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CONEERE~CE 0~ HEMOGLOBIN 2-3 MAY 1957 Hazy Char fad Baa of The Division of Medico Sciences \ationa1 Academy of Sciences- ~at10na1 Research Council Life Ma Of of The ~~tion~1 Heart Institute ationa1 Institutes of Health Pubbcat10n 557 ation~1 Academy of Sciences ~at10na1 Research Council Washington, D. C. 1938
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PREFACE The Conference on Hemoglobin was held by the Division of Medical Sciences, National Academy of Sciences-National Research Council, as a result of discussions within its Subcommittee on Blood and Related Problems. The members of the Subcommittee felt that the time was ripe for a compre- hensive review of research on hemoglobin, similar to that which had been held in Cambridge, England, in 1948 in honor of Sir Joseph Barcroft. Chairmen of the various sessions were Drs. John T. Edsall, Irving M. London, and George E. Cartwright, each of whom has been helpful in editing his respective section of the Proceedings. Within the Division of Medical Sciences, staff responsibility for planning and conducting the Conference was borne by Dr. Margaret H. Sloan and Dr. fames R. Weisizer with the help of Mrs. Mary Freeman and Mrs. Phyllis Danielson. The manuscript and illustrations of these Proceedings were prepared for publication by Mr. Frank At. Holz, under the supervision of Dr. Weisiger. Some papers have been revised by the editors, and much of the discussion has been rearranged in the interest of logical association and continuity. Special acknowledgment is due to Dr. Hans T. Cahnmann, Dr. Harvey A. Itano, and Dr. James V. Neel for their assistance with various sections. The Subcommittee felt that, insofar as possible, the contents of this volume should represent the material actually available to the participants at the time of the Conference. On this basis the editors have regretfully excluded one paper that was not received in time for presentation. On the other hand, a paper that could not be presented for lack of time is included, since copies had been circulated in advance of the meeting. It has also proved necessary to omit the remarks of one discussant, from whom the editors were unable to obtain illustrations needed for their interpretation. In conclusion, the Division wishes to express its deep appreciation for the willing help and encouragement of the National Heart Institute, whose grant (No. H-2896) underwrote the costs of holding the Conference and of publishing these Proceedings. to . 111
FOREWORD R. KEITH CANNAiNT Chairman, Division of Medical Sciences National Academy of Sciences-National Research Council "The subject of this book is the red blood of vertebrates." With this sentence Lawrence J. Henderson introduced his classical monograph entitled "Blood: A Study in General Physiology." He then proceeded to justify his choice of subtitle by giving the following description of his purpose: "We shall study this substance as a physico-chemical system and as a tissue, seeking in its properties the exemplification of the general properties of protoplasm. In its physiological function and relation with other parts of the body we shall look for an illustration of organic integration and adaptation. We shall also study it compara- tively, from species to species, in rest and activity, in health and disease. So far as is possible these studies will be quantitative and mathematical." It was Claude Bernard who gave to general physiology its first charter. He cc.nceived it as the science of "the elementary condition of the phenomena of life" and its goal the description of this elementary condition in the language of physical science. To Bernard we also owe the concept of blood as the "milieu interieur" of the body. It is a happy circumstance of history, therefore, that a succession of general physiologists- Bohr, Krogh, Haldane, Barcroft, Van Slyke, Hen- derson, and others too numerous to name have found in blood the biological system most accessible to controlled investigation and most amenable to exacting physico-chemical description. Prom their world we have inherited those quantitative and mathematical descriptions of the respiratory, osmotic, and ionic functions of the blood that so elegantly illustrate the harmonious organization and integration of components that we have learned to regard as the special characteristics of living systems in general. The subject of the Conference of which this book is the record is also "the red blood of vertebrates." It is true that attention was deliberately re- stricted to a single component of blood. This, however, may be defended as a logical and appropriate refinement of approach not only because hemoglobin is the predominant constituent of the system but also because its own unique . v
