Table Of ContentMETHODS
OF ENZYMATIC
ANALYSIS
Edited by
H a ns U l r i ch B e r g m e y er
in collaboration with Karlfried Gawehn
Second English Edition
Translated from the Third German Edition
V o l u me 2
V e r l ag C h e m ie W e i n h e im
A c a d e m ic P r e s s, I n c. N ew Y o rk a nd L o n d on
A Subsidiary of Harcourt Brace Jovanovich, Publishers
Translated by Dermot H. Williamson, with the editorial assistance of Patricia Lund
Medical Research Council External Staff
Metabolic Research Laboratory
Nuffield Department of Clinical Medicine
The Radcliffe Infirmary Oxford, England
Editor's note
The methods published in this book have not been checked experimentally by the editor. Sole responsibility
for the accuracy of the contents of the contributions and the literature quoted therein rests with the authors.
Readers are therefore requested to direct all enquiries to the appropriate authors (author's addresses are
listed on pp. XVII-XXXII).
Editions of the book to date :
a) German editions with the title "Methoden der enzymatischen Analyse"
1. Auflage 1962, one volume
2. neubearbeitete und erweiterte Auflage 1970, two volumes
3. neubearbeitete und erweiterte Auflage 1974, two volumes
b) English editions:
1st Edition 1963, one volume
2nd printing, revised, 1965
3rd printing, 1968
4th printing, 1971
2nd Edition (translated from the Third German Edition), 1974, four volumes
With 263 figures and 546 tables.
ISBN: 3-527-25370-X (Set Verlag Chemie)
3-527-25596-6 (Vol. 2 Verlag Chemie)
0-12-091302-X (Vol. 2 Academic Press)
Library of Congress Catalog Card Number: 73-75657
©Verlag Chemie GmbH, Weinheim/Bergstr., 1974
All rights reserved (including those of translation into foreign languages). No part of this book may be
reproduced in any form - by photoprint, microfilm, or any other means - nor transmitted or translated
into a machine language without written permission from the publishers.
Registered names, trademarks, etc., used in this book, even without specific indication thereof, are not
to be considered unprotected by law.
Composition: Helmut Becker, 6232 Bad Soden, Germany
Printed in the United States of America
Preface
The first German edition of this book appeared in 1962, and the first English edition in 1963.
In the mean time the English edition has been twice reprinted in a revised form. The develop
ment of enzymatic analysis has been so overwhelming that a completely new edition of "Methods
of Enzymatic Analysis" was necessary. So in 1970 a second completely revised and enlarged
German edition appeared. The translation was started soon, but it was interrupted by the work
on a further 3rd German edition. So it was possible to edit two identical versions: the 3rd
German and the 2nd English edition. Both include the latest developments in analytical bio
chemistry.
The numerous new enzymatic methods of analysis, the advances made in the various assay
techniques, the methods based on completely new principles, the mechanization of the labora
tory and many other topics made the need for a new edition of this book more pressing.
Although many of the developments are still in progress and the new techniques are just
beginning to be accepted so that a complete picture cannot be given, it was decided to bring
out a new edition.
Like its predecessor, this edition is directed to the worker in the analytical laboratory. Once
again authors with considerable practical experience have made available their methods. The
individual contributions therefore contain the important details from a practical standpoint.
The division of the work into four parts has been maintained, because this has proved suc
cessful. The composition of the individual chapters is essentially the same. The principles on
which the measurements are based can be easily seen in the equations given in each chapter.
A prominent feature of each method is the pipetting protocol, which is printed as a Table with
data on the "Concentration in assay mixture". The accepted abbreviations for enzymes and
metabolites now require no further explanations (a list of abbreviations is to be found on
p. XXXV). In addition, every cited enzyme is defined by its system name and number of the
Enzyme Commission of the International Union of Biochemistry, Recommendations 1972. With
a few necessary exceptions only International Units are used (abbreviation U; for the "Inhibitor
Unit", the symbol is IU).
Methods which have in the mean time become obsolete or out of date have been omitted;
new methods have taken their place.
An innovation is the information on the assays for enzyme activity in Section Β "Biochemical
Reagents". The inclusion of these is at the request of many biochemists, who have previously
looked in vain for these methods in Section C.
