HEMOGLOBIN AND RED CELL STRUCTURE AND FUNCTION

ADV ANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY Editorial Board:

Nathan Back

N. R. Di Luzio

Alfred Gellhorn

Bernard Halpern

Ephraim Katchalski

David Kritchevsky

Abel Lajtha

Rodolfo Paoletti

Volume 1

Chairman, Department of Biochemical Pharmacology, School of Pharmacy, State University of New York, Buffalo, New York

Chairman, Department of Physiology, . Tulane University School of Medicine, New Orleans, Louisiana

University of Pennsylvania Medical School, Philadelphia, Pennsylvania

College de France, Director of the Institute of Immuno.Biology, Paris, France

Department of Biophysics, The Weizmann Institute of Science, Rehovoth. Israel

Wistar Institute, Philadelphia, Pennsylvania

New York State Research Institute for Neurochemistry and Drug Addiction, Ward's Island, New York

Institute of Pharmacology, University of Milan, Milan,Italy

THE RETICULOENDOTHELIAL SYSTEM AND ATHEROSCLEROSIS Edited by N. R. Di Luzio and R. Paoletti· 1967

Volume 2 PHARMACOLOGY OF HORMONAL POLYPEPTIDES AND PROTEINS

Edited by N. Back, L. Martini, and R. Paoletti· 1968

Volume 3 GERM·FREE BIOLOGY: Experimental and Clinical Aspects

Edited by E. A. Mirand and N. Back· 1969

Volume 4 DRUGS AFFECTING LIPID METABOLISM

Edited by W. L. Holmes, L. A. Carlson, and R. Paoletti· 1969

Volume 5 LYMPHATIC TISSUE AND GERMINAL CENTERS IN IMMUNE RESPONSE

Edited by L. Fiore·Donati and M. G. Hanna, Jr .• 1969

Volume 6 RED CELL METABOLISM AND FUNCTION

Edited by George J. Brewer· 1970

Volume 7 SURFACE CHEMISTRY OF BIOLOGICAL SYSTEMS

Edited by Martin Blank· 1970

Volume 8 BRADYKININ AND RELATED KININS: Cardiovascular, Biochemical, and Neural Actions

Edited by F. Sicuteri, M. Rocha e Silva, and N. Back· 1970

Volume 9 SHOCK: Biochemical, Pharmacological, and Clinical Aspects

Edited by A. Bertelli and N. Back· 1970

Volume 10 THE HUMAN TESTIS

Edited by E. Rosemberg and C. A. Paulsen· 1970

Volume 11 MUSCLE METABOLISM DURING EXERCISE

Edited by B. Pemow and B. Sahin· 1971

Volume 12 MORPHOLOGICAL AND FUNCTIONAL ASPECTS OF IMMUNITY

Edited by K. Lindahl.Kiessling, G. AIm, and M. G. Hanna, Jr.· 1971

Volume 13 CHEMISTRY AND BRAIN DEVELOPMENT

Edited by R. Paoletti and A. N. Davison· 1971

Volume 14 MEMBRANE·BOUND ENZYMES

Edited by G. Porcellati and F. di Jeso • 1971

Volume 15 THE RETICULOENDOTHELIAL SYSTEM AND IMMUNE PHENOMENA

Edited by N. R. Di Luzio and K. Flemming· 1971

Volume 16A THE ARTERY AND THE PROCESS OF ARTERIOSCLEROSIS: Pathogenesis

Edited by Stewart W oU • 1971

Volume 16B THE ARTERY AND THE PROCESS OF ARTERIOSCLEROSIS: Measurement and Modification

Edited by Stewart Wolf· 1971

Volume 17 CONTROL OF RENIN SECRETION

Edited by Tatiana A. Assaykeen • 1972

Volume 18 THE DYNAMICS OF MERISTEM CELL POPULATIONS

Edited by Morton W. Miller and Charles C. Kuehnert • 1972

Volume 19 SPHINGOLIPIDS, SPHINGOLIPIDOSES AND ALLIED DISORDERS

Edited by Bruno W. Volk and Stanley M. Aronson. 1972

Volume 20 DRUG ABUSE: Nonmedical Use of Dependence-Producing Drugs

Edited by Simon Btesh • 1972

Volume 21 VASOACTIVE POLYPEPTIDES

Edited by N. Back and F. Sicuteri • 1972

Volume 22 COMPARATIVE PATHOPHYSIOLOGY OF CIRCULATORY DISTURBANCES

Edited by Colin M. Bloor • 1972

Volume 23 THE FUNDAMENTAL MECHANISMS OF SHOCK

Edited by Lerner B. Hinshaw and Barbara G. Cox. 1972

Volume 24 THE VISUAL SYSTEM: Neurophysiology, Biophysics, and Their Clinical Applications

Edited by G. B. Arden. 1972

Volume 25 GLYCOLIPIDS, GLYCOPROTEINS, AND MUCOPOLYSACCHARIDES OF THE NERVOUS SYSTEM

Edited by Vittorio Zambotti, Guido Tettamanti, and Mariagrazia Arrigoni. 1972

Volume 26 PHARMACOLOGICAL CONTROL OF LIPID METABOLISM

Edited by William L. Holmes, Rodolfo Paoletti, and David Kritchevsky • 1972

Volume 27 DRUGS AND FETAL DEVELOPMENT

Edited by M. A. Klingberg, A. Abramovici, and J. Chemke .1972

Volume 28 HRMOGLOBIN AND RED CELL STRUCTURE AND FUNCTION

HEMOGLOBIN AND RED CELL STRUCTURE AND FUNCTION

Proceedings of the Second International Conference on Red Cell Metabolism and Function h~ld at the University of Michigan Ann Arbor, April 27-29, 1972