V1 FOREWORD properties so largely determine the characteristics of some of the major physiological functions of blood. The purpose of the Conference was to . ~ refine the physico chemical characterization of the various forms of hemo- g]obin as a contribution to a better understanding of the "elementary con- dition" of blood as the internal environment of the body. In this sense the Conference is offered as an exercise in general physiology. Ten years ago a Symposium on Hemoglobin was held in Cambridge, England, as a memorial to Joseph Barcroft. Its proceedings were duly pub- lished in a notable volume. It is our hope that the papers and discussions recorded in the book which is now in your hands have recaptured something of the spirit and the zest of the earlier meeting. In the intervening years much progress has been made. Experimental tools that were only beginning to attract attention ten years ago are now the standard equipment of the protein chemist. Concepts which were, at that time, novel and hypothetical are now the accepted foundation for current ideas of the structure and interactions of hemoglobin. New experimental devices have opened up new avenues of investigation and new knowledge has posed new questions. The center of interest continues to shift. In the last fees years the discovery that the hemoglobin of a single species may exist in a variety of related natural forms has stimulated wide interest in the chemical nature of these "abnormal" molecules and in their genetic and clinical signifi- cance. This new interest is reflected in the pages that follow. In one of his reports on his work on the respiratory function of the blood, Barcroft reflected wistfully that the introduction of his refined differential manometric methods for the analysis of the blood gases had served only "to make more certain the uncertainty of our position." It may be true that hemo- globin has been more precisely characterized from a physico-chemical point of view than any other protein. However, as we dwell with satisfaction on the deepening invasion of the territory of biology by the physical sciences, it may be well to remind ourselves that action and reaction are equal and opposite and to recall the admonition of A. V. Hill that "physics and chem- istry will only dominate biology by becoming biology."
CONTENTS Preface . . PAGE It orewordR. Keith Cannan Opening Remarks Irving M. London and Carl Moore PART I STRUCTURE OF HEMOGLOBIN Current Concepts of Structure of Hemoglobin John T. Edsall Scope of Conference. Heme proteins in nature. Current concepts of hemo- globin structure. Importance of imidazole and sulfhydryl residues. All or none denaturation and reactivity of functional groups. Mechanism for acid denaturation. Heme binding sites. Theory of positive and negative heme in- teraction. Association constants for Hb., to Hb4. Species differences in heme interactions. Changes in physical properties associated with ligation. Thermo- dynamics of ligation. Dielectric increments. Imidazole as the heme-linked acid group. Individual Velocity Constants in the Chain of Reaction of Sheep Hemo- globin with Dissolved Gases S. Ainsworth, Q. H. Gibson, and F. J. BY. Roughton ..................................... ·- X1V Stimulus of Barcroft Memorial Conference. Flash photolysis. Consecutive rate constants for oxygen, carbon monoxide and nitric oxide. Constants di- rectly measurable. Derived constants. Influence of pH and sulfhydryl groups on constants. Use of dithionite to scavenge oxygen. Nitric oxide as oxygen analogue. Discussion: John T. Edsall, f alter L. Hughes, F. J. f . Roz~ghton, Baste Riggs, Jacinto Steir~hardt, and Reinhold Benesch .......... Interaction of heme and sulfhydryl groups in methyl mercury binding. Com- parison of CO and oxygen as ligands. Effects of PCMB on oxygen affinity. Influence of ionized groups on interactions. Change of configuration on asso- ciation with third ligand. Reversible denaturation. Precautions in use of dithionite. Glucose oxidase and catalase as oxygen scavenger. Evidence for and against sulfhydryl as heme-linked group. 27 A Survey of the Evidence For and Against a Crevice Configuration for the Heme in Hemoglobin- Philip George and R. L. J;Lyster 33 Function of heme proteins as determined by heme binding. Structural fea- .. V~11
