwick and Schneider (5). This strategy includes tests done on native Hbs (IEF on polyacrylamide gel, electrophoresis on cellulose acetate at alkaline pH, and citrate agar electrophoresis), and tests on dissociated globins (electrophoreses of globin chains in 6 M urea at pH 6.0 and 9.0 or in the presence of Triton X-100). The electrophoretic mobility of the variants is measured according to a quantified method that is described next. In our laboratory, a wide collection of identified rare variants is stored in liquid nitrogen and may be used, in a last step, as specific controls. 2. Materials and Methods 2.1. Isoelectric Focusing IEF studies of Hb may be done on polyacrylamide or agarose gels containing free ampholytes. These gels can be homemade (6–7), but it is more convenient to use IEF plates polymerized on support films, which are commercially available from several manufacturers (e.g., Perkin-Elmer, Norwalk, CT; Wallac, Akron, OH; Amersham Pharmacia Biotech, Uppsala, Sweden; Serva, Heidelberg, Germany). Polyacrylamide gels were the first ones used (6). Agarose gels suitable for IEF became only available later, when chemical treatments were developed to remove or mask the charged agaropectin residues present in the raw material. Agarose gels exhibit stronger electroendosmosis than polyacrylamide gels. Pores within this gel are also larger, making them more suitable for large proteins. In addition, agarose gels are also selected for routine work because they are not toxic and do not contain catalysts, which could interfere with the Hb molecule and thus lead to separation artifacts. Later, procedures were developed to cast polyacrylamide gels with covalently bound immobilized pH gradients. This technique allows the preparation of gel plates with pH ranges of 0.3 g% (~0.19 mM heme or ~0.05 mM tetramer) (Fig. 3, [14]).

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Fig. 2. Comparison of fluorescence optics: (A) front-face; (B) right-angle optics. (From ref. 17.)

Front-face measurements may be simply, but suboptimally, achieved in a right-angle configuration with the use of small (millimolar) rounded cuvets or triangular cuvets (14,16–17,20), with the latter providing more sensitive detection. A front-face cell is designed for easy insertion into a standard cuvet holder for 1 × 1 cm cells and orients the sample for the optimal angle requirement (Fig. 4A). The small volume required for this cell (100–200 µL) becomes advantageous when studying heme-proteins with limited availability (e.g., scarce mutants or recombinant mutants). However, an instrument designed with a horizontal orientation of the light source slit may preclude use of this cell. Most companies now offer the option of temperature controlled front-face adapters designed specifically for the fluorometer.

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Fig. 3. Concentration dependence of Hb fluorescence emission intensity plateaus when using front-face optics. Excitation wavelength: 280 nm; oxyHbA: 0.07 mM tetramer, 0.05 M potassium phosphate buffer, 25°C. (From ref. 14.)

Novel variations in front-face optical designs provided further advantage in the study of heme-protein fluorescence. The rhombiform optical cell (Fig. 4B), designed by Horiuchi and Asai (21), simultaneously measures absorption and fluorescence. This allows direct and continuous measurements of the binding of a fluorescent allosteric effector to Hb (at a limited range of concentration), while assessing variations in the partial pressure of oxygen during deoxygenation. The solution is gently stirred for gas exchange. Caution must be exercised when stirring any protein solution, and especially Hbs, which may be subject to mechanical instability (e.g., HbS [22]). With the purpose of eliminating reflections and stray emissions (which may become significant for the relatively low fluorescence emission of heme-proteins), Bucci and colleagues (23,24) developed an optimized shielded cuvet as well as designed an optical cell with a front-face configuration that operates on a free liquid surface (Fig. 4C). This also avoids any possible protein conformational changes induced by a protein-solid interface. However, air-water interfaces do have the potential to induce protein unfolding for some proteins and Hb mutants (25–26). Absolute fluorescence and the determination of quantum yields are not possible with heme-proteins until one can design a true “blank”: the identical globin fold without the heme. Finding a true blank remains a challenge since the respective apoglobins (i.e., globin without the heme) are structurally dis-

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Fig. 4. Novel fluorescence optical designs for detection of heme-protein fluorescence. (A) Front-face optics is achieved by an insert placed in a standard right-angle cuvet holder. The base plate shown on the left is removable. The key feature is that the exciting light makes an angle of 34° with the normal to the cell face, or, by inverting, one may make the angle of incidence 56°. The central rays of the excitation and emission beams intersect normally at the center of the cuvet holder for either configuration.


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tinct from intact Hb or intact myoglobin and, therefore, unsuitable for this purpose. Apohemoglobin and apomyoglobin exhibit shifted fluorescence emission maximas consistent with findings that apohemoglobin is a dimer (27,28) and apomyoglobin is unfolded (29). Apohemoglobin derived from HbA exhibits a fluorescence emission maximum shifted ~14 nm to longer wavelengths. Obviously, in the quest for a blank, the buffer does not represent a true baseline for a heme-protein. Therefore, until a true blank is designed, absolute fluorescence quantum yields are unattainable. Yet, this limitation has not impeded the utility of heme-protein fluorescence analysis and interpretation. Since fluorescence polarization calculations are based on right-angle optics, only relative polarization and anisotropic measurements are meaningful with front-face fluorescence using adapters with quartz cuvets. Alternative frontface cell designs (Fig. 4B,C) may provide a means to reduce the distortions in anisotropic measurements that are introduced by front-face fluorometry (24). Front-face optics also have been used in time-resolved fluorescence measurements of hemoglobins (30). Generally, with state-of-the-art time-resolved fluorescence measurements, normal Hb exhibits a multiexponential decay that fits to three lifetimes. Since Trp itself exhibits a multiexponential decay, interpretation of the intrinsic fluorescence lifetimes (picosecond, subnanosecond, and nanosecond) remains controversial and is discussed at length in ref. 17. Nevertheless, the intrinsic and extrinsic fluorescence of Hb is an established property that is useful in probing structural perturbations in Hb. Noteworthy is that front-face fluorometry is clinically important for the detection of fluorescent components circulating in blood. The hematofluorometer was designed to measure blood levels of zinc protoporphyrin, which rises in lead poisoning and iron deficiency anemia, and blood levels of protoporphyrin IX, which may rise in porphyria diseases (e.g., erythropoietic protoporphyria) (31–33). Circulating bilirubin may also be detected by this method (34). Furthermore, front-face fluorometry has been useful in detecting the binding of porphyrins to Hb (35); correlating high zinc protoporphyrin levels in sickle Fig. 1. (continued) The front window of the cell is 0.5 mm thick and the sample thickness is 1 mm. This cuvet is advantageous for rare samples, requiring ~100–200 µL. (From ref. 19.) (B) Shown is the schematic of the rhombiform optical cell compartment. Components (a) and (b) are made of quartz, and (c) is the Hb sample. The solid line depicts the incident excitation light beam; the broken and dotted lines show the transmitted and the emitted light, respectively. θ is 52.4°, avoiding light direct excitation beam reflectance. (From ref. 21.) (C) Side view of free-surface cuvet. 1–3, Fixed quartz windows; 4, sliding quartz window; 5, metallic mirror; 6, body of the cover; 7, body of the cuvet; 8, supporting stem; 9, liquid sample; 10, O-rings. (From refs. 23 and 24.) (Composite figure from ref. 17.)

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cell patients with low HbF levels (36); and detecting circulating fluorescent drugs, such as the antibiotic tetracycline (37), and drugs used in inflammatory bowel disease, such as the aminosalicylic acid derivatives that circulate in blood (38–39).

1.2. Intrinsic Hb Fluorescence Emission Is Sensitive to Tertiary and Quaternary Structural Alterations While Tyr fluorescence may be distinguished in a heme-protein containing both Tyr and Trp (14,29), Trp fluorescence emission generally predominates as a result of a greater quantum yield and fluorescence resonance energy transfer from Tyr to Trp. In general, Tyr and Trp are excited with 280 nm excitation light, while 296 nm selectively excites Trp (40). The contribution by Tyr may be dissected out by the difference spectrum (296 nm excitation emission spectrum –280 nm excitation emission spectrum) (40). The emission maximum of Tyr is ~305 nm, which is blue shifted from the emission maximum of Trp. It is well established that the Trp emission maximum is a function of the microenvironment: Trp in a hydrophilic environment or exposed environment exhibits an emission maximum at 350–353 nm, whereas the maximum for a hydrophobic or buried Trp is at 330–332 nm; Trp in limited contact with water exhibits an emission maximum at 340–342 nm (41). This environmental sensitivity of Trp arises from a large dipole change on excitation (42). Experimentally, the exact emission maximum wavelength may vary (up to ~5 nm) with the specific instrument employed. Evidence that the fluorescence signal emanates from intact Hb is supported in part by the ~330-nm emission wavelength, which is that of a buried Trp, consistent with the location of β37 Trp. By contrast, if α14 Trp and β15 Trp, which lie close to the surface, were the primary emitter, a longer wavelength emission maximum (~345–355 nm) corresponding to partially or fully exposed Trp would be expected. Likewise, if the Hb fluorescence emission originated from an exposed Trp, it should be quenched by KI. This is not the case (14). Similarly, if the emission arose from an apoglobin, the emission maximum would be shifted to longer wavelengths than that observed. An example of a shifted emission maximum when Trp is in an aqueous microenvironment is seen in the recombinant Hb α96 Val → Trp designed by Ho and collaborators (43,44). This Hb exhibits low oxygen affinity, high cooperativity, and no unusual subunit dissociation (43). The steady-state fluorescence emission maximum of Hb α96 Trp is of higher intensity and ~5 nm shifted to longer wavelengths in comparison with HbA (Fig. 5), suggesting that the additional α96 Trp is exposed to an aqueous environment. This prediction (as opposed to the one formulated by molecular dynamics simulation [43]) was confirmed by the high-resolution X-ray crystal structure showing the addi-


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Fig. 5. Steady-state front-face fluorescence emission spectra of a Trp-plus Hb recombinant mutant compared with HbA: COHb α96 Val → Trp and COHbA. The solution was 0.05 M HEPES, pH 6.5, 25°C. Note the ~5-nm shift to longer wavelengths of the emission maximum (relative to COHbA in the same conditions). This wavelength shift indicates that the additional Trp is in a more exposed or hydrophilic environment, as confirmed by the high-resolution X-ray crystal structure (44). The emission maximum of HbA falls at ~320 nm, indicative that the primary emitter is that of a buried or hydrophobic Trp (e.g., β37 Trp). Both Hbs contain α14 Trp, β15 Trp, and β37 Trp, with the recombinant Hb containing the additional α96 Trp.

tional α96 Trp indole side chain directed away from the α1β2 interface and directed toward the water-filled central cavity (44).