Edited by

George J. Brewer Department of Human Genetics and Medicine (Simpson Memorial Institute) University of Michigan Ann Arbor, Michigan

~ PLENUM PRESS • NEW YORK - LONDON • 1972

The Second International Conference on Red Cell Metabolism and Function was sponsored in part by Office of Naval Research (Contract #NOOOI4-72-C-0338) Michigan Cancer Institute The National Foundation Abbott Laboratories

Library of Congress Catalog Card Number 72-86140

ISBN-13: 978-1-4684-3224-4 e-ISBN-13: 978-1-4684-3222-0 DOl: 10.1007/978-1-4684-3222-0

© 1972 Plenum Press, New York Softcover reprint of the hardcover 1st edition 1972 A Division of Plenum Publishing Corporation 227 West 17th Street, New York, N.Y. 10011

United Kingdom edition published by Plenum Press, London A Division of Plenum Publishing Company Davis House (4th Floor), 8 Scrubs Lane, Harlesden, London, NWIO 6SE, England

All rights reserved

No part of this publication may be reproduced in any form without written permission from the publisher

PREFACE

Hemoglobin and the red cell have continued to set a dizzying pace as the objects of research in the two and one-half year interval since the First International Conference on Red Cell Metabolism and Function. Most exciting perhaps, is a beginning molecular attack on sickle cell disease. The story of the inter­action of red cell metabolism and oxygen transport has continued to unfold, and we can now infer that patients with hypoxia usually utilize red cell metabolic adjustments to improve oxygenation. This puts the red cell squarely in the center of medical practice, since much of medicine-heart, pulmonary, and blood diseases -deals with inadequate oxygenation.

On April 27th through the 29th, 1972, crystallographers, chemists, biochemists, physiologists, geneticists, and physi­cians from many medical disciplines met in the Towsley Center for Continuing Medical Education at the University of Michigan, Ann Arbor to present new data, to review recent developments, and to try to piece together additional features of the red cell puzzle. The meeting was dedicated to Dr. Francis John Worsley Roughton, Professor Emeritus of Colloid Science, University of Cambridge, England, in recognition of his numerous excellent contributions to the understanding of hemoglobin and red cell function.

The program got off to a good start with a paper from M. F. Perutz, Nobel Laureate, on the structure of hemoglobin. Dr. Perutz also key-noted the Conference with a special lecture on heme-heme interaction. A number of fascinating papers were presented on various aspects of hemoglobin, its structure, its interaction with ligands such as oxygen, and its properties under varying conditions. Red cell metabolism was considered, in depth, from many viewpoints, including defects in uremia, inter­actions with serum phosphorous, male -female differences, the role of catalase, genetic selection for quantitative variation, and

vii

viii PREFACE

mechanisms of glycolytic response to altitude stress and to anemia. As with the first conference, a session was devoted to the continuing assessment of the importance of decline in red cell oxygen transport functional capacity during blood bank storage. A session was also devoted to consideration of carbonic anhydrase and carbon dioxide transport, and the interaction of this area with oxygen transport.

A high point of the conference was the session on sickle cell structure and function. Excellent papers were presented on cyanate, including results of some early clinical trials which look promising. A trial with oral urea in sickle cell disea.se indicating possible usefulness of this approach was presented. The antis ickling properties of carbamyl phosphate were also discussed. The present status of prenatal diagnosis of sickle cell disease, and the sickling phenomenon of deer erythrocytes, we re other interesting topics. The discussions in the general area of sickle cell disease and the mechanisms by which antis ickling agents act were quite interesting because of the diversity and expertise represented in the audience.

This volume contains the Proceedings of this second confer­ence. It includes the formal papers and much of the informal discussion after the papers. It represents a compilation of the present state of the art, and the status of current thinking, in the various areas dis cussed above.

The Editor would like to acknowledge the great dedication and assistance of Mrs. Nellie Gill in all phases of the Conference, and pUblication of the volume. My wife, Lucia Feitler Brewer, worked very hard on all aspects of Conference organization, while keeping one hand busy in the laboratory. The continued good spirits of these two in the face of ever-mounting details and problems before and during the Conference, buoyed the rest of us. I wish to thank Dr. Fred J. Oelshlegel, Jr. and Dr. John W. Eaton for their counsel. Thanks are also due Ms. Nancy Noble, Ms. Lorna Grindlay Moore, Mr. Eric Schoomaker, Mr. Conrad Knutsen, Mr. Barry Sander, Dr. James V. Neel, Dr. C.J.D. Zarafonetis, and Dean John Gronvall for their help and support. The staff of Postgraduate Medicine, in particular Mr. Jack Burmeister, Mr. Robert Richards, and Mrs. Lynne Bowbeer, we re quite helpful. Mrs. Rena J ones and Mrs. Marion Wolfe (and Mrs. Nellie Gill) dese rve much credit for struggling with the discussion tapes. Financial support from the Office of Naval Research, the National Foundation, the University of Michigan Cancer Research Institute, and Abbott Laboratories made the

PREFACE ix

Conference possible. I thank the Biochemical Journal and the Biochemical Society for us"e of the photograph of Dr. Roughton. Plenum Press published the Proceedings of the First Conference in record time, and judging from their helpfulness with this vol­ume, I anticipate another remarkable job.