. . . V111 CONFERENCE ON HEMOGLOBIN tures in ligation. Magnetic susceptibility. Water as a ligand. Anhydro-Hb. Steric eEects in ligation. Thermodynamic constants for ligands. Heme-linked groups; consequences of one and two protein links to heme. Thermodynamics of crevice configuration. Nature of bonds in hemoglobin. Discussion: Felix Horowitz, Jacinto Stei?zhardt, and John T. Edsall . Surface tension evidence for heme-globin interactions. Evidence against imi- dazole as heme-linked group. Significance of surface tension changes. Kinetics of reversible denaturation. Dilution effect in dissociation. Non-heme associ- ation. X-Ray Analysis of Haemoglobin ~ n F. Callus, Howard M. Dintzis, and Max F. Perutz ........................................ Principles of x-ray analysis. Signs and their determination by isomorphous substitution. Fourier projections. Three-dimensional analysis. Applications to me/hemoglobin. Substitutions with PCMB. External shape. Substitution and spacing of sulfhydryl groups. Calculation of signs. Discussion: Reinhold Berlesch, David B. Smith, f alter L. Hughes, M. 17. Perz:~z, and Norman Davidson ........ Homocysteine thiolactone for heavy metal substitution near amino groups. ERect of pH and ionic strength on globin association. N-terminal amino acid sequences of globin. Ease of extraction of hemes from hemoglobin in non- aqueous systems and the preparation of "native" globin. Evidence for location of -SH group in Mgb and Hb. Angular orientation of hemes as shown by paramagnetic resonance. PART II BIOSYNTHESIS OF HEMOGLOBIN The Biosynthesis of Porphyrin- David Shemin .................. Cellular synthesis from glycine and succinate. Systems for in vitro synthesis. Labelled acetate, succinate and glycine as heme precursors. "Active" succi- nate; probable mechanism of condensation. 6-aminolevulinate and porpho- bilinogen as intermedites. Participation and inhibition of pyridoxal phosphate. Other inhibitors. Need for acyl group on nitrogen. Conversion of porpho- bilinogen to porphyrin. Tripyrrylmethane as intermediate. Comparison to vitamin Be a. The Enzymatic Synthesis of Uroporphyrinogens from Porphobilinogen Lawrence Bogorad ...................................... Products from enzymatic condensation of porphobilinogen. IJroporphyrin iso- mers. Porphobilinogen deaminase. The oxidative step. The isomerase. Enzy- matic production of uroporphyrinogen III and conversion to protoporphyrin. Kinetic evidence for concerted action of both enzymes. Probable sequence of reactions and mechanism of isomerization. Tripyrrylmethane as intermediate. PAGE 48 50 61 66 74
CONTENTS Enzymatic Studies of Protoporphyrin Synthesis S. Granzck 1X PAGE ........ 84 Three groups of enzymes in synthesis. Conversion of glycine and succinate to 6-aminolevulinate. Inhibitors. Relation to citric acid cycle. Function of mito- chondria. Enhancement of synthesis. Conversion of 6-aminolevulinate to uro- porphyrinogen and coproporphyrinogen. Conversion to porphyrin. Study of plant organelles. Control devices for biosynthesis. Discussion: Herbert C. Schwartz 92 Biosynthesis in hemolysate system. Use of radioiron. Activity and stability of enzymes. Enzyme kinetics and sulfhydryl inhibitors. A dialyzable factor. The Role of Iron in Hemoglobin Synthesis- Clement A. Finch Iron uptake by reticulocytes. Transferrin. Storage iron. Relation of nucleus to iron incorporation. Restriction of iron supply. Transport of iron to mar- row. Use of radioiron. Excessive iron uptake in some anemias. Diseases asso- ciated with abnormal porphyrin synthesis. Block of heme synthesis and ac- cumulation of intermediates. Need for ferrokinetic studies at cellular level. The Role of Copper in Erythropoiesis George E. CartwriyEt, Clark J. Gubler, and Maxwell M. bYintrobe Copper-deficient diet. Nature of resulting anemia. Blood and tissue copper levels. :Responses to added copper. Effect of copper on iron absorption and transport. Effect of copper on erythrocyte survival time. }hole of copper in erythropolesis and basis for anemia. Erythrocuprein from human erythrocytes. Physical characterization. 95 100 Discussion: I. H.Scheinberg and G. E. Cartwrighf 110 Distinction between erythrocuprein and hemocuprein. Hemoglobin Synthesis in Vitro in Rabbit Reticulocytes - Henry Borsook Methods of study; incorporation of labelled amino acids into hemoglobin. Production of reticulocvtosis. Substances which enhance protein synthesis in -., ~vitro. Species variations. Nutritional aspects of in biro synthesis. Transferrin. Fructose-amino and tagatose-amino acids. Optimal electrolyte concentrations. Inhibitors. Simultaneous heme and globin synthesis. Dissociation in human disease. Discussion : Max F.Perutz .............................. Possibility of structural and metabolic PNA. 111 ...... 130 The Incorporation of Glycine into Globin and the Synthesis of Heme in Duck Ervthrocvtes and Rabbit ReticulocytesIrvin q M. London, J ~ Helen Morell,and Anton Kassenaar 131 In vitro system for study. Heme to globin ratios of radioactivity. Methods. Influence of media. Effects of heavy metals. Significance of variations in heme- globin ratios. Species diffeecnces. Effect of nucleosides. Mechanism of glycine incorporation into globin. Metabolic pools for heme and globin synthesis. Factors influencing ratio. Possible other uses of method.