1.2.1. Ligand Binding and Quaternary Structure Changes in Hb It is well established, by several different laboratories, that the steady-state fluorescence emission intensity is dependent on the R (oxy) → T (deoxy) transition (for a review, see refs. 16 and 17): an 18–25% increase in the fluorescence intensity is observed on deoxygenation (Fig. 6). This R → T transitional change in fluorescence intensity is sensitive to pH as modulated by inositol hexaphosphate (IHP) for carp Hb (45) and HbA (Fig. 7). These pH effects are observed in the range where Trp and Tyr fluorescence emission is pH insensitive (i.e., pH 3.0–11.0) (46). Therefore, the data presented in Fig. 7 are modulated by the R → T transition, which, to a degree, may be correlated with the Bohr effect.

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Fig. 6. Hb fluorescence is a sensitive reporter of the R → T transition. The frontface intrinsic fluorescence emission of HbA varies as a function of ligand binding. All solutions are 0.155 mM Hb tetramer (pH 7.35), 0.05 M phosphate, 25°C. The lowest curve is the buffer solution. (From ref. 57.)

Relative fluorescence comparisons of variant hemoglobins to HbA are instructive. Conformational changes in non-Trp-substituted Hb mutants are reflected by emission differences compared to HbA. For example, fluorescence emission intensity differences are seen among R-state HbC (β6 Glu → Lys), HbS (β6 Glu → Val), and HbA. Coupled with functional studies, circular dichroism, differential fluorescence perturbations by allosteric effectors, and UV resonance Raman studies, significant differences in the A-helix and the central cavity of the β6 mutants were revealed (47–50). Spectroscopic solution studies may reveal fluctuations that are not seen in the crystal structure because of lattice constraints. This serves as an example of the importance of spectroscopy to reveal fluctuations that occur in solution but that may be constrained in the crystal structure and, thus, not observed with ~2Å resolution. Fluorescence spectroscopy combined with resonance Raman spectroscopy and functional studies revealed site-specific tertiary and quaternary differences in the Tyr-substituted Hb, Hb Montefiore (α126 Asp → Tyr), which exhibits high oxygen affinity and low cooperativity (51). OxyHb Montefiore exhibits a ~40% increase in fluorescence compared with oxyHbA. This difference is more than would be expected with the additional Tyr. The difference spectrum using


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Fig. 7. The intrinsic fluorescence of HbA is sensitive to pH and is further modulated by IHP. (a) Plotted is the change in fluorescence intensity of deoxyHbA minus oxyHbA in the absence of IHP and presence of IHP (excitation: 280 nm; emission maximum: 330 nm). (b) Plotted is the difference between the change in the fluorescence intensity in the absence of IHP [deoxy – oxy] minus the fluorescence intensity change in the presence of IHP [deoxy – oxy]. Note that Trp or indole fluorescence intensity is not sensitive to pH within this range (46,81) (from Lin and Hirsch, unpublished data; ref. 82).

280-nm excitation exhibits a shoulder at ~308 nm, providing a spectral marker for α126 Tyr. Confirmation of this marker is shown by the difference emission spectrum which arises with 296-nm excitation selective for tryptophans, that does not show the ~308-nm shoulder. The large difference in the fluorescence compared to HbA indicates a conformational change or a change in the fluctuation properties of this Hb. The molecular alteration appears to be more significant in the T state than in the R state since the difference spectrum when subtracting deoxyHb Montefiore from deoxyHbA is significantly greater than

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Fig. 8. oxyHb intrinsic fluorescence spectra as a function of synthetic allosteric effector DCBAA titration. A titration of oxyHbA with DCBAA, up to a molar ratio of 10:1 (DCBAA:Hb tetramer ratio), is monitored by decreases in the intrinsic fluorescence emission of Hb (pH 7.35, 0.1 M HEPES). Excitation wavelength: 280 nm (unpublished data; see ref. 53).

the difference spectrum of the oxy forms. The T-state fluorescence difference may result from the alteration in the important contact between α126 Asp and the α1β2 interface residues found in normal Hb, specifically β35 Tyr and β34 Val and the C-terminal 141 Arg. The probable perturbation of this contact by α126 Tyr and conformational alteration is likely to extend to the nearby β37 Trp that serves as the reporter of this altered T state. This destabilized T state may bind oxygen with higher affinity than deoxyHbA (51). Binding of allosteric effectors and their perturbation of Hb conformation may be monitored by front-face fluorometry. For example, the synthetic allosteric effector 3,4-dichloro-benzyloxy acetic acid (DCBAA) decreases the oxygen affinity of Hbs by binding specifically at the deoxyHb surface/crevice residue α14 Trp (52). The crystal structure of DCBAA bound to deoxyHbA demonstrates site-specific binding to the A-helix of deoxyHb, specifically α14 Trp (52). Thus, binding of DCBAA to Hb as monitored by Hb intrinsic fluorescence changes serves as a specific reporter of the A helix at α14 Trp. This is seen by probing distally to the β6 site of mutation in R states of HbC and HbS. The differences in oxyHb intrinsic fluorescence in response to DCBAA are shown in Fig. 8; oxyHbC and oxyHbS show a minimal decrease in the intensity of the intrinsic fluorescence emission maximum on titration with DCBAA. These titration results show that the fluorescence intensity changes of oxyHbC and oxyHbS, with increasing concentration of DCBAA, are just at the level of resolution of the instrument. By contrast, oxyHbA exhibits a larger decrease in fluorescence intensity (~6%) with clear resolution of the fluorescence intensity changes as a function of DCBAA titration. Consistent with the findings by


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Mehanna and Abraham (52), the oxyHbA titration data indicate that DCBAA binds to R-state HbA in a 6:1 mole ratio. The titration curve (Fig. 8) exhibits a change of slope at a ratio of 2:1, which may imply two sites with different affinity and capable of allosteric change. The ability of DCBAA to serve as a fluorescence quencher is shown by the significant decrease in fluorescence of liver alcohol dehydrogenase on addition of DCBAA (53). The above data further demonstrate differences in the A-helix of HbC and HbS. A further conclusion that may be drawn from these studies is that the relatively small intrinsic fluorescence quenching of Hb on binding of DCBAA implies that α14 Trp is a small contributor to the overall intrinsic fluorescence signal emanating from HbA. Changes in the relative fluorescence intensity in the presence of allosteric effectors may also be explained as a function of the R → T transition. The 2,3diphosphoglycerate (DPG) analogs—IHP (e.g., Fig. 7) and the fluorescent 1,3hydroxypyrene-trisulfonate (HPT) (see Subheading 1.3.)—both induce changes in fluorescence intensity and have been useful in interpreting Hb fluorescence, comparing structural perturbations in Hb variants, as well as obtaining binding constants (54). Since the natural allosteric effectors, such as DPG and chloride, alter the conformation of Hb that is revealed as a perturbation in the fluorescence emission (49,55), it is important to strip Hb of these factors before embarking on such titration studies. The method to strip Hb is described in Subheading 3. Hb-reducing agents such as sodium dithionite should not be used for deoxygenation, because dithionite exhibits significant UV absorption and fluorescence that overlaps with Hb excitation and intrinsic emission. However, dithionite interference is minimal in the visible light range and may be used to deoxygenate Hb bound with an extrinsic fluorophore that emits visible light (56). Deoxygenation without dithionite is carried out by gently blowing nitrogen or helium over an Hb solution in a closed system for up to 90 minutes if needed, without met (Fe+3) Hb formation.

1.2.2. Hb Oxidation Intrinsic fluorescence emission intensity increases significantly (greater than twofold) on oxidation to methemoglobin compared with oxyHb and deoxyHb (54,57). In addition, the chemical oxidation of Hb may be monitored as a function of the generation of a fluorescent heme degradation end product using H2O2 as the oxidizing agent. The fluorescent end-product exhibits an emission maximum at 465 nm with 321-nm excitation (58–60). The coupling of frontface fluorometry with this method permits comparison of the oxidation propensity of various Hb mutants at concentrations at which the Hb remains a tetramer (55).

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1.2.3. Hb Dissociation Hb fluorescence studies using right-angle optics require low Hb concentrations. Generally, when right-angle optics are employed, low protein concentrations (on the order of micromolar) are required. However, in the case of intact HbA (a tetramer composed of two identical α- and two identical β-chains) significant dissociation to dimers occurs at low concentration in the oxy or R-state forms: under conditions of moderate ionic strength (~0.1 M NaCl), KD = 1.0 × 10–6 M. In the case of deoxyHb, dissociation is significantly less (KD = 2.0 × 10–11 M) (61–62). Note that a high salt concentration will shift the dissociation equilibrium to dimers (27,63–64). The percentage of dimer dissociation (α) is calculated by (64): α = {KDM4[1 + (16c/KDM4)] – KDM4}/8c

in which KD is the dissociation constant in molarity, M4 is the molecular weight of the tetramer, and c is the concentration in grams/liter. Dimerization results in emission maximum shifts to longer wavelengths as a result of Trp exposure to a more hydrophilic environment (65–66). This concentration-dependent dissociation (1) complicates the interpretation and comparison of Hb fluorescence studies performed under different solution conditions, and (2) highlights the advantage of using front-face optics which reduces inner-filter effects that arise in a strongly absorbing solution. Reversible protein dissociation and unfolding may be investigated using high hydrostatic pressure (up to ~2 bBar) coupled to fluorescence (67). A number of laboratories have utilized steady-state and anisotropic fluorescence measurements in conjunction with the application of high pressure to study the dissociation properties of variant hemoglobins (68–72). In summary, Hb in its native state is a tetramer, and unwarranted solution conditions (e.g., dilute solutions, high salt concentration) can shift the equilibrium to the preponderance of dimers that differ in structure and function; some Hb variants are more prone to dissociation (73); and the apoprotein (i.e., globin without hemes) results in a distinct structure without resemblance to the native tetramer. Dissociation and apoglobin formation may be revealed by front-face fluorometry. The fluorescent moiety 1-anilinonaphthalene-8-sulfonic acid (ANS), known to bind to the empty heme pocket, was first used to demonstrate that the fluorescence arises from intact Hb (13).