THE EDITOR

May 19, 1972

DEDICATION

OF THE SECOND INTERNATIONAL CONFERENCE ON

RED CELL METABOLISM AND FUNCTION TO

DR. FRANCIS JOHN WORSLEY ROUGHTON PROFESSOR EMERITUS OF COLLOID SCIENCE

UNIVERSITY OF CAMBRIDGE, ENGLAND

Dr. Roughton was born on June 6, 1899, in Kettering, an industrial town in the midlands of England, His Roughton ances­tors were busy practicing doctors in the town for five genera­tions - father to son - from 1737 to 1933. As a boy he hoped, in due course, to become the sixth of the series, but his physical cons titution - notably a cardiac anomaly - made such an arduous livelihood unwise. His father, the last of the five generations of Kettering doctors, urged him in 1912 ins tead to become a scientist with a special lean towards chemistry and its borders with medical sciences, for which he saw a great future. It is interesting to recall that Dr. Roughton's great grandfather William, in a letter to home in 1799 from St. Bartholomew's Hospital, London, where he was a medical student, apologized for his extravagance in spending ten shillings and sixpence (about 2 1/2 dollars at the current rate of exchange) on Lavoisier's Treatise of Chemistry, which he considered lias indispensably necessary for the understanding of the modern physiology".

In the course of his genealogical researches, to which he is almost as devoted as to his scientific work, Dr. Roughton was thrilled to discover in 1955 that he is directly descended, through his Roughton great grandmother, from Daniel Harvey, one of the brothers of William Harvey, the discoverer of the circula­tion of the blood. William Harvey, though married, was child­less so that as one of his numerous seven great nephews, Dr.

xi

xii DEDICATION

Roughton is as closely related to the illustrious William Harvey as anyone now living.

After a conventional British schoolboy education, Dr. Roughton won an open competitive scholarship at Trinity College, Cambridge, in mathematics, physics, and chemistry. To these subjects he added at the University, biology and physiology, including biochemistry which at that time had not attained full independence. In 1920, only a few days before his 21st birthday, an accident, fortunate for him, brought him suddenly into the

front line of research. Joseph Barcroft had just published the result of a pioneer "simulated altitude" experiment in which he lived for 10 days in a glass chamber with the partial pressure of oxygen being steadily reduced to a value corresponding to an altitude of about 15,000 feet. Barcroft found no evidence of development of oxygen secretion by the lung, as his colleague, J. S. Haldane of Oxford, had claimed as an important feature of acclimatization to high altitude. Also included in the class ical paper of Barcroft et al. were calculations of the diffus ion con­stant of the lung which Roughton, though only a student of 20, promptly spotted as erroneous. Barcroft, 30 years older, equally promptly and generously took his very junior colleague into partnership to rectify the error, and several joint papers followed. From 1921 to 1923 Roughton not only worked on his own on the diffus ion problem, but also went into a 5 -year part­nership with Hamilton Hartridge leading in the winter of 1922 to the development of the Hartridge-Roughton method for mea­suring the velocity of rapid reactions. These reserrches won for Roughton a competitive prize fellowship at Trinity College for 6 years (1923-1929). During this period he attained rapid promotion in Cambridge University, first as a University Lecturer in Physical Biochemistry and then (1926-1947) to a similar post (the equivalent of an Associate Professorship) in Physico-Chemical Physiology. Dr. Roughton spent much of World War II on the East Coast of the United States, helping in war science projects (a) in the Harvard Fatigue Laboratory at Boston, mainly on problems to do with ca rbon monoxide which were of considerable military importance, and (b) in the Physiological Laboratory of the Medical School of Columbia University, New York, both in the fields of' carbon monoxide and shock. In 1946 he was offered the chairmanship of the Depart­ment of Colloid Science at Cambridge, which he only accepted after great hesitation, being worried by the title of the subject. In practice, however, it was interpreted as a mixture of surface chemistry and biophysical chemistry and these were, in fact,

DEDICATION

the general themes during Dr. Roughton1s 20 year tenure of the chair, from which he retired at the age limit in the fall of 1966.

Dr. Roughton1s researches during the past half-century have in general followed the initial 1921-1926 pattern of forming powerful and fairly lengthy partnerships, wherein he has had a substantial part to contribute. The early work on diffusion broadened to studies of mixed diffusion plus chemical reaction

processes, for example in analysing - both theoretically and experimentally - the factors determining the rate of penetration of oxygen and carbon monoxide into the red cell. This work has not only proved of importance to the detailed study of oxygen diffusion processes in the lung and the blood, but also has won for Dr. Roughton an important reputation in chemical engineer­ing circles.