x CONFERENCE ON HEMOGLOBIN Discussion: Felix Haurowitz, Hurt Salomon, Henry Borsook, and Jean- Claude Savoie ~ Glycine uptake by red cells. State of intracellular glycine. Incorporation of glycine by bone marrow. Dissociation of heme and globin synthesis. Effect of irradiation. Sites of heme formation. Specific activity of residue-bound hemin. PART III ABNORMAL HEMOGLOBINS Electrophoretic Analyses of Abnormal Human HemoglobinsHarvey A. Itan 0 ................................................. Comparison of moving boundary and zone electrophoresis. Nomenclature of human hemoglobins. Net charge. Proof of normal heme. Basis of mobility dif- ferences in globin. Limitation of electrophoretic method. Use of "reference mixtures." Quantitative aspects. Analysis of hemolysates; precautions in handling. Inherited and acquired abnormal hemoglobins. Relative propor- tions of abnormal hemoglobins in heterozygotes. Artifacts during aging of cells. Significance of louder composition. Homogeneity of products of a single genotype. Summary of advantages of electrophoresis. Need for studies of complete amino acid sequence. Discussion: Jaci7lto Steinhard~t, Harvey J. Itano, F. I. Ax. Roughton, PAGE 140 144 Reinhold Benesch, V. M. Ingram, and John H. Taylor 154 Difficulties in use of ferricyanide. Advantages in use of me/hemoglobin. Pos- sible artifacts from slow dissociation. Advantages of nitric oxide. Avoidance of ferricyanide oxidation of -SH groups. Dithiomethemoglobin properties. Zone Electrophoresis and the Minor Hemoglobin Components of Normal Human Blood Henry J. Kunkel 157 Limitations of zone electrophoresis. Supporting media. Comparative quanti- ties of minor components. A2 in thalassemia. Turnover in A`, and fast A fractions by radioiron. Comparison with other results. Discussion: Eves Derrien, Henry J. Kunkel, and Jerome Vinograd .. "Protein X" as a minor component. Possible artifacts. Evidence against "pro- tein X" as A2. Findings with crystalline hemoglobins. A Method for the Characterization of Abnormal Human Hemoglobins Based Upon Differences in Chromatographic Behavior on Amberlite IRC 50 T. H. J. Huisman .................... Column chromatography of nine hemoglobins. Comparison of results with electrophoresis and chromatography. Discussion: M artin M orrison ................................. Quantitative analysis of hemoglobins on IRC columns. Identification of com- ponents. Effect of pH on mobilities and elusion schedule. Effect of partial oxidation of hemes. Lack of dissociation. Results with thalassemia. 162 165 166
CONTENTS 'the Alkali Denaturation ProceduresAmok I. Chernoff Alkali resistance of fetal hemoglobin. Quantitation of hemoglobin F. Effect of heme configuration on denaturation. Nature of alkali-resistant fraction. Sig- nificance of alkali-resistant hemoglobin in pathological states. Sensitivity of method. Comparison of results by other methods. X1 PAGE - 172 Discussion: Abner Robinson and A.llI. Josephson 176 "Electrochromatographic" separation of various hemoglobins. Additional minor components. Advantages of method. Immunologic Aspects of the Human HemoglobinAmoz I. Chernoff 179 Lack of immunologic specificity of adult hemoglobins. Production of potent, specific antibodies. Limitations of method. Use of Ouchterlony's technique. Interpretation of results. Discussion: S. J. Singer and A1. I. Chernoff ........ Immunologic difference between Hb A and Hb S. Studies on the Heterogeneity of Adult and Fetal Hemoglobins by Saltin~- ~ . ~ . . ~ . . ~ . O , ~ But, Shall ~enaturat~on and iviov~ng Boundary Electrophoresis Yves Derrien ............................................. Subfractions of minor components obtained by salting out. Tests for validity of results. "Protein X" in alkali-resistant fraction. Isoleucine contents. Com- parison with electrophoretic results. Comparative evaluation of methods. 182 183 Discussion: Harvey /.Itano and Yves Derrien 199 Sources of error in salting-out and electrophoresis. EIeterogeneity of Hemoglobin and Methods of Isotopic Biosynthesis Georyes Schapira, Jean-Cla?