1.3. Extrinsic Fluorescence The site-specific labeling of proteins with extrinsic fluorescent probes allows spectroscopic probing of side chains of amino acid residues that are nonfluorescent and may serve as a ruler to measure intra- and intermolecular distances. Front-


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face fluorometry permits direct monitoring of the probe bound to a hemeprotein. For example, iodoacetamidofluorescein covalently modifies β93 Cys (the nearest neighbor to the proximal His of the heme) and is responsive to the R → T transition (74). Fluorescence quenching studies of HPT when bound to Hb demonstrated that HPT serves as a DPG analog, binding noncovalently (one per Hb tetramer) to the central cavity (11,75–77). DPG is the natural allosteric effector of the red blood cell that binds to Hb in a 1:1 ratio, effectively lowering the oxygen affinity so that sufficient oxygen is released to tissues with low oxygen saturation. Direct monitoring of HPT fluorescence is a means to explore the Hb central cavity and has been useful in demonstrating (1) structural alterations distal from the site of mutation, such as HbS (β6 Glu → Val) and HbC (β6 Glu → Lys) (48); and (2) central cavity differences anticipated in crosslinked Hbs designed as potential therapeutic oxygen carriers (78). Another fluorescent DPG analog, 1,3,6,8-pyrenetrisulfonate, may be advantageous for time-resolved fluorescence studies probing the DPG pocket; this application revealed a long-range communication from the positively charged substitution in the middle of the central cavity of Hb Presbyterian (β108 Asn → Lys) to the DPG-binding pocket that lies at the entrance to the ββ cleft (79). In summary, intrinsic and extrinsic fluorescence properties of Hb are valuable reporters of site-specific conformational changes. 2. Materials 1. 2. 3. 4. 5. 6. 7. 8.

Purified hemoglobins. Fluorescence spectrophotometer with UV excitation and emission monochrometers. Front-face adapter or specially designed cuvets for front-face optics. Cuvet washer. 100% Ethyl alcohol contained in glass. Nitric acid. Anaerobic glove box or glove bags. Fluorescence probes (e.g., 5-iodoacetamidofluorescein, cat. no. I-3; Molecular Probes, Eugene OR; or 1,3-hydroxypyrene trisulfonate, H-1529; Sigma, St. Louis, MO).

3. Methods (see Notes 1–9) 3.1. Purification of Hemoglobins For reasons already noted, it is critical to this technique that the Hb be homogeneously purified, and that a concentration be selected wherein it remains intact in its tetrameric native state containing all four hemes. Depending on the Hb mutant, Hb may be separated and purified by liquid column chromatography using anion- and cation-exchange resins. The red cell

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lysate (hemolysate) contains 95% Hb. It also contains a small percentage of other molecules, cofactors that interact with Hb, and a variety of enzymes and proteins including minor Hbs (e.g., HbF, HbA2, HbA1a1, HbA1a2, HbA1b, HbA1c). Thus, it is imperative that these other proteins and compounds be removed. This is specifically addressed by Pin et al. (70). Therefore, for meaningful comparisons, the proteins should be purified and handled according to one method of choice (see ref. 80). (Hb purification is further developed in Chapters 3, 6, 10, and 14.) Purified Hbs are stripped (i.e., DPG and various ions are removed) by Sephadex G-25 gel filtration chromatography. Stripping twice ensures the preparation of fully stripped Hbs, which may be assessed by oxygen equilibrium methods: it is known that stripped Hb has a higher oxygen affinity compared with nonstripped Hb.

3.2. Covalent Modification with Fluorescent Probes As an example, iodoacetamidofluorescein (5-IAF) (cat. no. I-3, Molecular Probes) covalently binds to sulfhydryl groups. β93 labeling of Hb may be effected by a slight modification of a procedure described earlier (74). A purified Hb solution (~3–5 g%, 5 mL) in the presence of an ~5-IAF:1 heme in the desired buffer is incubated for 3 hours at 4°C with gentle hand rotation every 20–30 min. After 3 h, the solution is added to a Sephadex G-25 column (50-mL volume) equilibrated in the desired buffer. The solution is collected, repurified, and concentrated in Centricon 10 (YM 10, Amicon) three times or until no fluorescence is found in the dialysate as detected by a fluorometer. Isoelectric focusing and mass spectrometry are used to assess complete modification and to ensure purity. An inability to titrate with paramecuricbenzoic acid is another method to verify that the reactive —SH groups are all bound. However, if available, mass spectrometry is the method of choice for determination of site-specific and complete modification. Functional alterations that may arise as a result of site-specific labeling are examined by oxygen-equilibrium methods. 4. Notes 1. Quartz cuvets are required for UV excitation light and emission of intrinsic fluorescence. 2. At millimolar Hb concentrations, the Raman band should not interfere with the emission spectrum as observed with dilute aqueous solutions. 3. Do not store liquid buffers or Hb solutions in plastic tubes for any length of time (hours); plastic derivatives that scatter and fluoresce may be introduced into the solutions. 4. The choice of buffer is important, with 0.05 M HEPES is optimal. Phosphates may bind in the DPG pocket of the central cavity. Tris buffers should be avoided


5.

6.

7.

8.

9.

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because Tris exhibits fluorescence (although this may not be the case with ultrapurified Tris). Therefore, the buffer baseline should always be recorded to ensure no background fluorescence from the buffer or from any residual material in the cuvet. Cuvets may be soaked with 50% nitric acid for several minutes up to a few hours. If soaked longer or if higher concentrations of nitric acid are used longer than a few minutes, etching of the cuvet is possible. The use of detergents is not recommended because they may contribute to the fluorescence background. The cuvet should be rinsed thoroughly with distilled water, followed by a final rinse with 100% ethyl alcohol, and dried on a vacuum-cuvet washer. Pipeting samples into the cuvet should be carefully done without scratching. If necessary, soft tubing may be added to the end of a needle or hard pipet tip. The cuvets should be held at the edges to avoid fingerprints. As for any fluorescence study, the same cuvet should be used for relative comparisons. Moreover, the cuvet must be placed in the identical position during measurements. Excitation sources such as intense lasers or light arising from a synchrotron may cause heating, which can interfere with the stability of the protein. This may be avoided by rotating the cuvet, replacing the sample after every third scan, or recording spectra in a closed flowing cell. Fluorescence intensity is temperature dependent (i.e., the higher the temperature, the lower the intensity), and, therefore, temperature must be kept constant during the course of the experiment and for relative comparisons. For studies of temperature-sensitive mutants, the fluorescence difference as a function of temperature between the wild-type protein and the temperature-sensitive variant must be factored in.

Acknowledgments This work was supported in part by the American Heart Association, Heritage Affiliate Grant-in-Aid No. 0256390T; and the National Institutes of Health R01 HL58038 and RO1 HL58247. References 1. Weber, G. (1953) Rotational brownian motion and polarization of the fluorescence of solutions. Adv. Protein. Chem. 8, 415–459. 2. Teale, F. W. J. and Weber, G. (1957) Ultraviolet fluorescence of the aromatic amino acids. Biochem. J. 65, 476–482. 3. Teale, F. W. J. and Weber, G. (1959) Ultraviolet fluorescence of proteins. Biochem. J. 72, 156. 4. Teale, F. W. J. (1960) The ultraviolet fluorescence of proteins in neutral solution. Biochem. J. 76, 381–388. 5. Stryer L. (1978) Fluorescence energy transfer as a spectroscopic ruler. Annu. Rev. Biochem. 47, 819–846.

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72. Hirsch, R. E., Friedman, J. M., Harrington, J. R., and Scarlata, S. F. (1994) Stability of a potential blood substitute, HbXL99α under high pressure. Biochem. Biophys. Res. Commun. 200, 1635–1640. 73. Sharma, V. S., Newton, G. L., Ranney, H. M., Ahmed, F., Harris, J. W., and Danish, E. H. (1980) Hemoglobin Rothschild (β 37(C3)Trp replaced by Arg): A high/low affinity hemoglobin mutant. J. Mol. Biol. 144, 267–280. 74. Hirsch, R. E., Zukin, R. S., and Nagel, R. L. (1986) Steady-state fluorescence emission from the fluorescent probe, 5-iodoacetamidofluorescein, bound to hemoglobin. Biochem. Biophys. Res. Commun. 138, 489–495. 75. MacQuarrie, R. and Gibson, Q. H. (1971) Use of a fluorescent analogue of 2,3diphosphoglycerate as a probe of human hemoglobin conformation during carbon monoxide binding. J. Biol. Chem. 246, 5832–5835. 76. MacQuarrie, R. and Gibson, Q. H. (1972) Ligand binding and release of an analogue of 2,3-diphosphoglycerate from human hemoglobin. J. Biol. Chem. 247, 5686–5694. 77. Serbanescu, R., Kiger, L., Poyart, C., and Marden, M. C. (1998) Fluorescent effector as a probe of the allosteric equilibrium in methemoglobin. Biochim. Biophys. Acta 1363, 79–84. 78. Gottfried, D. S., Juszczak, L. J., Fataliev, N. A., Acharya, A. S., Hirsch, R. E., and Friedman, J. M. (1997) Probing the hemoglobin central cavity by direct quantification of effector binding using fluorescence lifetime methods. J. Biol. Chem. 272, 1571–1578. 79. Gottfried, D. S., Manjula, B. N., Malavalli, A, Acharya, A. S., and Friedman, J. M. (1999) Probing the diphosphoglycerate binding pocket of HbA and HbPresbyterian (β 108 Asn → Lys). Biochemistry 38, 11,307–11,315. 80. Schroeder, W. A. and Huisman, T. H. J. (1980) The Chromatography of Hemoglobin, Marcel, Dekker, New York. 81. White, A. (1959) Effect of pH on fluorescence of tyrosine, tryptophan, and related compounds. Biochem. J. 71, 217–220. 82. Lin, M. J., Rao, M. J., Friedman, J. M., Acharya, A. S., and Hirsch, R. E. (1991) Inositol hexaphosphate induced pH sensitive conformational changes of the α1β2 interface of hemoglobin. Biophys. J. 59(2), 290a.

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10 Nucleation and Crystal Growth of Hemoglobins The Case of HbC Peter G. Vekilov, Angela Feeling-Taylor, and Rhoda Elison Hirsch 1. Introduction Hemoglobin (Hb) crystallization is of significance both in vivo and in vitro. Hb crystals form in red blood cells (RBCs), as occurs in the case of patients expressing βC-globin (β6 Glu → Lys). In vitro, high-resolution structural determination by crystallographic methods requires the growth of Hb crystals to approx ~1 mm in diameter, which may be induced by a variety of precipitants. The first indication that oxyHb and deoxyHb exhibit two distinct conformational states and different crystal habits was first observed in 1938 by Haurowitz (1), who noted cracking of the horse deoxyHb crystal on oxygenation. Years later, Perutz and colleagues (2–4) obtained the high-resolution structure of deoxy (T-state) and oxy (R-state) human HbA. Since then, crystal diffraction methodology has continued to provide high-resolution details of structural alterations that occur on point mutations or peptide changes in natural Hb mutants or engineered recombinant variants (see Chapter 1).

