Clinical Chemistry

291 Pages·1976·6.12 MB·English

by  Donald T. Forman and Richard W. Mattoon (Eds.)

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6- Clinical Chemistry
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Clinical Chemistry
Donald T. Forman, EDITOR
Evanston Hospital and Northwestern
University School of Medicine
Richard W. Mattoon, EDITOR
1 Abbott Laboratories
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9 Based on the
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02 Annual Lecture Series
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Chicago, Ill.,
February 13-April 3, 1975
36
ACS SYMPOSIUM SERIES
AMERICAN CHEMICAL SOCIETY
WASHINGTON, D. C. 1976
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e Library of Congress Data
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J Clinical chemistry.
ate: (ACS symposium series; 36 ISSN 0097-6156)
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n Includes bibliographies and index.
o 1. Chemistry, Clinical—Addresses, essays, lectures.
ati I. Forman, Donald T., 1932- . II. Mattoon, Rich
ublic aCrhdi cWagiol bSuerc, ti1o9n1.2 - IV. A. mIIeIr.i caAnm eAriscsaonci aCtihoenm iocafl CSloicnieictyal.
P Chemists. Chicago Section. V. Series: American Chemi

cal Society. ACS symposium series; 36.
[DNLM: 1. Chemistry, Clinical—Congresses. QY90
C641 1975]
RB40.C57 616.07'56 76-49983
ISBN 0-8412-0345-8 ACSMC8 36 1-293
Copyright © 1976
American Chemical Society
All Rights Reserved. No part of this book may
be reproduced or transmitted in any form or by
any means—graphic, electronic, including photo
copying, recording, taping, or information storage
and retrieval systems—without written permission
from the American Chemical Society.
PRINTED IN THE UNITED STATES OF AMERICA
ACS Symposium Series
Robert F. Gould, Editor
Advisory Board
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6.f Kenneth B. Bischoff
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76- Ellis K. Fields
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1, Philip C. Kearney
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Joseph V. Rodricks
Roy L. Whistler
Aaron Wold
FOREWORD
01 The ACS SYMPOSIUM SERIES was founded in 1974 to provide
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w a medium for publishing symposia quickly in book form. The
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3 format of the SERIES parallels that of the continuing ADVANCES
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6- IN CHEMISTRY SEMES except that in order to save time the
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19 papers are not typeset but are reproduced as they are sub
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b mitted by the authors in camera-ready form. As a further
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02 means of saving time, the papers are not edited or reviewed
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0. except by the symposium chairman, who becomes editor of
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oi: the book. Papers published in the ACS SYMPOSIUM SERIES
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19 major part and include reports of research as well as reviews
e 1, since symposia may embrace both types of presentation.
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PREFACE
HT his book is the result of the eigth annual series of lectures for con
tinuing education sponsored by the Chicago Section of the American
Chemical Society. It is also the first volume in the ACS Symposium
Series to be sponsored by a local section of the ACS. The purpose of
the lecture series and this volume is to present topics that have a clinical
chemical basis but are also of interest to specialists in other clinical
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0 areas. Indeed, several contributors to this volume represent the disci
pr
6. plines of pediatrics, endocrinology, and hematology. The material in
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00 cluded is appropriate to both the clinical aspects of the subject and to
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7 analytical and technological advances. Chapters in this book are con
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k- cerned with etiology of disease and diagnosis as well as technical ad
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1/ vances which can be applied more practically in the very near future.
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1 We believe the chapters are excellent examples of the integral part that
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oi: 1 the clinical chemist plays in modern medicine. The chapters on com
d petitive protein binding assays and drug interference in laboratory test
76 | ing were not originally presented as lectures but were added because
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1, 1 of the current interest in these subjects.
ne The editors hope that this book will be as enlightening and useful
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e: J to the reader as the lecture series was to the participating audience.
at It is a great pleasure to thank our contributors and publisher for their
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on excellent cooperation, without which this volume would not have been
cati possible. A special note of grateful acknowledgment goes to Florence
ubli Forman and Debra H. Forman for their meticulous editorial assistance.
P