~de Dreyfus, and Jacques Arch 201 Problems of establishing protein homogeneity or heterogeneity. Advantages of isotopic method. Comparison of specific activities of heme and globin, syn- thesized intvievo and in vitro. Time curves of in ciao synthesis. Anemia and acute leukemia. Interpretation of results. Heterogeneity of erythrocytes. Ef- fect of pathological environment. Extra-medullary synthesis. Heterogeneity of hemoglobin in a single cell. Starch Electrophoresis of Hemoglobin: Findings in Thalassemia Syn- drome - Park S. Gerald 212 Studies of thalassemia patients and their families. Criteria for diagnosis. Distinctions from hereditary microcytoses. Thalassemia trait. "Lepore ab- normality." "Pseudo-thalassemia" form of hemoglobin H disease. Discussion : J. M. Josephson, A. G. Ilotulsky, J. L. Cook, I. M. London, H. G. Kunkel, M artin Morrison, and Alfred Chanutin .. 215 A2 levels in thalassemia trait, pernicious anemia and Hb H disease. Prop- erties of minor components. Methemoglobin reductase. Chromatography of
. ~ X11 CONFERENCE ON HEMOGLOBIN thalassemia hemolysates. Identification of minor components by alternative techniques. Need for reference samples. Hemoglobin changes in stored blood. Observations on the Amino Acid Composition of Human Hemoglobins William H.Stein .......................................... Principles of method. Preparation of hemoglobin. Analytical results. Signifi- cance of absence of isoleucine. Limiting values for half-cystine. Problems of . . . . removing Impurities. PAGE 220 Discussion: BY. A. Schroeder 225 Confirmation of lack of isoleucine in electrophoretic Hb. N-terminal groups. Kinetics of hydrolysis of DNP-valyl peptides from the two chains. The Structural Basis of Difference in Electrophoretic Behavior of Human Hemoglobins I. Herbert Scheinbery .................. Dissociation constants of ionizable groups and relation of charge to mobility. Interpretation of variation of mobility with pH. Possible structural basis for differences in Hbs A, S and C. 227 Discussion : Reinhold Benesch 232 Possible ionization constants for -SH groups. The Chemical Difference Bet~veer1 Normal Human and Sickle Cell Anaemia Haemoglobins - V. M. Ingram ...................... Enzymatic degradation. Methods of fractionation and "fingerprinting;." Dis- tinction between Hb A and Hb S. Amino acid composition of unique peptides. Confirmation of identical half-hemoglobins. Relationship of structure to gene mutation. Findings with Hb C. 233 Discussion : Walter L. Hughes, Rein hold Benesch, Howard M. D~ntsis, Max F. Per2l to, V. ELI. I Gram, U7il loam H. Stein, Jo ha F. Taylor, Makio M?lrayama, and T. H. J. Huisman 238 Hughes: Sulfhydryl groups of hemoglobin from different species as deter- mined by silver and mercury titrations. Possibility of different types of -SH groups and metal binding bv other than -SH groups. Be?zesch: Steric hind- ~ , ~ ~ rance in metal binding. Relation of -SH bonding to heme-bound group. Dintzis: Metal birding when -SH groups are blocked. Permits: X-ray evidence for location of -SH groups. Comparison of silver and mercury. Ingram: A~n- ities of metals for non -SH groupings. Effect of denaturation on steric hind- rance. Confirmation of species differences. Stein: Limits on total -SH groups. Cysteic acid confirmatior~ of half-cystine values. Evidence for homogeneity of Hb A and of Hb S. Taylor: Relation of -SH groups to heme-heme inter- actions. Precautions in handling reagents and comparison of methods. Use of dithio derivatives. Be7zescl2: Affinities of silver and mercury. Specificity. Marayama: Temperature effects of "elation and metal binding. Results with abnormal hemoglobins. Spatial configuration of -SH groups as revealed by mercury blocking. Effect of dialysis. i7uis~rzar~: N- and C-terminal sequences and -SH content of Hb F. Comparison with fIb A. Dithio function in Hb F.