The rapid kinetic work, especially on the reactions of ligands with the heme groups in hemoglobin, has continued over the past 50 years, the last 20 of which have been occupied by a very fruitful, part-time partnership with Q. H. Gibson in this field. This work has won for him a still more prominent place in physical chemistry.

xiii

In 1931, stimulated by the revolutionary papers by Henriques from Copenhagen on the kinetics of the reactions of carbon dioxide in hemoglobin solution, Dr. Roughton began researches in this field, at first with Dr. R. Brinkman (of Holland) and Dr. R. Margaria (of Italy). In 1932 he succeeded, in brief partnership with an exceptionally gifted young biochemist, N. U. Meldrum, who died prematurely in the following year, in sep­arating from blood highly concentrated and catalytically active solutions of the enzyme carbonic anhydrase, which now - just 40 years later - forms a complete section of the present con­ference.

Although Henriques in 1928 thought he had completely dis­proved the existence of an enzyme such as carbonic anhydrase in blood, he did however bring forward the first reliable evidence for a rapid, reversible, carbamate reaction of carbon dioxide with hemoglobin to form HbC02 . In 1933/1935 Ferguson and Roughton developed a difficult chemical method of estimating this compound, the role of which in the normal respiration-ci.r­culation cycle was then evaluated. All this work was summarised in 1935 in a well-known article in Physiological Reviews entitled IIRecent Work on Carbon Dioxide Transport by the Blood l !.

Roughton continued researches both on carbonic anhydrase and on the carbamate problem intermittently during the next 25 years,

xiv DEDICATION

but it was not until his partnership with L. Rossi-Bernardi began in 1961 that he became deeply involved with HbC0 2 again. Some of the exciting results of this partnership are presented in this volume.

The interpretation of the kinetics of oxygen with hemoglobin requires a knowledge of the individual equilibrium constants of the 4-stage Adair reaction of oxygen with hemoglobin. During the past 25 years Dr. Roughton has, with 5 very able collabora­tors, succeeded in making specially accurate measurements both in the low range (0 to 1. 5% saturation) and the top range (98.5 to 100% saturation) of the oxygen dissociation curves of hemoglobin, and recently - in partnership with Dr. J. W. Severinghaus - of whole human blood under physiological condi­tions of CO pressure, pH, and temperature. By this work, and with genero~s assistance from statisticians and computers, he has succeeded to a reasonable extent in escaping from the im­passe caused by four arbitrary constants in Adair's equation.

These six or seven lines of research in which Dr. Roughton has been a pioneer in the field of blood, physical chemistry and respiration, have brought him a number of honours. In his own country he was elected F. R. S. in 1936 and much more recently to honorary membership of the Physiological Society, to the Presidency of the Cambridge Philosophical Society (which pub­lishes Biological Reviews) and to the F. G. Hopkins lectureship and medal, the highes t honor of the Biochemical Society. In the United States, where he has worked on and off for nearly 10 years of his life, he gave a HarveySociety lecture in 1943 (not knowing at the time of his relationship to William Harvey). He is an honorary member of the American Physiological Society and of the New York Academy of Sciences. In Denmark, where his work is also well-known and affectionately appreciated, in view of the great help he has derived and acknowledges from the class ical work of Carl Faurholt on carbamates, he is an honor­ary member of the Royal Academy of Letters and Sciences.

I am sad to report, that the day after this conference was over, Professor Roughton, who was unable to attend because of health problems, died in his horne of a cerebral hemorrhage.

L. Rossi-Bernardi Unive rs ity of Milan, Italy

CONTENTS

Participants . . . . . . . . . . . Registrants • . . . . . . . . . . . . . Abbreviations

I. HEMOGLOBIN STRUCTURE AND FUNCTION

Chairman: M. F. Perutz

Structure of Haemoglobin M Milwaukee, a Mutant Form Exhibiting Interaction between Ferrous

xxi

xxv

xxxi

and Ferric Subunits • • • • • • • • • • • • • •• 3 M. F. Perutz, 'R. D. Pulsinelli, and H. M. Ranney

The Interaction Between Hemoglobin and Its "Oxygen-Linked" Ligand s • • • • • • • • • • •

O. Brenna, M. Luzzana, M. Pace, M. Perrella, F. Rossi, L. Rossi-Bernardi, and F. J. W. Roughton

The Interaction of Sickle Hemoglobin with DPG, C02' and with Other Hemoglobins: Formation of Asyunnetrical Hybrids ••••••••••

H. Franklin Bunn

Enhancement of the Acid and Alkaline Bohr Effects of Hemoglobin by Organic Phosphates

A. Riggs and T. Imamura

Functional Non-Equivalence of a and ~ He~es in Human Hemoglobins • • • • • • . . . . . .

C. Ho and T. R. Lindstrom

xv

19

41

55

65

xvi CONTENTS

II. REIr CELL METABOLISM AND FUNCTION

Chairman: H. Franklin Bunn

Hemoglobin Ca sper G8 (3 106 Leu - Pro: Further Evidence that Hemoglobin Mutations are Not Random • • • • . • • • • •

R. T. Jones, R. D. Ko1er, M. Duerst, and Z. Stock1en

Potential Effects of Hemoglobin Concentration on Red Cell Metabolism Together with Observations on Red Cell Metabolic Differences Between Men and Women •

G. J. Brewer, F. J. Oe1sh1ege1, Jr., E. B. Schoomaker, and C. A. Knutsen

Catalase Activity and Red Cell Metabolism J. W. Eaton, M. Boraas, and N. L. Etkin