In spite of strict editing of the individual chapters the whole book is far more than twice the size
of the first edition. In particular the Section on "Experimental Techniques" had to be extended
considerably. New but essential sections have been included in the individual chapters, though
this has not been without risk, as for example, the information on the "Accuracy and Precision
of Method". None of the authors were aware of the content of the large chapter reviewing the
"Statistical Analysis, Control and Assessment of Experimental Results". That is why the
presentation of the data in the individual methods is not uniform. The values presented mostly
concern "Precision in the Series". The presentation of the data on the reliability of enzymatic
analytical methods in a standard way only recently has become accepted as a routine.
The book had to be divided into four volumes and therefore the order of sections A to D had
to be changed. To make it easier for the reader each volume contains the complete index, the
VI Preface
list of authors and abbreviations and the complete list of contents. It is hoped that these
volumes will continue to be true laboratory books.
I wish to particularly thank all the contributors for their understanding and willing co-operation
in complying with the strict layout of the text. I thank the reviewers of the first edition for the
well-meaning and constructive criticisms, which I have taken note of in this edition. Thanks
are also due to my colleagues for numerous discussions, to Dr. D. H. Williamson for the
excellent translation and for many suggestions during this work, to my co-workers for their
help and to Verlag Chemie for the fruitful collaboration. I am especially obliged to Mr. K.
Gawehn. Without his help it would have been impossible to produce this English edition in
a form identical to the 3rd German edition.
Tutzing/Oberbayern (Germany), March 1974 Hans Ulrich Bergmeyer.
From the Preface to the 1st Edition
Today enzymes are much more widely used as analytical tools than in the past. New methods
have been worked out for the use of those enzymes which are now available in a high state of
purity, and existing techniques have been improved.
This laboratory manual contains the working directions for carefully tested procedures.
The analytical methods have been contributed by authors who have had many years of ex
perience in their particular field of study. Consequently, the reader is certain to have reliable
experimental directions which represent the latest advances in this branch of science.
Any type of laboratory can make good use of this book, since it is designed on strictly practical
lines. The individual chapters are arranged according to the substances to be determined (not
according to the enzymes used). Grouping by substrates is employed since today the reagents
are commercially available (with the exception of a few special enzymes). For these exceptions
a short resume of isolation techniques is included. The possibility of attempting the preparation
of these enzymes is then easily judged by the reader, bearing in mind the facilities available to
him
Tutzing/Oberbayern (Germany), March 1963 Hans Ulrich Bergmeyer
VI Preface
list of authors and abbreviations and the complete list of contents. It is hoped that these
volumes will continue to be true laboratory books.
I wish to particularly thank all the contributors for their understanding and willing co-operation
in complying with the strict layout of the text. I thank the reviewers of the first edition for the
well-meaning and constructive criticisms, which I have taken note of in this edition. Thanks
are also due to my colleagues for numerous discussions, to Dr. D. H. Williamson for the
excellent translation and for many suggestions during this work, to my co-workers for their
help and to Verlag Chemie for the fruitful collaboration. I am especially obliged to Mr. K.
Gawehn. Without his help it would have been impossible to produce this English edition in
a form identical to the 3rd German edition.
Tutzing/Oberbayern (Germany), March 1974 Hans Ulrich Bergmeyer.
From the Preface to the 1st Edition
Today enzymes are much more widely used as analytical tools than in the past. New methods
have been worked out for the use of those enzymes which are now available in a high state of
purity, and existing techniques have been improved.
This laboratory manual contains the working directions for carefully tested procedures.
The analytical methods have been contributed by authors who have had many years of ex
perience in their particular field of study. Consequently, the reader is certain to have reliable
experimental directions which represent the latest advances in this branch of science.
Any type of laboratory can make good use of this book, since it is designed on strictly practical
lines. The individual chapters are arranged according to the substances to be determined (not
according to the enzymes used). Grouping by substrates is employed since today the reagents
are commercially available (with the exception of a few special enzymes). For these exceptions
a short resume of isolation techniques is included. The possibility of attempting the preparation
of these enzymes is then easily judged by the reader, bearing in mind the facilities available to
him
Tutzing/Oberbayern (Germany), March 1963 Hans Ulrich Bergmeyer
Contributors
Abeles, Robert H. Ashwell, Gilbert
Graduate Department of Biochemistry National Institutes of Health
Brandeis University Department of Health, Education and
Waltham, Massachusetts 02154, USA Welfare
p. 2200 Bethesda, Maryland 20014, USA
p. 1365, 1368
Aebi, Hugo
Aw, Swee E.