CONTENTS PART IV GENETIC ASPECTS OF ABNORMAL HEMOGLOBINS Genetic Aspects of Abnormal HemoglobinsJames V. Heel ........ Genetics of Hb S and Hb C. Possible allelism of S and C. Inheritance of abnormal hemoglobin genes. Genetics of thalassemia. Probable non-allelism of Hb S and thalassemia. Study of family with genes for Hb S. Hb C and thalassemia. Suggested genetic terminology. Conditions regulating formation of Hb F. Biochemical genetics. Nature of gene action. Population genetics of abnormal hemoglobins. Gene selection pressure. Frequency of mutation. . . . x~ PAGE: 253 Discussion: Salome G. faelsch, James V. Neel, Harvey ~1. Itano, 1. Herbert Scheinbery, and In R. Bergren 272 Primary gene products; reservations regarding hemoglobin. Amino acid se- quence determination by genes. Need for genetic study in clinical hematology. Incidence of HL S. Recent findings with Hb H and other abnormal hemo- globins. PART V CEINICAL CHARACTERISTICS ASSOCIATED WITH ABNORMAL HEMOGLOBINS Clinical Manifestations of Sickle Cell Diseases Ernest [Y. Smith and C. L. Conley ............................................. Sickle cell anemia, sickling, and genetic variants of sicklemia. Relation to oxyger~ ter~sion. Blood viscosity, anoxia and other indications. Clinical cases. Vascular stasis and tissue infarctions. Body configurations. Renal changes. Retinal changes. 276 The Thalassemia Syndromes Amoz I. Chernoff 283 Definition of the syndrome. Relation to Hb F. Possible hereditary pattern. Re- ~riew of earlier genetic concepts. Association of thalassemia with other de- fects. Clinical findings; criteria for diagnosis. Mechanism of suppression of Hb A. Relation to Hb E disease. Electrophoretic findings. Characteristics of Hb E diseases. Status of study. Hemoglobin C. Diseases - Helen At. Ranney .................... Hb C trait. Combination with fIb S. Hb C disease; clinical features; electro- phoretic findings. Hb C and thalassemia; genetics; clinical aspects. Problems of diagnosis. A clinical case. Discussion. HYolfgang BY. Zuelzer . ~ Various lob C combinations. "Normal" variants. Closing Remarks : Georpe E. Cart~right and Carl Moore Attendance List ... Index of Authors and Discussants 287 295 296 297 303
CONFERENCE ON HEMOGLOBIN OPENING REMARKS Dr. Irving M. London: In opening this Conference on Hemoglobin, I should like to welcome you and to express the appreciation of Dr. Edsall, Dr. Cartwright, and myself to all who have come. We are grateful to you, and we particularly appreciate the participation of our friends from other lands who have come long distances to be with us today. We are deeply indebted to those members of the professional and secre- tarial staff of the Division of Medical Sciences of the National Academy of Sciences-National Research Council who have carried the responsibility and burden of the administrative organization of this Conference. . I should like now to introduce Dr. Carl Moore, Professor of Medicine at Washington University and Chairman of the Subcommittee on Blood and Related Problems of the Division of Medical Sciences, which is sponsoring this Conference. Dr. Carl Moore: My function is a very brief and a very pleasant one to express again to all of you the appreciation of the Subcommittee for your willingness to participate in this session and the thanks of the Academy-Re- search Council's Division of Medical Sciences in general for joining us in this discussion. I also want to express our extreme gratitude to the National Heart Institute of the National Institutes of Health, who supplied the sup- port which made the session possible. In order not to take up any more time, I will merely introduce the first speaker on the program. After he has finished his presentation, he will take over as chairman of the morning session. Dr. John T. Edsall of Boston will speak to us on the current ideas on the structure of hemoglobin. rev