Red Cell Hexosemonophosphate Shunt Deficiency in Uremia .... . . . . . . . •

H. Jacob, Y. Yawata, and R. Howe

Effect of Inorganic Phosphate on Red Cell Metabolism: In Vitro and In Vivo Studies •• •••••

M. C. Brain and R. T. Card

Studies of the Metabolic Basis of the ATP-DPG Differences in Genetically Selected

79

99

121

133

145

High and Low ATP-DPG Rat Strains •••••••• 155 N. A. Noble and G. J. Brewer

III. CARBONIC ANHYDRASE AND CARBON DIOXIDE TRANSPORT

Chairman: Richard E. Tashian

Introductory Remarks at the Beginning of Session III R. E. Tashian

Functional Aspects of the Three-Dimensional Structure of the Active Site of Carbonic Anhydrase •••• • • • •

I. Waara, S. Lovgren, A. Li1jas, K. K. Kannan, and P.-C. Bergsten

The Effect of Temperature on the Catalytic Activity of Bovine Carbonic Anhydrase

J. C. Kernohan

167

169

189

CONTENTS

Carbonic Anhydrase, Red Cell Membranes, and C02 Transport • • • • • • . •

T. Enns

Effect of Ch1ortha1idone Binding on the Electrophoretic Properties of Human Red Cell Carbonic Anhydrase Isozymes

R. E. Tashian and Y.-S. L. Yu

The Role of Carbonic Anhydrase in the Control of Intracellular pH • • • •

W. J. Waddell

The Contribution of Carbamate in Human Adult and Foetal Blood to the C02 Exchange during the Respiratory Cycle • • . • • • • • •

C. Bauer

IV. SICKLE CELL STRUCTURE AND FUNCTION

Chairman: Donald L. Rucknage1

xvii

201

209

215

225

Introductory Remarks at the Beginning of Session IV • • •. 239 M. F. Perutz

Thermal (or Endothermic) Aggregation of Sickle Cell Hemoglobin (Hb S) During Sickling • • • •

M. Murayama

Chemical and Biological Aspects of the Inhibition of Red Blood Cell Sickling by Cyanate •

J. M. Manning, Ao Cerami, P. No Gillette, F. G. de Furia, and D. R. Miller

Preliminary Clinical Trials with Cyanate ••••• P. N. Gillette, C. M. Peterson, J. M. Manning, and A. Cerami

Carbamyl Phosphate Modification of Hemoglobin S Structure Resulting in Altered Sickling

L. M. Kraus, A. Rasad, and A. P. Kraus

Oxygen Affinity Independent Action of Cyanate and 2,3 DPG on Sickling •• ••••

M. C. Jensen, H. F. Bunn, G. C. Ha1ikas, and D. G. Nathan

Evaluation of Oral Urea in the Management of Sickle Cell Anemia ••••

J, Mo Lusher and M. I. Barnhart

243

253

261

279

297

303

xviii

The Sickling.Phenomenon of Deer Erythrocytes H. Kitchen and W. J. Taylor

. . . . . . . Further Studies on the Antenatal Detection of

Sickle Cell Anemia and Other Hemoglobinopathies ••••••••

H. H. Kazazian, Jr., M. M. Kaback, A. P. Woodhead, C. O. Leonard, and W. S. Nersesian

V. ADAPTA TION TO HYPOXIA

Chairman: Mikael Rorth

Adjustments of the Oxygen Transport System During Residence at High Altitude ••••••

L. H. Hartley

Red Cell Metabolism and Oxygen Affinity of Healthy

. . . . .

CONTENTS

325

337

349

Individuals during Exposure to High Altitude 361 M. Rorth, S. F. Nygaard, and H. H. Parving

Enzymatic Mechanisms of Red Cell Adaptation to Anemia 377 F. J. Oelshlegel, Jr., G. J. Brewer, J. A. Penner, and E. B. Schoomaker

Red Cell Metabolic Changes in Acute and Chronic Exposure to High Altitude •••• 397

L. G. Moore, G. J. Brewer, and F. J. Oe1sh1ege1, Jr.

Effects of Cyanate in Rabbits • D. R. Harkness, S .• Roth, P. Goldman, and M. Goldberg

VI. THE EFFECT OF OXYGEN AFFINITY ON

OXYGEN TRANSPORT: BLOOD STORAGE

Chairman: Stanley Ba1cerza~

Studies on the Ability of Stored Blood to Transport Oxygen In Vivo • • • • • • • • • • •

S. Balcerzak, J. Guy, E. Metz, and P. Bromberg

The Two Bohr Effects: Physiological Consequences of Ligand Interaction with Hemoglobin •

B. Wranne, R. Woodson, and J. Detter . . . . .

415

433

449

Rejuvenation and Freezing of Outdated Human Red Cells • •• 457 C. R. Valeri and C. G. Zarou1is

CONTENTS

Effects of Adenine on Stored Human Red Cells G, R. Bartlett

Hemoglobin Function During Blood Storage XV: Effects of Metabolic Additives Inosine and Methylene Blue on p50 and 2,3-DPG •••••

R. B. Dawson and W. F. Kocholaty

The Effect of Massive Transfusion of Stored Blood

xix

479

495

on Oxygen Transport - A Preliminary Report 511 S. V. Kevy, L, N. Button, and R, M. Filler

Index • . . . . . . . . . . • . . . . . . . . . . Q • • •• 517

PARTICIPANTS

Stanley P. Balcerzak, University Hospital, Columbus, Ohio

Grant Bartlett, Laboratory for Comparative Biochemistry, San Diego, California

Christian Bauer, Physiologisches Institut, Medizinische Hochschule, Roderbruchstrasse, Hanover, Ger.many

M. C. Brain, Department of Medicine, McMaster University Hamilton, Ontario, Canada.

George J. Brewer, Department of Human Genetics, University of Michigan, Ann Arbor, Michigan.