Medizinisch-Chemisches Institut der
Department of Biochemistry
Universitat Bern
Faculty of Medicine
Buhlstrasse 28
Singapore 3, Republic of Singapore p. 909
CH-3000 Bern, Switzerland p. 673
Bachrach, Uriel
Anggard, Erik
Department of Bacteriology
Department of Pharmacology The Hebrew University-Hadassah
Karolinska Institute Medical School
S-10401 Stockholm 60, Sweden p. 1877 Jerusalem, Israel p. 1740, 1744
Anderson, Norman G. Bassler, Karl-Heinz
Molecular Anatomy Program Physiologisch-Chemisches Institut der
Oakridge National Laboratory Johannes Gutenberg-Universitat
Oakridge, Tennessee 37830, USA D-6500 Mainz, Germany p. 1381
and the Molecular Anatomy Institut,
P.O. Box 17 Baginski, Eugene S.
Oakridge, Tennessee 37830, USA p. 213
St. Joseph Mercy Hospital
Pontiac, Michigan 48053, USA p. 876
Appel, Walter
Beaucamp, Klaus
Zentrallaboratorium der
St.-Vincentius-Krankenhauser Boehringer Mannheim GmbH
Sudendstrasse 32 Biochemica Werk Tutzing
D-7500 Karlsruhe 1, Germany p. 949, D-8132 Tutzing/Obb., Germany
950, 954, 958, 964, 967, 978, 986,1041, 1058 p. 523, 1656
Bechtler, Gunter
Aprison, Μ. H. Eppendorf Geratebau
Section of Neurobiology Netheler & Hinz GmbH
The Institute of Psychiatric Research Barkhausenweg 1
Indiana University Medical Center D-2000 Hamburg 63, Germany
Indianapolis, Indiana 46202, USA p. 1690 p. 611, 733, 758, 1106
XVIII Contributors
Bergmeyer, Hans Ulrich Boulanger, Paul
Boehringer Mannheim GmbH Laboratoire de Chimie Biologique
Biochemica Werk Tutzing Faculte de Medicine et Pharmacie
D-8132 Tutzing/Obb., Germany p. 94,95, Lille, France p. 1648
103, 121, 131, 158, 221, 308, 417, 425, 523,
557, 574, 579, 590, 613, 624, 727, 735, 739, Brand, Karl
742, 752, 760, 764, 784, 864, 1100, 1149, Max-Planck-Institut
1172, 1176, 1196, 1205, 1222, 1233, 1243, fur Ernahrungsphysiologie
1304, 1323, 1492, 1496, 1506, 1517, 1520, Rheinlanddamm 201
1528, 1538, 1577, 1643, 1696, 1704, 1772, D-4600 Dortmund, Germany p. 396,
1786, 1791, 1813, 1919, 1951, 1967, 2008, 399, 710
2078, 2097, 2127, 2132, 2246
Brin, Myron
Department of Biochemical Nutrition
Bernt, Erich
Hoffmann-La Roche Inc.
Boehringer Mannheim GmbH Nutley, New Jersey 07110, USA p. 703
Biochemica Werk Tutzing
D-8132 Tutzing/Obb., Germany p. 158, Brock, David J. H.
308, 557, 574, 579, 590, 613, 624, 727, 735,
Department of Human Genetics
739, 742, 752, 760, 764, 774, 784, 864, 868,
Western General Hospital
1100, 1172, 1176, 1196, 1201, 1205, 1215,
Edinburgh 4, Scotland p. 1844
1304, 1499, 1506, 1577, 1643, 1696, 1704,
1772, 1890, 1951, 2246
Brosnan, John T.
Department of Biochemistry
Memorial University of
Beutler, Hans-Otto
Newfoundland,
Boehringer Mannheim GmbH St. Johns,
Biochemica Werk Tutzing Newfoundland, Canada p. 2266
D-8132 Tutzing/Obb., Germany p. 523
1314, 1708
Brown, David H.
Bevill, Rardon D. Department of Biological Chemistry
School of Medicine
Department of Molecular Biology
Washington University
Albert Einstein College of Medicine
St. Louis, Missouri 63110, USA
1300 Morris Park Avenue
p. 1251, 1257
Bronx, New York 10461, USA
p. 2209, 2217 Brown, Joseph G.