Evanston Hospital and Northwestern University
School of Medicine
Chicago, Ill. DONALD T. FORMAN
Abbott Laboratories
North Chicago, Ill. RICHARD W. MATTOON
September 20, 1976
ix
1
Separation and Characterization of Hemoglobins
TITUS H. J. HUISMAN
Departments of Cell & Molecular Biology and Medicine, Laboratory of Protein
Chemistry, Comprehensive Sickle Cell Center, Medical College of Georgia,
and Veterans Administration Hospital, Augusta, Ga. 30902
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36. Human red cells are Ideally suited for studies of genetic
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0 disorders mainly because they can easily and at any degree of
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7 frequency be obtained in relatively large quantities. It is,
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k-1 therefore, not surprising that in the course of time many
1/b genetic disorders involving this cell have been detected. It
02 is interesting that some of these result in a severe hemolytic
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0. anemia, while other genetically imposed changes might not
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oi: affect the life nor the function of the cell. An excellent
d example is the hemoglobin molecule; in some variants the
6 | chemical change does not influence the properties of this
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1 protein, whereas in others the change occurs at a critical
e 1, site affecting its stability and/or functional properties.
un Identification and characterization of these variants have
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e: become routine procedures in some laboratories. The results
Dat of these studies have led to extensive investigations of
n patients with unexplained hereditary hemolytic anemias, of
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ati structure-function relationships of normal hemoglobins and of
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bli variants found in patients with an unexplained alteration in
Pu their oxygen transport system, of the different types of

genetic polymorphism, and of the incidence of selected variants
in several populations. It therefore appears appropriate to
summarize general aspects of the normal hemoglobins and of
selected hemoglobin variants, to review methods used to identify
and separate these proteins, and to discuss some procedures
useful in the characterization of possible chemical changes in
this molecule.
Normal and Abnormal Hemoglobins
Hemoglobin is a large complex protein molecule; the atomic
model of oxyhemoglobin at 2.8 A resolution has been reported in
1968 by Perutz and coworkers, and a similar model of deoxy-
hemoglobin in 1970 (1,2). The molecule is a spheroid, approx-
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2 CLINICAL CHEMISTRY
Imately 65 λ X 55 Χ X 50 It is composed of four polypeptide
chains each resembling quite closely the myoglobin chain. The
three dimensional structure of the subunits is held together by
weak noncovalent bonds. The polar amino acid side chains are
in contact with the solvent, and the nonpolar residues are
located in the interior of the molecule or in regions which
form the contacts between chains. The heme group is located
in a pocket in each chain; residues in contact with heme are
invariable Ot.e. are the same in different mammalian hemoglo­
bins) and the bonds between heme and chain are hydrophobic
interactions. Contacts between like chains (α-α; 3-3) are
limited to salt bridges involving the terminal residues but
contacts between unlike chains (a-3) are many. The largest
number of contacts is between the αχ and 3l (34 residues with
1 110 atoms being within 4 X). These are primarily contributed
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h0 by amino acid residues of the B, G, and Η helices. The αΐ-&2
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6. contact is formed by 19 residues (in helices C, F, and G) and
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00 is of importance for the functional interactions between sub-
6- units. The effect of deoxygenation causes an alteration in
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19 distance between iron atoms. The most pronounced change is in
bk- the 31-32 distance, which increases 6 to 7 &. The αχ-3\ contact
21/ undergoes only slight change whereas drastic changes seem to
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0.1 occur in the 04-32 contact. Figures 1 and 2 (from reference 3)
1 give further details on the structures of the α-and 3-chains of
oi: Hb-A and list the residues that participate in the contacts
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19 The major type of human adult hemoglobin (Hb-Ao) is com­
1, posed of two pairs of identical chains, and its formula can be
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n written as ct232- The primary structure of the two chains has
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e: J been known for several years. The second adult hemoglobin is
at Hb-A2 and it constitutes 2.5 to 3.0 percent of the total hemo­
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n globin. Hb-A2 also consists of four polypeptide chains, two
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ati of these are a-chains and the other two (the δ-chains) differ
blic from the 3-chains of Hb-Ao by amino acid substitutions in 10
Pu positions. The formula of Hb-A2 can be written as 0262· A
small amount (less than one percent) of fetal hemoglobin (Hb-F)
is also present in red cells of the human adult. The electro-
phoretically fast moving fraction Hb-A3 (which separates as
Hb-Ai by chromatography) is a mixture of at least four distinct
minor fractions. One of these fractions (Hb-Aj) is present in
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adult red cell hemolysates for 3.5 to 4.5 percent and is com­
posed of two normal α-chains and two 3-chains each with the
NH2 terminal group being blocked by a carbohydrate. A second
fraction (Hb-Ai) has been identified as a mixed disulfide of
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one molecule of Hb-Ao and two glutathione residues. Increased
quantities of Hb-Ai are found in aged red cell hemolysates.
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Fetal hemoglobin is the major hemoglobin of the red cells
of the fetus and the newborn. It has the characteristic
property of being resistant to denaturation by alkali. Hb-F is
composed of two α-chains (the same as in Hbs A and A2) and two
HUISMAN Separation and Characterization of Hemoglobins
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Advances in Clinical Chemistry
Figure 1. Two-dimensional presentation of the a-chain of human hemoglobin.
·, residues in contact with the heme group; ©, residues that participate in the
<χι-βι contact; ©, residues that participate in the αι-βι contact (3).