1.1. HbC Forms Crystals in RBCs HbC is the second most commonly encountered abnormal Hb in the United States and, next to HbS and HbE, the third most prevalent hemoglobin structural variant worldwide (5,6). Approximately 3 of 100 African Americans carry the HbC gene. Individuals homozygous for HbC exhibit a mild hemolytic anemia, not considered a life-threatening disease. However, double heterozygotes for both HbS and HbC have sickle cell (SC) disease, which results in a reduced life expectancy and significant morbidity. It is life-threatening after the age of From: Methods in Molecular Medicine, vol. 82: Hemoglobin Disorders: Molecular Methods and Protocols Edited by: Ronald L. Nagel © Humana Press Inc., Totowa, NJ

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20, and some patients have severe retinal, osteonecrotic, and pulmonary complications (for a review, see ref. 5). It has long been known that HbC (β6 Glu → Lys) forms tetragonal crystals in red cells of CC patients (individuals homozygous for the expression of βC-globin) without resulting in morbid pathophysiology. In the venous blood of splenectomized patients, ~3% of RBCs contain crystals with all of the Hb recruited into a single crystal (7–9). Unknown was the Hb state in which the intraerythrocytic crystal formed. In 1985, it was first demonstrated by video-enhanced microscopy, (1) that these intraerythrocytic HbC crystals formed in the oxygenated (R) liganded state in cells that contained no detectable fetal hemoglobin (HbF), and (2) that deoxygenation (or switching to the T-state) resulted in dissolution of the intraerythrocytic crystal (10). The lack of vasoocclusion in CC patients could be explained as follows: Deoxygenation in the microcirculation (secondary to oxygen delivery to tissues) will result in the dissolution of the oxyHbC crystals, avoiding vasoocclusion by allowing the CC red cell to regain its pliability, necessary to navigate through narrow capillaries. Individuals coexpressing HbS and HbC exhibit a moderately severe disease, SC disease, arising from the polymerization of deoxyHbS induced by the increased intracellular hemoglobin concentration (6,11). Yet, along with RBCs containing HbS polymers, intraerythrocytic crystals are also detected in SC patients (12). To date, little is known about the mechanism of oxy (R-state) HbC crystallization nor why the oxy form of HbC crystallizes in the red cell whereas the deoxy form of HbS polymerizes in the red cell. Our laboratories have taken several different approaches to elucidate the mechanisms of liganded HbC crystallization.

1.2. In Vitro Batch Nucleation Studies Since CC erythrocytes containing HbF did not contain crystals (10), in vitro batch nucleation were undertaken to determine the effects of co-habiting hemoglobins in the RBC on HbC crystallization (13). In vitro, purified HbC forms tetragonal crystals within 15–30 min in concentrated phosphate buffer (1.8 M) at 30°C (Fig. 1). The size of the crystal is dependent on nucleation kinetics (i.e., the faster the nucleation rate, the smaller the crystal). Similar solution conditions were first employed by Adachi and colleagues (14,15) to study deoxyHbS nucleation and polymerization. A lag phase (on the order of 15–30 minutes) is always seen to precede any observation of crystal formation from a purified HbC solution using the methods outlined below (Fig. 2A,B). In agreement with the general expectations about any nucleation process (16–18), the length of the lag phase for nucleation and crystallization is dependent on the supersaturation of the solution (19,20).


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Fig. 1. Tetragonal oxy or CO (R-state) HbC crystals form in concentrated potassium phosphate buffer (1.8 M, pH 7.35). The Hb concentration is 2 g% (magnification: ×1000).

These batch crystallization methods demonstrated in vitro that HbF inhibits nucleation (Fig. 2) (13), while HbS (sickle cell Hb, β6 Glu → Val) accelerated nucleation and is incorporated into the crystal (21). HbA simply serves as a diluent in these nucleation studies (13,21). The value of using in vitro batch crystallization to identify contact sites (Table 1) and the effects on crystallization of binary mixtures of Hb and cell components soon became apparent (13,21–26). Interestingly, intraerythrocytic crystal morphology is altered in heterozygous individuals expressing HbC and other point mutated globins: those expressing HbC and Hb Korle-bu (β73 Asp → Asn) form cubic crystals (22), while those expressing HbC and Hb αG-Philadelphia (β68 Asn → Lys) form unusually long, narrow crystals (24). Different intertetrameric contacts are implied and may give rise to the altered morphology in a manner analogous to that proposed by Gallagher et al. (27).

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Fig. 2. (a) Effects of HbA and HbF on kinetics of nucleation. (A) Dependence of the total number of crystals nucleated in fixed solution volume on concentrations of HbA (upper curve) and HbF (lower curve). (B) Time dependence of number of crystals nucleated in a crystallization cell in presence of concentrations of HbA and HbF indicated on plots. Note that HbA and HbF increase the time lag and decrease the number of nucleated crystals; that is, both proteins decrease the rate of nucleation, with HbF having a markedly stronger action. (From ref. 13).

The question of why R-state HbC gives rise to crystals, in contrast to polymer formation when HbS is deoxygenated (T state), is addressed by a variety of approaches. Front-face fluorescence and ultraviolet resonance Raman spectroscopic studies, employed to probe intratetrameric R-state differences in HbS and HbC compared with HbA, at nonaggregating concentrations, suggest intramolecular alterations in the A-helix position and in the central cavity. (For methodological details of front-face fluorescence, see Chapter 9.) Video-enhanced differential interference contrast (DIC) microscopy compared aggregation/crystallization pathways of oxyHbC and deoxyHbC in concentrated salt conditions. It was demonstrated that R-state HbC exhibits a large propensity to crystallize in a tetragonal habit, whereas under similar conditions, deoxyHbC is driven to form a variety of morphological aggregates (e.g., radial arrays and macroribbons) while hexagonal crystal formation is a rare and least favored pathway (28).

1.3. In Vitro Solubility Studies Further quantification of R-state HbC crystal growth parameters, such as protein solubility and its dependence on temperature, was assessed by a novel scintillation method (29). An exploded view of the new scintillation arrangement is shown in Fig. 3A. The solution is contained in a silica microcell. This


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Table 1 Nucleation and Crystallization Effects by Binary Mixtures of HbC with Hb Variants HbC compound Heterozygote

Site-specific substitution

Effect on oxyHbC nucleation and crystallization

HbS Hb Korle-bu HbF Hb N-Baltimore Hb Riyadh HbαGPhiladelphia

β6 Glu→Val β73 Asp→Asn β87 Thr→Gln β95 Lys→Glu β120 Lys→Asn α68 Asn→Lys

Accelerates Accelerates Inhibits Accelerates Inhibits Accelerates

Hb J-Baltimore

β16 Gly→Asp

Accelerates

Crystal morphology Tetragonal Cubic-like — Tetragonal — Tetragonal and numerous 3X elongated Tetragonal (in vitro)

Reference 13 21 22 23 23 24

25

cell is surrounded by a machined brass jacket that sits on a thermoelectric heat pump (Peltier cooler) connected to a programmable controller. The use of Peltier elements to maintain temperature increases the temperature’s stability to about ± 0.02°C and allows temperature ramps at rates of up to 5°C/min. The temperature of the brass block is monitored with a type-T thermocouple. The attached controller facilitates programmed temperature changes. A laser beam from a self-contained laser diode assembly is directed through the solution. Light scattered normally to the incident beam is detected by an integrated detector/amplifier photodiode through a polished rod capping the microcell. A small segment of latex rubber tubing envelops the microcell and the cap and minimizes evaporation of the solvent. A beam splitter between the laser and cell diverts some of the laser’s output to a second integrated photodiode, whose signal is used to correct for intensity fluctuations of the laser. Backscatter of light, which passes through the cell, is minimized by painting the inside of the brass block black and by a light trap in the cavity formed by a rotating horseshoe magnet. This magnet drives a small nickel wire (0.6 mm in diameter, 5–7 mm long) used as a stirring bar inside the lower part of the solution, not illuminated by the laser. The output from the two integrated photodiodes is amplified and filtered with a four-pole Butterworth-style circuit located on a circuit board mounted to the nylon block, which minimizes the signal path length. Low-noise, low-drift/offset (0.6 mV/°C, 25 mV) precision op-amps, along with low-temperature coefficient (24 h should be done at 140 mg/mL of concentrated HbC. Be sure to recalculate HbC concentration values; CO gas tends to further concentrate the mixture. For this reason, buffers are made by first bubbling CO gas into H2O and then by adding calculated portions of buffer/precipitants. Adding CO gas directly into the HbC solution results in incorrect concentration values for the solution. 2. The concentration of the solution must remain low to prevent nucleation so that the crystals do not interfere with the laser light.


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3. Erythrocytes can be fixed by treating cover slips with full-strength poly-L-lysine. Droplets of RBCs are added and incubated. The RBCs are rinsed two to three times with PBS and fixed with 1% glutaraldehyde for imaging (2 to 3% glutaraldehyde has also been used).