Department of Pharmacology
Birchmeier, Heidi School of Medicine
Laboratoire Central Washington University
Hopital Cantonal St. Louis, Missouri 63110, USA p. 1565
CH-1011 Lausanne, Switzerland p. 721
Bucher, Theodor
Institut fur Physiologische Chemie und
Bodansky, Oscar Physikalische Biochemie der
Sloan Kettering Institute Universitat Munchen
for Cancer Research Goethestrasse 33
New York, Ν. Y. 10021, USA p. 768 D-8000 Munich, Germany p. 254
Contributors XIX
Buttner, Hannes Dahl, Katharina von
Medizinische Hochschule Hannover Boehringer Mannheim GmbH
Institut fur Klinische Chemie Abt. Stoffwechsel
Roderbruchstrasse 101 Sandhofer Strasse
D-3000 Hannover-Kleefeld, Germany D-6800 Mannheim 31, Germany p. 819
p. 318
Ceriotti, Giovanni Dahlqvist, Arne
Laboratorio Centrale di Analisi Department of Nutrition,
Ospedale Civile di Padova Chemical Center
1-35100 Padova, Italia p. 691 University of Lund
S-22007 Lund 7, Sweden p. 916
Chase, James F. Α.*
Department of Biochemistry Decker, Karl
University of Cambridge Albert Ludwigs-Universitat Freiburg
Tennis Court Road Medizinische Fakultat
Cambridge, England p. 1758 Biochemisches Institut
Hermann-Herder-Strasse 7
D-7800 Freiburg i. Br., Germany p. 1127,
Coddington, Alan
1228, 1988, 2001, 2017, 2022, 2172, 2221,
School of Biological Sciences 2225
University of East Anglia
Norwich, NOR 88 C, England
p. 1928, 1932 Dubach, Ulrich C.
Medizinische Universitatspoliklinik
Cohen, Patricia S.
Abt. fur Innere Medizin
Medical Laboratory Assoc. Hebelstrasse 1
Birmingham, Alabama 35233, USA CH-4056 Basel, Switzerland p. 699
p. 793
Cooperman, Jack M.
Eberhard, Arnold
Department of Pediatrics
Klinisch-Chemisches Institut der
Hematology and Nutrition Laboratories
Rhein.-Westf. Techn. Hochschule
New York Medical College
D-5100 Aachen, Germany p. 1165
New York, Ν. Y. 10029, USA p. 1556
Egami, Fujio
Czok, Rudolf
Sandoz Forschungsinstitut GmbH Mitsubishi-Kasei Institute
of Life Sciences
Brunnerstrasse 59
11, Minamiooya
A-1235 Wien-Liesing, Austria
Machida-shi,
p. 1424, 1446
Tokyo, Japan p. 2260
Dagley, Stanley
Department of Biochemistry
Eggleston, Leonard V.*
University of Minnesota
St. Paul, Minnesota 55101, USA Metabolic Research Laboratory
p. 1562 Nuffield Department of Clinical Medicine
The Radcliffe Infirmary
* deceased Oxford, England p. 1308
XX Contributors
Eggstein, Manfred Friebe, Ursula
Medizinische Universitatsklinik (IV) Biochemisches Institut der
Otfried-Muller-Strasse Universitat Freiburg
D-7400 Tubingen, Germany /?. 1825 Hermann-Herder-Strasse 7
D-7800 Freiburg i. Br., Germany
p. 1935
Eisenberg, jr., Frank
Fried, Lygia W.
The National Institutes of Health
Department of Biochemistry
Public Health Service
Creighton University
United States Department of Health,
School of Medicine
Education and Welfare
Omaha, Nebraska 68131, USA
Bethesda, Maryland 20014, USA
p. 644, 1945
p. 1337
Fried, Rainer
Fasold, Hugo Department of Biochemistry
Creighton University
Institut fur Biochemie der
School of Medicine
J. W. Goethe-Universitat Frankfurt
Omaha, Nebraska 68131, USA
Sandhofstrasse
p. 644, 1945
D-6000 Frankfurt/M.-Niederrad, Germany
Friedmann, Herbert C.
p. 1625, 1640
The University of Chicago
Fishman, William H. Department of Biochemistry
School of Medicine Chicago, Illinois 60637, USA p. 824,
Tufts University 1963, 2179, 2182
Boston, Massachusetts 02111, USA
p. 929 Fritsch, Wolf-Peter
I. Medizinische Klinik der Universitat
Foa, Piero P.