H. Franklin Bunn, Thorndike Memorial Laboratory, Harvard Medical School, Boston, Massachusetts.

James Manning, Rockefeller University, New York, N. Y.

R. Ben Dawson, School of Medicine, University of Maryland, Baltimore, Maryland 21201

John Eaton, Department of Anthropology, Washington Univers ity St. Louis, Missouri.

Theodore Enns, Scripps Institution of Oceanography, University of California, La Jolla, California.

Peter Gillette, Rockefeller University, New York, N. Y.

Donald Harkness, Veterans Administration Hospital, Miami, Florida.

L. Howard Hartley, Harvard Medical Unit, Boston City Hospi­tal, Bos ton, Mas sachusetts.

Chien Ho, University of Pittsburgh, Department of Biophysics and Microbiology, Pittsburgh, Pennsylvania.

xxi

xxii PARTICIPANTS

Harry S. Jacob, Univers ity of Minnesota Medical School, Minneapolis, Minnesota.

Michael Jensen, The Children's Hospital Medical Center, Boston, Mas sachusetts.

Richard T. Jones, Department of Biochemistry, University of Oregon Medical School, Portland, Oregon.

Haig H. Kazazian, Jr., Johns Hopkins Hospital, Baltimore, Maryland.

John C. Kernohan, Biochemistry Department, The University of Dundee, Dundee, Scotland.

Sherwin V. Kevy, The Children's Hospital Medical Center, Bos ton, Mas sachusetts.

Hyram Kitchen, The Center for Laboratory Animal Resources, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan.

Lorraine Kraus, University of Tennessee Medical Units, Memphis, Tennessee.

Anders LUjas, Department of Biological Sciences, Purdue University, West Lafayette, Indiana.

Jeanne Lusher, Children's Hospital of Michigan, Detroit, Michigan.

Lorna Grindlay Moore, Department of Human Genetics, Univer­sity of Michigan, Ann Arbor, Michigan.

Makio Murayama, National Institutes of Health, Bethesda, Maryland.

Nancy Noble, Department of Human Genetics, Univers ity. of Michigan, Ann Arbor, Michigan.

Fred J. Oelshlegel, Jr., Department of Human Genetics, University of Michigan, Ann Arbor, Michigan.

M. F. Perutz, MRC Laboratory of Molecular Biology, Hills Road, Cambridge, England.

Austen Riggs, Department of Zoology, University of Texas, Austin, Texas.

Mikael Rorth, Department of Clinical Chemistry, Rigshospitalet, Copenhagen, Denmark

PARTICIPANTS xxiii

L. Rossi-Bernardi, Universita Di Milano, Cattedra Di Enzimolo­gia, Milano, Italy.

Donald L. Rucknagel, Department of Human Genetics, Univers ity of Michigan, Ann Arbor, Michigan.

Richard E. Tashian, Department of Human Genetics, University of Michigan, Ann Arbor, Michigan.

C. Robert Valeri, Naval Blood Research Laboratory, Chelsea, Mas sachusetts.

WilliaUl J. Waddell, Dental Research Center, Univers ity of North Carolina, Chapel Hill, North Carolina.

Robert D. Woodson, University Hospital, University of Washing­ton, Seattle, Washington.

REGISTRANTS

Junius AdaITls, Veterans AdITlinistration West Side Hospital, Chicago, Illinois.

Clarence Alfrey, The Methodist Hospital, Houston, Texas.

Marion 1. Barnhart, Wayne State University School of Medicine, Detroit, Michigan.

Sandra Bittenbender, The Children1s Hospital Medical Center, Boston, Mas sachusetts.

John A. Black, University of Oregon Medical School, Portland, Oregon.

K. Boriboon, Children1 s Hospital of Michigan, Detroit, Michigan.

ThOITlas Bradley, Veterans AdITlinistration Hospital, San Fran­cisco, California.

J. Phillip Brewer, Box 579, Greeley, Colorado.

Philip A. BroITlberg, Ohio State University Hospital, ColuITlbus, Ohio.

Lawrence Button, The Children1s Hospital Medical Center, Boston, Mas sachusetts.

David G. CaITleron, DepartITlent of Zoology, Montana State University, BozeITlan, Montana.

Donald C. CaITlpbell, Mayo Clinic, Rochester, Minnesota.

Robert T. Card, DepartITlent of Medicine, McMaster University, HaITlilton, Ontario, Canada.

Alfred Chanutin, BiocheITlical Laboratory, School of Medicine, University of Virginia, Charlottesville, Virginia.

SaITluel Charache, The Johns Hopkins Hospital, BaltiITlore, Maryland.

xxv

xxvi REGISTRANTS

Flossie Cohen, Children's Hospital of Michigan, Detroit, Michi­gan.