Acknowledgments This work was supported in part by the American Heart Association, Heritage Affiliate Grant-in-Aid No. 0256390T; the National Institutes of Health R01 HL58038 and NHLBI 1F31 HL09564; and the Universities Space Research Association Research Contract 03537.000.013. We would like to acknowledge Dr. S.-T. Yau for his efforts with the atomic force microscope imaging. References 1. Haurowitz, F. (1938) Das Gleichgewicht zwischen Hamoglobin und Sauerstoff. Z. Physiol. Chem. 254, 266–274. 2. Perutz, M. F. and Mathews, F. S. (1966) An x-ray study of azide methaemoglobin. J. Mol. Biol. 21(1), 199–202. 3. Perutz, M. F. and Lehmann, H. (1968) Molecular pathology of human haemoglobin. Nature 219(157), 902–909. 4. Perutz, M. F. (1969) The Croonian Lecture, 1968. The haemoglobin molecule. Proc. R. Soc. Lond. B. Biol. Sci. 173(31), 113–140. 5. Nagel, R. L. (1991) The distinct pathobiology of SC disease: therapeutic tmplications, in Hematology/Oncology Clinics of North America, vol. (Nagel, R. L., ed.), W. B. Saunders, Philadelphia, pp. 433–451. 6. Nagel, R. L. and Steinberg, M. H. (2000) Hb SC and Hb C disease, in Disorders of Hemoglobin: Genetics, Pathophysiology, Clinical Management, (Steinberg, M. H., Forget, B. G., Higgs, D. R., Nagel, R. L., eds.), Cambridge University Press, MA. 7. Diggs, L. W. and Kraus, A. P. (1954) Intraerythrocytic crystals in a white patient with hemoglobin C in the absence of other types of hemoglobin. Blood 9, 1172–1184. 8. Kraus, A. P. and Diggs, L. W. (1956) In vitro crystallization of hemoglobin occurring in citrated blood from patients with hemoglobin. C. J. Lab. Clin. Med. 47, 700–705. 9. Charache, S., Conley, C. L., Waugh, D. F., Ugoretz, R. J., and Spurrell, J. R. (1967) Pathogenesis of hemolytic anemia in homozygous hemoglobin C disease. J. Clin. Invest. 46(11), 1795–811. 10. Hirsch, R. E., Raventos-Suarez, C., Olson, J. A., and Nagel, R. L. (1985) Ligand state of intraerythrocytic circulating HbC crystals in homozygote CC patients. Blood 66(4), 775–777. 11. Bunn, H. F., Noguchi, C. T., Hofrichter, J., Schechter, G. P., Schechter, A. N., and Eaton, W. A. (1982) Molecular and cellular pathogenesis of hemoglobin SC disease. Proc. Natl. Acad. Sci. USA 79(23), 7527–7531. 12. Lawrence, C., Fabry, M. E., and Nagel, R. L. (1991) The unique red cell heterogeneity of SC disease: crystal formation, dense reticulocytes, and unusual morphology. Blood 78(8), 2104–2112.

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13. Hirsch, R. E., Lin, M. J., and Nagel, R. L. (1988) The inhibition of hemoglobin C crystallization by hemoglobin F. J. Biol. Chem. 263(12), 5936–5939. 14. Adachi, K. and Asakura, T. (1981) Aggregation and crystallization of hemoglobins, A., S, and, C., Probable formation of different nuclei for gelation and crystallization. J. Biol. Chem. 256(4), 1824–1830. 15. Adachi, K., Segal, R., and Asakura, T. (1980) Nucleation controlled agregation of deoxyhemoglobin. S. J. Biol. Chem. 255, 7595–7603. 16. Kashchiev, D. (1969) Solution of the non-steady state problem in nucleation kinetics. Surf. Sci. 14, 209–220. 17. Kashchiev, D., (1995) Nucleation, in Science and Technology of Crystal Growth (van Eerden J. P. and Bruinsma, O. S. L., eds.), Kluwer Academic, Norwell, MA, pp. 53–56. 18. Mutaftschiev, B. (1993) Nucleation theory, in Handbook of Crystal Growth, vol. 1 (Hurle, D. T. J., ed.), Elsevier, Amsterdam, pp. 189–247. 19. Weber, P. (1991) Physical principles of protein crystallization, in Advances in Protein Chemistry, vol. 41 (Afinsen, C. B., Richards, F. M., Edsal, J. T., and Eisenberg, D. S., eds.), Academic, New York. 20. Weber, P. C. (1997) Overview of protein crystallization methods, in Methods in Enzymology, vol. 276. (Carter, C. W. Jr. and Sweet, R. M., eds.), Academic, New York, pp. 13–22. 21. Lin, M. J., Nagel, R. L., and Hirsch, R. E. (1989) The acceleration of hemoglobin C crystallization by hemoglobin S. Blood 74, 1823–1825. 22. Nagel, R. L., Lin, M. J., Witkowska, H. E., Fabry, M. E., Bestak, M., and Hirsch, R. E. (1993) Compound heterozygosity for hemoglobin C and Korle-Bu: moderate microcytic hemolytic anemia and acceleration of crystal formation [corrected] [published erratum appears in Blood 1994; 83(10):3105]. Blood 82(6), 1907–1912. 23. Hirsch, R. E., Witkowska, H. E., Shafer, F., Lin, M. J., Balazs, T. C., Bookchin, R. M., and Nagel, R. L. (1997) HbC compound heterozygotes [HbC/Hb Riyadh and HbC/Hb N-Baltimore] with opposing effects upon HbC crystallization. Br. J. Haematol. 97(2), 259–265. 24. Lawrence, C., Hirsch, R. E., Fataliev, N. A., Patel, S., Fabry, M. E., and Nagel, R. L. (1997) Molecular interactions between Hb alpha-G Philadelphia, HbC, and HbS: phenotypic implications for SC alpha-G Philadelphia disease. Blood 90(7), 2819–2825. 25. Hirsch, R. E., Shafer, F., Wajcman, H., Fataliev, N., and Nagel, R. L. (1999) HbJBaltimore Favors Crystallization of OxyHbC, but chain assembly abnormalities blunt the phenotype of the compound heterozygote. Blood 92(10)(Suppl. 1, Pt. 1), 11a. 26. Adachi, K., Lai, C. H., Konitizer, P., Donahee, M., and Campbell, A., S. S. (1994) Crystallization of recombinant hemoglobins with basic amino acid substitutions (Lys and Arg) at the 6 position. Blood 84, 1309–1313. 27. Gallagher, D. T., Eisenstein, E., Fisher, K. E., et al. (1998) Polymorphous crystallization and diffraction of threonine deaminase from Escherichia coli. Acta. Cryst. D. 54, 467–469. 28. Hirsch, R. E., Samuel, R. E., Fataliev, N. A., Pollack, M. J., Galkin, O., Vekilov, P. G., and Nagel, R. L. (2001) Differential pathways in oxy and deoxy HbC aggregation/crystallization [in process citation]. Proteins 42(1), 99–107.


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29. Feeling-Taylor, A. R., Banish, R. M., Hirsch, R. E., and Vekilov, P. G. (1999) Miniaturized scinitillation technique for protein solubility determinations. Rev. Sci. Instr. 70(6), 2845–2849. 30. Cabrera, N. and Vermileya, D. A. (1958) The growth of crystals form solution, in Growth and Perfection of Crystals (Doremus, R. H., Roberts, B. W., and Turnbul, D., eds.), Wiley, New York. 31. Voronkov, V. V. and Rashkovich, L. N. (1994) Step kinetics in the presence of mobile adsorbed impurity. J. Crystal Growth 144, 107–115. 32. Dewan, J. C., Feeling-Taylor, A., Puius, Y. A., et al. (2002) Structure of mutant human carbonmonoxyhemoglobin C (βE6K) at 2.0 Å resolution. Acta. Crystallogr. D58, 2038–2042. 33. Rosenberger, F., Howard, S. B., Sowers, J. W., and Nyce, T. A. (1993) Temperature dependence of protein solubility—determination and application to crystallization in X-ray capillaries. J. Crystal Growth 129, 1–12. 34. Hansma, H. G. and Hoh, J. H. (1994) Biomolecular imaging with the atomic force microscope. Annu. Rev. Biophys. Biomol. Struct. 23, 115–139. 35. Hansma, P. K., Cleveland, J. P., Radmacher, M., et al. (1994) Tapping mode atomic force microscopy in liquids. Appl. Phys. Lett. 64(13), 1738–1740. 36. Möller, C., Allen, M., Elings, V., Engel, A., and Müller, D. J. (1999) Tappingmode atomic microscopy produces faithful high-resolution images of protein surfaces. Biophys. J. 77, 1150–1158. 37. Noy, A., Sanders, C. H., Vezenov, D. V., Wong, S. S., and Lieber, C. M. (1998) Chemically sensitive imaging in tapping mode by chemical force microscopy: relationship between phase lag and adhesion. Langmuir 14, 1508–1511. 38. Yau, S.-T., Petsev, D. N., Thomas, B. R., and Vekilov, P. G. (2000) Molecularlevel thermodynamic and kinetic parameters for the self-assembly of apoferritin molecules into crystals. J. Mol. Biol. 303(5), 667–678. 39. Fitzgerald, P. M. and Love, W. E. (1979) Structure of deoxy hemoglobin C (beta six Glu replaced by Lys) in two crystal forms. J. Mol. Biol. 132(4), 603–619. 40. Volmer, M. (1939) Kinetik der Phasenbildung. Steinkopff, Dresden. 41. Giesen, M., Schulze Icking-Konert, G., Stapel, D., and Ibach, H. (1996) Step fluctuations on Pt(111) surfaces. Surface Sci. 366, 229–238. 42. Malkin, A. J., Kuznetsov, Y. G., Land, T. A., DeYoreo, J. J., and McPherson, A. (1996) Mechanisms of growth of protein and virus crystals. Nat. Struct. Biol. 2, 956–959. 43. McPherson, A., Malkin, A. J., and Kuznetsov, Y. G. (2000) Atomic force microscopy in the study of macroimoleular crystal growth. Annu. Rev. Biomol. Struct. 20, 361–410. 44. Yip, C. M. and Ward, M. D. (1996) Atomic force microscopy of insulin single crystals: direct visulization of molecules and crystal growth. Biophysical J. 71, 1071–1078. 45. Thomas, B. R., Vekilov, P. G., and Rosenberger, F. (1998) Effects of microheterogeneity on hen egg white lysozyme crystallization. Acta Crystallogr. Sect. D 54, 226–236.