Moorenstrasse 5
Department of Research D-4000 Dusseldorf 1, Germany p. 1046
Sinai Hospital
Detroit, Michigan 48235, USA p. 876 Fritz, Hans
Institut fur Klinische Chemie und
Forster, Edith Klinische Biochemie der Universitat
Nussbaumstrasse 20
II. Med. Universitatsklinik der
D-8000 Munich, Germany p. 1064
Johannes-Gutenberg-Universitat
Langenbeckstrasse 1
Fromm, Herbert J.
D-6500 Mainz, Germany p. 1923
Iowa State University of Science
and Technology
Forster, Georg Department of Biochemistry and
Schweizerische Pflegerinnenschule Biophysics
Samariterstrasse 5 Ames, Iowa 50010, USA p. 1354
CH-8032 Zurich, Switzerland p. 784
Gale, Ernest F.
University of Cambridge
Frei, Jorg
Department of Biochemistry
Laboratoire Central Sub.-Dept. of Chemical Microbiology
Hopital Cantonal Cambridge, CB 2 1 QW, England
CH-1011 Lausanne, Switzerland p. 721 p. 1662
Contributors XXI
Garland, Peter Bryan Goldberg, Nelson D.
Department of Biochemistry Department of Pharmacology
University of Dundee, University of Minnesota
Dundee, Scotland p. 1981,1993, 2015 Medical School
Minneapolis, Minnesota 55455, USA
p. 1573, 1600, 1608
Gawehn, Karlfried
Boehringer Mannheim GmbH Graham, Jr., L. T.
Biochemica Werk Tutzing Institute of Psychiatric Research
D-8132 Tutzing/Obb., Germany p. 158, Indiana University Medical Center
425, 1263, 1492, 1496, 2172, 2234, 2239 Indianapolis, Indiana 46207, USA
p. 1690
Gerlach, Ulrich Grassl, Marianne
Medizinische Klinik und Poliklinik der Boehringer Mannheim GmbH
Westfalischen Wilhelms-Universitat Biochemica Werk Tutzing
D-4400 Munster/Westf., Germany D-8132 Tutzing/Obb., Germany /?. 308,
p. 31, 569, 871 425, 1268, 1296, 1331, 1682, 1686, 2073,
2145, 2149, 2153, 2158, 2162, 2166, 2168
Giang, Paul A.
Analytical Chemistry Laboratory
Greenberg, Elaine
Agr. Environmental Quality Institute
University of California
Beltsville, Maryland 20705, USA
Department of Biochemistry
p. 2249
and Biophysics
Gibbs, Martin Davis, California 95616, USA p. 2204
Department of Biology
Brandeis University Greiling, Helmut
Waltham, Massachusetts 02154, USA
Klinisch-Chemisches Institut der
p. 409, 881, 1385
Rhein.-Westf. Techn. Hochschule
Gitzelmann, Richard D-5100 Aachen, Germany p. 1157, 1165
Labor fur Stoffwechselforschung der
Universitats-Kinderklinik Gruber, Wolfgang
Steinwiesstrasse 75 Boehringer Mannheim GmbH
CH-8032 Zurich, Switzerland p. 1291 Biochemica Werk Tutzing
D-8132 Tutzing, Obb., Germany p. 1323,
1890, 2078, 2097, 2127
Giusti, Guiseppe
Clinica Delle Malattie Infetive
Gundlach, Gerd
deirUniversita
I'Facolta di Medicina e Chirurgia Universitatsklinik, Urologie
Via D. Cotugno, 1 (Osp. Gesu e Maria) Landeskrankenhaus
1-80135 Napoli, Italia p. 1086, 1092 D-6650 Homburg/Saar, Germany
p. 1625
Goedde, Heinz W. Gutmann, Ingeborg
Institut fur Humangenetik der Boehringer Mannheim GmbH
Universitat Biochemica Werk Tutzing
Butenfeld 32 D-8132 Tutzing/Obb., Germany p. 774,
D-2000 Hamburg 54, Germany 1149, 1172, 1185, 1323, 1464, 1499, 1517,
p. 1394, 1514 1585, 1791