Melvin L. Conway, 600 E. Lafayette, Detroit, Michigan.

M. Robert Cooper, Bowman Gray School of Medicine, Winston­Salem, North Carolina.

Nannie K. M. de Leeuw, Royal Victoria Hospital, Montreal, Quebec, Canada.

Dennis A. Diederich, University of Kansas Medical Center, Kansas City, Kansas.

Gregory S. Duboff, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan

Miles J. Edwards, Department of Medicine, Univers ity of Oregon Medical School, Portland, Oregon.

Valerie Evans, Pontiac General Hospital, Department of 'Lab­oratories, Pontiac, Michigan.

Virgil F. Fairbanks, Mayo Clinic, Rochester, Minnesota.

C. Stephen Foster, National Institutes of Health, Bethesda, Maryland.

Milton H. Freedman, Baptist Profess ional Building, Atlanta, Georgia.

Gian F. Gaetani, Department of Pediatrics, University of North Carolina, School of Medicine, Chapel Hill, North Carolina,

Thomas A. Garrett, Travenol Laboratories, Inc., Morton Grove, Illinois.

Gary Geller, University of Minnesota, St. Paul, Minnesota.

Ronald O. Gilcher, Central Blood Bank of Pittsburgh, Pittsburgh, Pennsylvania.

John Gilman, 138 W. Gorham St., Madison, Wisconsin.

Owen C. Grush, Department of Pediatrics, Emory University Medical School, Atlanta, Georgia.

Jerry Guy, University Hospital, Columbus, Ohio.

Saburo Hara, Meharry Medical College, Nashville, Tennessee.

David J. Harris, Ecological Investigations Program, Center for Disease Control, Kansas City, Kansas.

REGISTRANTS xxvii

L. F. Hass, Department of Biological Chemistry, M. S. Hershey Medical Center, Hershey, Pennsylvania.

James A. Hogan, St. Barnabas Medical Center, Livingston, New Jersey.

Harold J. Hommerson, Butterworth Hospital, Grand Rapids, Michigan.

H. E. Hynes, Scott & White Clinic, Temple, Texas.

Takashi Imamura, Department of Zoology, University of Texas at Austin, Austin, Texas.

William Isaacs, Ibadan, Nigeria.

Rodger S. Izzo, Travenol Laboratories, Inc. Morton Grove, Illinois.

Rouben M. Jiji, University of Maryland Hospital, Baltimore, Maryland.

Violet Jiji, University of Maryland Hospital, Baltimore, Mary­land.

Aaron M. Josephson, Travenol Laboratories, Inc. Morton Grove, Illinois

Yuet Wai Kan, The Children's Hospital Medical Center, Boston, Massachusetts.

Robert L. Kaufman, Department of Pediatrics, St. Louis Children's Hospital, St. Louis, Missouri.

Panpit P. Klug, Department of Medicine, George Washington University Medical Center, Washington, D. C.

Alfred P. Kraus, Section of Hematology, University of Ten­nessee, Memphis, Tennessee.

James E. Krook, Duke Hospital, Durham, North Carolina.

Wayne P. Krout, 440 New Castle Street, New Wilmington, Pennsylvania.

E. Stephen Kurtides, 3135 Park Place, Evanston, Illinois.

Lawrence S. Lessin, Department of Medicine, George Washing­ton University Medical Center, Washington, D. C.

Michael Liepman, 2048 Charlton, Ann Arbor, Michigan.

Patrick C. McCarthy, Department of Biology, Westminister College, New Wilmington, Pennsylvania.

xxviii REGISTRANTS

Paul R. McCurdy, D. C. General Hospital, Washington, D. C.

Earl N. Metz, University Hospital, Columbus, Ohio.

Nydia Meyers, Department of Human Genetics, University of Michigan, Ann Arbor, Michigan.

Harry J. Miller, 2500 Ridge Avenue, Evanston, Illinois.

William W. Miller, Department of Pediatrics, University of Texas Southwestern Medical School, Dallas, Texas.

Paul F. Milner, The Johns Hopkins Hospital, Baltimore, Mary­land.

Mary Jane Moore, Department of Anthropology, Univers ity of Wisconsin, Madison, Wisconsin.

Allan J. :Morris, Department of Biochemistry, Michigan State Univers ity, East Lansing, Michigan.

M. M. Mozen, Cutter Laboratories, Berkeley, California.

Stephen Murata, Medical World News, Chicago, Illinois.

James V. Neel, Department of Human Genetics, University of Michigan, Ann Arbor, Michigan

David J. Newman, Smith Kline & French Laboratories, Phila­delphia, Pennsylvania.

Mohammed P. Nisar, Monmouth Medical Center, Long Branch, New Jersey.

James M. Norton, Research Department, Maine lv1edical Center, Portland, Maine.

Vasudeva Paniker, Divis ion of Hematology, Vanderbilt Univer­s ity, Nashville, Tennessee.

Philip Paress, Division of Hematology, Coney Island Hospital, Brooklyn, N. Y.

Catherine W. Patrick, Pontiac General Hospital, Department of Laboratories, Pontiac, Michigan.

Robert Peloquin, Institut D'Hematologie De Montreal, Montreal, P. Quebec, Canada.

Michele Perrella, Universita Di Milano, Cattedra Di Enzimologia, Milano, Italy.