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46. Thomas, B. R., Vekilov, P. G., and Rosenberger, F. (1996) Heterogeneity determination and purification of commercial hen egg white lysozyme. Acta Crystallogr. Sect. D 52, 776–784. 47. Vekilov, P. G. (1993) Elementary processes of protein crystal growth, in Studies and Concepts in Crystal Growth (Komatsu, H., ed.), Pergamon, Oxford: pp. 25–49. 48. Vekilov, P. G., Monaco, L. A., and Rosenberger, F. (1995) Facet morphology response to non-uniformities in nutrient and impurity supply. I. Experiments and interpretation. J. Crystal Growth 156, 267–278. 49. Vekilov, P. G. and Rosenberger, F. (1996) Dependence of lysozyme growth kinetics on step sources and impurities. J. Crystal Growth 158, 540–551. 50. Stojanoff, V., Siddons, D. P., Monaco, L. A., Vekilov, P. G., and Rosenberger, F. (1997) X-ray topography of tetragonal lysozyme grown by the temperature controlled technique. Acta Crystallogr. Sect. D 53, 588–595. 51. Vekilov, P. G., Monaco, L. A., Thomas, B. R., Stojanoff, V., and Rosenberger, F. (1996) Repartitioning of NaCl and protein impurities in lysozyme crystallization. Acta Crystallogr. Sect. D 52, 785–798. 52. Hirsch, R. E., Rybicki, A. C., Fataliev, N. A., Lin, M. J., Friedman, J. M., and Nagel, R. L. (1997) A potential determinant of enhanced crystallization of Hbc: spectroscopic and functional evidence of an alteration in the central cavity of oxyHbC. Br. J. Haematol. 98(3), 583–588. 53. Monaco, L. A. and Rosenberger, F. (1993) Growth and etching kinetics of tetragonal lysozyme. J. Crystal Growth 129, 465–484. 54. Samuel, R. E., Salmon, E. D., and Briehl, R. W. (1990) Nucleation and growth of fibres and gel formation in sickle cell haemoglobin. Nature 345, 833–835. 55. Schroeder, W. A. and Huisman, T. H. J. (1980) The Chromatography of Hemoglobin, Marcel Dekker, Nw York. 56. Adachi, K. and Asakura, T. (1979) Gelation of deoxyhemoglobin A in concentrated phosphate buffer. Exhibition of delay time prior to aggregation and crystallization of deoxyhemoglobin, A., J. Biol. Chem. 254(24), 12,273–12,276. 57. Adachi, K. and Asakura, T. (1979) Nucleation-controlled aggregation of deoxyhemoglobin S: possible difference in the size of nuclei in different phosphate concentrations. J. Biol. Chem. 254(16), 7765–7771. 58. Adachi, K. and Asakura, T. (1979) The solubility of sickle and non-sickle hemoglobins in concentrated phosphate buffer. J. Biol. Chem. 254(10), 4079–4084.

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11 Semisynthesis of Hemoglobin Seetharama A. Acharya and Sonati Srinivasulu 1. Introduction 1.1. Recombinant DNA Technology and Total Chemical Synthesis for Generation of Mutant Forms of Proteins Protein engineering, generation of mutant or modified forms of protein, has become the first step for studying the correlation of structure and function of proteins. Design and generation of novel protein molecules with tailor-made properties is the long-range goal of such studies. Such novel protein molecules are now designed and generated by recombinant DNA technology, as long as these protein molecules contain only the naturally occurring 20 amino acids residues (1,2). However, if unnatural amino acids needs to be introduced, cell-free protein expression system and special manipulation of the tRNA is necessary (3,4). Incorporation of the unnatural amino acid residues into a protein in a site-specific fashion could also be achieved through total chemical synthesis. Gutte and Merrifield (5) achieved the total chemical synthesis of RNase-A using solid-phase synthesis, starting from the carboxyl end of the molecule and building, one residue at a time, to its amino terminus. Hoffman et al. (6) introduced an alternate approach that involved the synthesis of a limited number of medium-size, protected segments of the molecule and condensing them to generate the full-length protein. This approach, generally referred to as a segment condensation approach (6), has gained significant attention in recent years. The molecular size of the proteins that biochemists desire to design and assemble are larger compared to RNase A. Accordingly, interest in developing newer and simpler approaches of segment condensation has increased.

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1.2. Semisynthesis of Proteins Semisynthesis of a protein can be considered as a specialized case of total chemical synthesis of protein by the segment condensation approach, where a significantly large portion of the protein is derived from the wild-type protein, and only a small segment of the protein is chemically synthesized with the desired changes in the covalent structure. This segment is then assembled with the complementary segment from the wild-type protein to generate a mutant form of the protein. The assembly may involve splicing of the two segments to establish chain contiguity. These semisynthetic reactions are referred to as covalent semisynthesis. However, establishing chain contiguity may not be necessary if the complementary segments exhibit strong noncovalent interaction among them that facilitates the assembly of the native folding of the protein (fragment-complementing systems). The latter represents the case of noncovalent semisynthesis.

1.2.1. Noncovalent Semisynthesis So far, in choosing the appropriate segment of the parent protein suited for chemical synthesis, information on the permissible discontinuity region of the protein has provided the road map (7). Many proteins have been converted into functionally active fragment-complementing systems either by limited proteolysis or site-specific chemical cleavage of the protein (8–10). In the modified protein, generally referred to as the fragment-complementing system, two (or more) segments of the protein are held together by strong noncovalent interactions, and accordingly, the modified protein conserves most of the conformational aspects of the parent protein. However, the interacting segments of the fragment-complementing system could be separated under denaturing conditions. In addition, a functional unit having the conformational and functional properties similar to that of the starting material could be generated by reassembling the complementary segments of the protein under the physiological conditions. In such a system, the amino acid sequence of segments of the protein is readily accessible for chemical manipulation through peptide synthesis. The modified peptide segment could be assembled with other segments of the system. The assembled product carrying a chemically synthesized segment is a semisynthetic protein and is analogous to the fragment-complementing system of the parent protein (11).

1.2.2. Covalent Semisynthesis Over the years, it has been established that it is now possible to induce the protease that generated the fragment-complementing system of a protein to religate the discontinuity in the polypeptide chain of the new semisynthetic

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fragment-complementing system (12). The approach of splicing the discontinuity has been referred to as covalent semisynthesis. There are many examples of such covalent semisynthetic reactions in the literature to date (13–15). A recent modification of this approach introduced by Proudfoot et al. (16) is to have a very reactive group on the α-carboxyl group of the discontinuity site. In such a fragmentcomplementing system, the nucleophilic attack of the activated carboxyl group by the α-amino group of the discontinuity site established the contiguity to the polypeptide chain. Since the noncovalent interaction of the complementary segments facilitating the generation of native-like conformation in the fragmentcomplementing system is pivotal to the splicing reaction, this reaction is referred to as a conformationally assisted protein ligation reaction (17).

1.3. α-Globin Semisynthetic Reaction The V8 protease catalyzes the splicing of the complementary segments of α-globin (α1–30 and α31–141) (18), and has been used by our laboratory group for the preparation of many chimeric α-globins. A schematic representation of the generation of chimeric (semisynthetic) α-globin by exchanging one of the complementary segments of human α-globin with that of the animal α-globin is shown in Fig 1. Similarly, an exchange between the two animal α-globin chains could also be carried out to generate animal-animal chimeric α-globin chain. The V8 protease–catalyzed splicing reaction (semisynthetic reaction) is novel and very distinct from the previously described covalent protein semisynthetic reactions. This ligation reaction is facilitated by the conformation aspects of the product, rather than that of the “native-like” conformational aspects of reactants (i.e., the fragment complementing system). The continuity of the polypeptide chains established in the mixture of the complementary segments facilitates the induction of α-helical conformation into the contiguous chain in the presence of the organic cosolvent. This secondary structure of the ligated segment protects the Glu30 Arg31 peptide bond from proteolysis (19,20). There are several unique features of this α-globin semi synthetic reaction. First, there is the absence of noncovalent interactions between the two reacting peptides that establish a native-like structure in the fragment-complementing system. Second, the splicing reaction requires the presence of 30% propanol (or other α-helix-inducing organic solvents). Third, an extensive excision of the amino-terminal and the carboxyl-terminal region of α1–30 and α31–141 can also be made without influencing the equilibrium yields of the splicing reaction. Therefore, reaction has been exploited for the generation of semisynthetic hemoglobins (Hbs) (21–24).

1.4. Semisynthesis of Hbs Hb tetramer contains four polypeptide chains, two copies each of two subunits, the α- and β-chains. Each of these chains is composed of the protein part,

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Fig. 1. Schematic illustration of V8 protease–catalyzed slicing and splicing of α-globin to generate chimeric (semisynthetic) α-chains. The complementary segments (α1–30 and α31–141). of α-chains with single or multiple sequence differences in either the α1–30 or α31–141 segments or in both segments are spliced together to reduce or increase the number of sequence differences in the α-chain as compared with that of human α-chain.

globin, and the non-protein part, heme. Thus, the generation of tetrameric Hb from semisynthetic α-globin involves the assembly of the heme with semisynthetic α-globin, and the hybridization of the semisynthetic α-chain (hemebound polypeptide chain) with the appropriate β-chain to generate the α2β2 structure. Accordingly, the protocol for semisynthesis of Hb involves two major sections: (1) covalent semisythesis of desired variant or chimera of α-globin, and (2) assembly of semisynthetic α-globin with heme and β-chain to generate the tetramer through the alloplex intermediate pathway (25) (see Note 1). 2. Materials 2.1. Preparation of α and β-Globin Chains of HbA and/or Animal Hb 1. 2. 3. 4. 5.

Chromatographically purified HbA or HbS (refer to Chapter 3 for procedures). p-Hydroxymercuribenzoate (HMB) (see Note 2). 0.1 N NaOH (Fischer). 0.1 N Acetic acid (Fischer Scientific). Chromatographic columns (Rainin or Pharmacia), 1.5 × 30 cm for loads of 50–200 mg of protein, and 2.5 × 50 cm for loads of 1–2.5 g and higher. 6. Ion-exchange resin, CM-52 cellulose (Whatman). 7. Phosphate buffers: 10 mM phosphate buffer, pH 6.5, and 15 mM phosphate buffer, pH 8.3.

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8. 0.5% Acid acetone (prepared by mixing 5 mL of concentrated HCl with 995 mL of acetone). 9. Corex centrifugation flasks (200–250 mL). 10. Sorval centrifuge RC-5C, LKB fraction collector, and a manual linear gradient marker when using a single-pump system or gravity for chromatography. Alternatively, a fast protein liquid chromatogrphy (FPLC) or an Acta system from Pharmacia can be used.

2.2. Preparation of α1–30 and α31–141 from Human and/or Animal α-Globins 1. 10 mM Ammonium acetate buffer, pH 4.0. 2. Lyophilized α-globin (about 100 mg). 3. Staphylococcus aureus V8 protease (Pierce, Rockford, IL). The V8 protease from Pierce comes as a lyophilized sample. Generally, a stock solution of the enzyme is made in water and stored at –80°C. One milligram of the enzyme is dissolved in water; the exact concentration of the enzyme is determined spectrophotometrically. The absorbance of a 1 mg/mL solution at 280 nm is 0.67. 4. Reverse-phase C4 column, analytical reverse-phase high-performance liquid chromatography (RP-HPLC) column (Vydac) for analytical runs, and semipreparative or preparative columns, for isolation of the complementary segments of α-globin using RP-HPLC. 5. Sephadex G-50, for purification of the complementary fragments of α-globin under denaturing conditions (0.1% trifluoroacetic acid [TFA] in water). 6. Urea (98+% purity) (Sigma, St. Louis, MO). 7. Buffer A: 5 mM phosphate buffer, pH 7.0, containing 8 M urea and 50 mM β-mercaptoethanol. 8. Buffer B: 25 mM phosphate buffer, pH 7.0, containing 8 M urea and 50 mM β-mercaptoethanol. 9. Buffer C: 50 mM phosphate buffer, pH 7.0, containing 8.0 M urea and 50 mM β-mercaptoethanol. 10. HPCL setup (we use a Shimadzu system), for the RP separation of the complementary fragments 11. LKB fraction collector, FPLC or Acta (both from Pharmacia).

2.3. V8 Protease–Catalyzed Ligation of α1–30 with α31–141 and Purification of Semisynthetic α-Globin 1. An α1–30 segment that is to be spliced, generated either by chemical synthesis incorporating the desired sequence differences (mutation) or by the V8 protease digestion of an animal α-globin carrying a number of sequence differences. 2. Desired α31–141 segment from the α-globin of either human or mammal. 3. 50 mM Ammonium acetate buffer, pH 6.0 containing 30% n-propanol. 4. V8 protease: a stock solution prepared as discussed in Subheading 2.2. is used. 5. C-4 RP-HPLC column (Vydac). 6. CM-52 cellulose (Whatman).

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7. Sephadex G-50. 8. Urea.

2.4. Assembly of Semisynthetic Hb and Its Purification 1. Mutant semisynthetic α-globin, or internally or externally deleted semisynthetic α-globin, or semisynthetic chimeric α-globin. 2. Chromatographically purified HMB βA- or βS-chains. 3. Catalase. 4. 100 mM EDTA in water (37.2 mg/mL of water). 5. 100 mM Dithiothreitol (DTT) (15.5 mg/mL) (Sigma). 6. Hemin (Sigma) solution: Dissolve 5 mg of hemin in 200 µL of 0.1 N NaOH. 7. Sodium cyanide: 1 mg in 100 µL of water. 8. Hemin dicyanide solution: 200 µL of hemin solution mixed with 60 µL of sodium cyanide solution and made up to 5 mL with double-distilled water (see Note 3). 9. Chromatographic columns: 1.5 × 50 cm and 0.9 × 30 cm (Rainin). 10. Sephadex G-25. 11. CM-52 cellulose (Whatman). 12. 100 mM Tris-HCl buffer, pH 7.4. 13. Globin-dissolving buffer: 50 mM Tris-HCl buffer, pH 7.4, containing 8 M urea, 1 mM EDTA, and 2 mM DTT.

2.5. Chemical and Functional Characterization of the Semisynthetic Hb 1. C4 column (Vydac), for RP-HPLC analysis of semisynthetic Hb. 2. Mass spectral analysis of semisynthetic α-globin of semisynthetic Hb isolated by RP-HPLC (API-III Triple-Quadrupole Mass Spectometer, Perkin-Elmer Sciex®). 3. Isoelectric focusing of semisynthetic Hb (using the instrument from Isolab). 4. Hem-O-Scan or Hem-Ox-Analyzer, for analysis of O2 affinity of semisynthetic Hb.

3. Methods 3.1. Preparation of α- and β-globin Chains from HbA and/or Animal Hb

3.1.1. α- and β-HMB Chains of HbA or HbS Reaction of HbA or HbS with HMB around pH 6.0 in the presence of 200 mM NaCl generates HMB α- and β-chains (26), which can be separated by CM-cellulose chromatography. Reaction of HbA or its mutant or chemically modified forms with HMB is carried out at a final concentration of 0.1 mM (6.5 mg/mL) in 20 mM phosphate buffer, pH 6.0, and containing 200 mM NaCl. A stock solution of Hb, generally at a concentration of 2.5 mM, is diluted with distilled water to a concentration of 0.2 mM (approx 12–15 mg/mL). When all the reagents are added,

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and the pH is readjusted to 6.0, the volume of the reaction mixture is nearly doubled and protein concentration will be approx 6.5 mg/mL. A volume of 1.0 M phosphate buffer, pH 6.0, equivalent to one-fiftieth of the volume of the 0.2 mM Hb solution is added to this solution. Similarly, an amount of 1.0 M NaCl in 20 mM phosphate buffer, equivalent to one-fifth of the final volume (1:2.5 with respect to the 0.2 mM Hb solution) of the reaction mixture is added to this solution. Enough HMB, to make the final concentration of the reagent in the final reaction mixture 1.0 M, is dissolved in a minimum amount of 0.1 N NaOH. Once the reagent is completely dissolved, giving a clear solution, it is neutralized with 0.1 N acetic acid until a light whitish turbidity appears in the solution; it should not precipitate. This freshly prepared HMB solution is added completely to the Hb solution and mixed gently. The final volume of the reaction mixture is made up by adding water (the final concentration of Hb is 0.1 mM in tetramer). The pH of the reaction mixture is adjusted to 6.0 with dilute acetic acid. The reaction mixture is kept on an ice bath in a cold room overnight (generally 16–20 h). After the overnight HMB reaction with Hb, generally some precipitation is seen in the reaction mixture. Such a precipitate is removed by centrifuging of the reaction mixture (7000 rpm for 20 min in a Sorval centrifuge at 4°C), and the supernatant, which contains the HMB α-and β-chains, is dialyzed against three changes of 10 mM phosphate buffer, pH 6.0. The sample is then concentrated to approx 50 mg/mL. The concentrated sample is chromatographed on a CM-cellulose column (0.9 × 30 cm), equilibrated to pH 6.0. The chromatogram is developed with a linear gradient generated from an equal volume of 10 mM phosphate buffer, pH 6.0 (starting buffer), and 15 mM phosphate buffer, pH 8.5 (final buffer). A 0.9 × 30 cm column can easily purify the chains from a load of 200 mg of HMB-reacted Hb employing a gradient generated by 250 mL each of starting (10 mM phosphate buffer, pH 6.0) and final buffers (15 mM phosphate buffer, pH 8.5). Elution of the protein is monitored by following the absorption of the effluent at 540 nm. The HMB β-chain elutes first from the column, and the HMB α-chain elutes at the end of the gradient. The identity as well as the purity of the HMB chains as they elute from the CM-cellulose column are assessed by RP-HPLC of the samples (the HMB globin chains as well as the HMB-free globin chains elute at the same position in RP-HPLC). The chromatographically purified HMB α-and β-chains are pooled and concentrated and stored at –80°C until needed.

3.1.2. α-and β-Globin Chains of Hb The heme-free α-and β-globin chains can be prepared from the respective HMB α-and β-chains by acid acetone preparation (27). Alternately, the α- and β-globin chains can be prepared by CM-52 cellulose–urea column chromatography

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(28) of the total globin prepared by the acid acetone precipitation of the Hb chosen for the study (see Note 4). In our experience, both methods yield globin chains that work well in the semisynthetic reaction. Since the HMB reaction of animal Hbs has not been successful in generating the α-and β-chains, we generally follow CM-52 cellulose–urea column chromatography for preparation of the globin chains of animal Hbs. 3.1.2.1. ACID ACETONE PRECIPITATION OF TOTAL HB

Under strong acidic conditions, heme dissociates from the globin chain, and acetone precipitates the globin chains (27). Thus, the heme remains in solution, and globin chains precipitate. The protocol for the precipitation involves diluting of the Hb sample first with water to a concentration of about 10 mg/mL and keep it cold in an ice bath. The Hb solution is transferred to Corex centrifuging tubes or flasks. To this Hb solution 10 vol of acid acetone is slowly added. Until the addition of nearly 7 to 8 vol, the solution remains clear; afterward the addition of acid acetone results in precipitation of the protein as fluffy white material. The remaining volume of the acid acetone is added to the solution while the solution is mixed by using a glass rod. After the mixing of the Hb solution and acid acetone are completely mixed, the tube (or flask) is left in the ice bath for 30 min. All the protein precipitates as fluffy material. The sample is centrifuged to pellet down the precipitated globin. The supernatant, which is colored because it contains the heme, is decanted and discarded. The globin pellet is redissolved in 0.1 M acetic acid (in a volume equivalent to that of the original Hb solution, 10 mg/mL), and the acid acetone protocol is repeated two or three times until no more heme is extracted into the acid acetone phase. The final pellet of globin is dissolved in 0.1 M acetic acid (about 1 mg/mL) and lyophilized. Globin lyophilizes as white fluffy material. 3.1.2.2. CM-52-CELLULOSE-UREA CHROMATOGRAPHY OF ACID ACETONE PRECIPITATED GLOBIN

Thirty grams of CM-52 cellulose is first equilibrated with buffer A (50 mM phosphate buffer, pH 7.0, containing 8.0 M urea and 50 mM β-mercaptoethanol). The equilibrated resin is packed into a 2.5 × 15 cm column and further equilibrated by washing the column with 5 bed vol of buffer A. This column can resolve 150–200 mg of acid acetone–precipitated globin. The lyophilized sample of acid acetone–precipitated globin is dissolved in buffer A (10–15 mg/mL) and dialyzed against the same buffer (20 times over the volume of the protein solution) for 3 to 4 h. This dialyzed sample is loaded onto the CM-52 column prepared fresh, and the protein sample is eluted with a linear gradient of buffers B and C (250 mL each). The protein elution is moni-

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tored by measuring the optical density of the fractions at 280 nm. The proteincontaining fractions are pooled and their identity is established by RP-HPLC. The pooled fractions containing α- and β-globins are dialyzed extensively to get rid of both the urea and the salts and then lyophilized. The lyophilized material is stored at –20°C until needed.

3.2. Preparation of α1–30 and α31–141 from Human and/or Animal α-Globin Chains The human α-globin is readily and quantitatively digested by V8 protease at pH 4.0 and 37°C to generate α1–30 and α31–141 (29). The animal α-globin is also digested in the same fashion (30,31).

3.2.1. V8 protease Digestion of Human or Animal α-Globin The α-globin sample is taken in 10 mM acetate buffer, pH 4.0, at a concentration of 0.5 mg/mL and placed in a 37°C water bath. When the α-globin solution is equilibrated to 37°C , protease digestion is initiated by adding the required amount of V8 protease. A substrate-to-enzyme ratio of 200:1 (w/w) is used for the digestion (2.5 µL of V8 protease solution [1 mg/mL]/1 mL of α-globin solution). Aliquots of the reaction mixture are analyzed by RP-HPLC to determine completion of the digestion (Fig. 2, inset A). When the digestion is complete, the ratio of the integrated area of α1–30 to α31–141 is nearly 1:3. Complete digestion of α-globin takes nearly 1–3 h. Once digestion is complete, the digest is lyophilized.