David H. Petering, Department of Chemistry, University of Wisconsin, Milwaukee, Wisconsin.

REGISTRANTS

Robert M. Petitt, Mayo ClLnic, Rochester, Minnesota.

Peter W. Rand, Research Department, Maine Medical Center, Portland, Maine.

Y. Ravindranath, Children l s Hospital of Michigan, Detroit, Michigan.

xxix

1. Drummond Rennie, Presbyterian-St. Luke1s Hospital, Depart­ment of Medicine, Chicago, Illinois.

Richard Rice, Saint Vincent Hospital, Worcester, Massachu­setts.

Robert D. Ringle, Ohio State University, Columbus, Ohio.

Julius Rutzky, William Beaumont Hospital, Royal Oak, Michi­gan.

Thomas H. Schmitz, Travenol Laboratories, Inc. Morton Grove, Illinois.

Joel M. Schwartz, Coney Island Hospital, Brooklyn, N. Y.

Ruth Andrea Seeler, Department of Pediatrics, Cook County Hospital, Chicago, Illinois.

George F. Sheldon, Department of Surgery, San Francisco General Hospital, San Francisco, California.

Charles E. Shields, Houston, Texas.

Gregory K. Snyder, Department of Medicine, University of Florida, Gainesville, Florida.

L. Michael Snyder, Saint Vincent Hospital, Worcester, Massachusetts.

Satish Srivastava, City of Hope Medical Center, Duarte, California.

James M. Stengle, National Institutes of Health, Bethesda, Maryland.

Ralph Stern, Carnegie Institution of Washington, Department of Embryology, Baltimore, Maryland.

Paul V. Strumia, Laboratory of Clinical Pathology, the Bryn Mawr Hospital, Bryn Mawr, Pennsylvania.

Kouichi R. Tanaka, Department of Medicine, Harbor General Hospital, Torrance, California.

xxx REGISTRANTS

Basil Tatsis, The Long Island Jewish Medical Center, Depart-ment of Hematology, Jamaica, New York.

Garson H. Tishkoff, American Red Cross, Lansing, Michigan.

Howard N. Ward, 204 Medical Arts Building, Topeka, Kansas.

Lowell Weitkamp, University of Rochester, Rochester, New York.

William L. White, 200 First St. S. W., Rochester, Minnesota.

John C. Wiltsie, Mayo Clinic, Rochester, Minnesota.

William P. Winter, Department of Human Genetics, University of Michigan, Ann Arbor, Michigan.

Alan Wright, AMA Audio News Journal, Chicago, Illinois.

Yoshihito Yawata, Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota.

Solomon J. Zak, Hematology Section, Veterans Administration Hospital, Minneapolis, Minnesota.

Alvin Zipursky, Department of Pediatrics, McMaster Univer­sity, Hamilton, Ontario, Canada.

Wolf W. Zuelzer, Children's Hospital of Michigan, Detroit, Michigan.

CO pCdZ co Hg IJ.M mM M gm % % 1J.1 ml L ACD CPD mole IJ.mole romole mg IJ.g g Kg m

em rom N hb hct rbc

ABBREVIA TrONS

oxygen oxygen pressure pressure of oxygen at which hemoglobin or red cells are half saturated with Oz carbon dioxide carbon dioxide pressure carbon monoxide mercury micromolar millimolar molar grams per 100 ml per cent microliter milliliter liter acid-citrate-dextrose preservative solution citrate-phosphate-dextrose preservative solution mole micromole millimole milligram microgram gram kilogram meters (for des ignating altitude) centimeter millimeter number in a sample hemoglobin hematocrit red blood cell

xxxi

xxxii

ATP,ADP, & AMP G-6-P NADP & NADPH GSSG & GSH G-6-PD 6-PG 6-PGD TK TA FDP DHAP GA-3-P NAD & NADH Pi 1,3-DPG 3-PG 2-PG DPG PEP PK LDH PGI PFK FA TPI

ABBREVIATIONS

adenos ine triphosphate, diphosphate and monophos­phate, respecttvely

glucose 6 -phosphate oxidized and reduced nicotinamide adenine dinucleo­

tide phosphate, respectively oxidized and reduced glutathione, respectively

glucose-6-phosphate dehydrogenase 6 -phosphogluconate 6-phosphogluconate dehydrogenase transketolase trans aldolase fructose-I, 6-diphosphate dihydroxyacetone phosphate glyceraldehyde 3-phosphate oxidized and reduced nicotinamide adenine dinucleo­tide, respectively inorganic phosphate 1,3-diphosphoglycerate 3-phosphoglycerate 2-phosphoglycerate 2,3-diphosphoglycerate phosphoenol pyruvate pyruvate kinase lactate dehydrogenase phosphoglucose isomerase phosphofructokinase fructoaldolase triosephosphate isomerase

GA-3-PD glyceraldehyde-3-phosphate dehydrogenase diphosphoglyce rate mutase 2, 3-diphosphoglycerate phosphatase 3-phosphoglycerate kinase

DPGM 2,3-DPGP 3-PGK PG-2, 3-M phosphoglycerate"-2, 3-mutase E PGM G-I-P G-l,6-DP F-6-p

enolase phosphoglucomutase glucose -l-phosphate glucose-l, 6-diphosphate fructose -6-phosphate