3.2.2. Purification of Complementary Fragments of α-Globin V8 protease digestion of 5–10 mg of α-globin has been routinely purified by our laboratory using RP-HPLC. A semipreparative Vydac C-4 column is used for such a purification. For a large-scale purification of the complementary segments of α-globin, size-exclusion chromatography (SEC) on Sephadex G-50 under denaturing conditions has been found very convenient. The lyophilized digest of α-globin is dissolved in 0.1% TFA in water (20–30 mg/mL). This is loaded onto a Sephadex G-50 column (1.5 × 70 cm) equilibrated with 0.1% TFA, and size-exclusion chromatographic separation of the fragments is achieved by eluting the column with 0.1% TFA at a flow rate of 0.5 mL/min. A load of 100 mg of V8 protease digest of α-globin is well resolved into complementary fragments in about 6 h. A typical purification profile of one such preparation is shown in Fig. 2. The identity and purity of the resolved complementary segments of α-globin can be established by RP-HPLC (Fig. 2, insets B and C). The fractions representing α1–30 and α31–141 are pooled and lyophilized. The purity of α31–141 is further established by tryptic digestion and RP-HPLC of the tryptic peptides. The absence of the tryptic peptides αT1, αT2, and αT3 in the tryptic digest establishes the purity of this fragment.

186


Fig. 2. Large-scale purification of α1–30 and α31–141 from a V8 protease digest of human α-globin on a sephadex G-50 column (1.5 × 100 cm). The size-exclusion chromatography was developed using 0.1% TFA. Elution of the complementary segments from the column was followed by monitoring the absorption of the fractions at 280 nm. The high molecular weight fraction (fraction a) has been identified as α31–14 (inset B) and the lower molecular weight fraction as α1–30 (inset C). (Inset A) The RP-HPLC pattern of the total V8 protease digest is shown. Insets B and C are the RP-HPLC patterns of pooled fractions of a and b isolated from the Sephadex G-50 column. RP-HPLC analysis was carried out using a Vydac C4 column employing a linear gradient of 5–70% acetonitrile in 0.1% TFA in 130 min. The flow rate was 1 mL/min, and elution of the fragments was monitored at 210 nm.

3.2.3. Chemical Synthesis of Mutant α1–30 Segment The desired α1–30 segments with deletion/replacement/addition have also been generated by chemical synthesis. The fragments that are chemically synthesized include α1–30 (H2OQ), α1–30 (K16E), and α1–30des23–26. Prior to using these synthetic peptides, they were subjected to purification by RP-HPLC using a semipreparative column.

3.3. V8 Protease–Mediated Splicing of α1–30 Carrying Desired Structural Modification with α31–141 Splicing of the desired complementary segments is carried out in 50 mM ammonium acetate buffer (pH 6.0) containing 30% n-propanol. n-propanol

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induces α-helical conformation into the contiguous segments generated through protease-mediated splicing (18). Other organic cosolvents, such as propanediol and butanediol, are also suitable for the splicing reaction.

3.3.1. Splicing of α1–30 with α31–141 Nearly a 1.2-fold molar excess of α1–30 or its mutant form over the desired α31–141 is mixed in 0.1% TFA and lyophilized to obtain a fluffy material. The lyophilized material is taken up in 50 mM ammonium acetate buffer (pH 6.0) containing 30% n-propanol, and a clear solution should be obtained. If a clear solution is not obtained with this level of organic solvent, the concentration of n-propanol can be increased to obtain a clear solution. If this also does not facilitate solubility of the mixture of the complementary segments, other organic solvents such as propanediol or butanediol can be tried. The final concentration of the complementary fragments is kept at about 10 mg/mL. The solution is kept at 4°C, and the semisynthetic reaction is initiated by adding the required amount of a stock solution of the V8 protease. An enzyme-to-substrate ratio of 1:200 is maintained in the reaction mixture. Progress of the ligation of the complementary segments is monitored by RP-HPLC analysis of the sample at different time intervals. The decrease in the amount of α1–30 in the reaction mixture as a function of time reflects the progress of the splicing reaction. Nearly 50% of the α1–30 is spliced with α31–141 within the initial 24 h of the reaction. Nonetheless, the incubation is generally continued up to 48 h. Once the reaction attains equilibrium, the reaction mixture is lyophilized and stored at –20°C until further processing.

3.3.2. Purification of Unreacted α1–30 from Ligated and Unligated α31–141 The unreacted α1–30 in the reaction is generally recovered in order to subject it to a second cycle of the splicing reaction. Recovery of the unreacted α1–30 is achieved by subjecting the lyophilized sample to an SEC on a Sephadex G-50 column as described in Subheading 3.2.2. The unreacted α1–30 and the mixture of the semisynthetic chimeric α-globin and α31–141 are pooled separately and isolated by lyophilization. The α1–30 is subjected to a second round of the splicing reaction using a new sample of α31–141 and processed similarly.

3.3.3. Purification of Semisynthetic α-Globin The high molecular weight fraction from the SEC of the spliced sample on Sephadex G-50 column is a mixture of α31–141 and the semisynthetic mutant or chimeric α1–141. The resolving power of size-exclusion chromatographic column is not sufficient to resolve the two, and accordingly, both elute together from the column. However, semisynthetic α-globin can be purified by CM-52 cellulose–urea column chromatography (28), as explained in Subheading

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3.1.2.2. The fractions from the chromatography containing the purified chimeric α-globin and α31–141 or mutant α1–141 are extensively dialyzed against 0.1% acetic acid and lyophilized. The lyophilized material is stored at –20C°.

3.4. Assembly of Semisynthetic Hb and its Purification To generate a functional tetrameric molecule, the semisynthetic (chimeric) α-globin has to be assembled with the complementary β-chain in the presence of heme (used as hemin dicyanide) (see Note 3). Depending on the objectives of the investigation, we have assembled the semisynthetic α-globin with either βA- or βS-chains. For these assembly reactions, the alloplex intermediate pathway, originally developed by Yip et al. (25) is used. This protocol takes advantage of the propensity of the β-chains of Hb (with free thiols) to interact noncovalently with heme-free α-globin to generate a tetramer, referred to as alloplex intermediate (only the β-chains of the tetramer containing heme). When hemin dicyanide is added to this, it occupies the heme-binding pocket of the molecule. The dithionite reduction of such a complex generates a functional tetramer.

3.4.1. Regeneration of the Sulfhydryl Groups of HMB β-Chain The HMB βA- or βS-chain, as established by the experimental protocol, is diluted into 50 mM Tris-HCl buffer, pH 7.4, containing 1 µg/mL of catalase, 2 mM DTT, and 1 mM EDTA to get a solution of 5 mg/mL. This solution kept at 4°C for about 45 min. The DTT removes the HMB groups from the thiol group of Cys-residue, regenerating the free sulfhydryl group.

3.4.2. Regeneration of Sulfhydryl Groups of the Semisynthetic α-Globin The semisynthetic α-globin is dissolved in 50 mM Tris-HCl buffer (pH 7.4) containing 8.0 M urea, 2 mM DTT, and 1 mM EDTA to get a solution of 5 mg/mL. This solution is incubated for 30–45 min at room temperature.

3.4.3. Preparation of Half-Filled Molecules (Alloplex Intermediate) Half-filled molecules are prepared by slowly adding the α-globin and β-chain solutions to the dilution buffer (50 mM Tris-HCl buffer, pH 7.4, containing 1 mM DTT, 1 mM EDTA, and 1 µg/mL of catalase). The two solutions are added to the dilution buffer (nearly 10 times the volume of the solution of semisynthetic α-globin) simultaneously with constant stirring at 4°C so that the urea concentration is lowered to 0.8 M. After the dilution, the final concentration of the protein is about 1 mg/mL (0.5 mg/mL of each chain). This mixture is incubated at 4°C for 30 min to facilitate the formation of half-filled molecules.

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3.4.4. Generation of Cyanomet Semisynthetic Hb To the solution of half-filled molecules generated in Subheading 3.4.3., an equivalent amount of freshly prepared hemin dicyanide is added dropwise, to generate a 1.1-fold molar excess of hemin dicyanide in solution over that of globin. This addition is carried out with mild shaking of the sample. The reaction between hemin dicyanide and half-filled molecules should then be allowed to proceed for about 60 min at 4°C. This will generate the semisynthetic molecule in the cyanomet form. This solution containing the cyanomet form of semisynthetic Hb is subjected to an extensive dialysis against 50 mM Tris-Hcl buffer, pH 7.4. During dialysis, some amount of precipitation occurs. This is removed by centrifuging the sample at 4°C for 20 min at 7000 rpm. The clarified dialyzed sample is concentrated by ultrafiltration to a concentration of 20 mg/mL.

3.4.5. Dithionite Reduction of Cyanomet Form of Semisynthetic Hb The semisynthetic Hb generated in Subheading 3.4.4. is in the ferric state. To convert it to the ferrous state, the sample has to be reduced by either an enzymatic procedure (32) or a chemical method (33). In our laboratory, we have routinely used the sodium dithionite to reduce the methemoglobin sample (MetHb). This is done using a Sephadex G-25 column under anaerobic conditions. Preswollen Sephadex G-25 column equilibrated with 10 mM phosphate buffer, pH 7.0 is packed into a column (1.5 × 50 cm) and the column is equilibrated with 10 mM phosphate buffer (pH 7.0) that is constantly purged with N2 gas. Once the column is washed with 2 to 3 bed vol of the buffer, the dithionite solution is freshly prepared. Sodium dithionite, approx 1.1 equivalents over the semisynthetic α-globin used in the reconstitution, is dissolved in 10 mM phosphate buffer, pH 7.0, degassed with N2 gas. The volume used to dissolve the sodium dithionite is equal to that of the semisynthetic cyanoMetHb solution that needed to be reduced. This solution is placed on the top of the Sephadex G-25 column very gently without disturbing the gel and allowed to enter the bed completely. Once the dithionite solution enters the column, the column is washed with a volume of buffer (degassed with nitrogen) equivalent to that of the dithionite solution. The column is then loaded with semisynthetic cyanoMetHb. About 2 to 3 mL of 10 mM phosphate buffer, pH 7.0, previously degassed with nitrogen gas is used to rinse the top of the column, and the chromatogram is developed with 10 mM phosphate buffer. During the elution, the buffer tank is continuously purged with N2 gas. The effluent is collected as a 1-mL fraction, and elution of Hb is monitored by measuring the absorption at 540 nm. The fractions containing Hb are analyzed for the concentration of MetHb. Samples of semisynthetic Hb containing

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