Methods of Biochemical Analysis, Volume 16

449 Pages·1968·13.468 MB·English

METHODS OF BIOCHEMICAL ANALYSIS
16
Volume
I
Advisory Board
W. E. COHN, Oak Ridge National Laboratory, Tennessee
R. CONSDEN, The Canadian Red Cross Memorial Hospital, Taplow, Maiden-
head, Berkshire, England
J. GROSS, Department of Experimental Medicine and Cancer Research, Hebrew
University Hadassah Medical School, Jerusalem, Israel
H. HOLTER, Carlsberg Laboratory, Copenhagen, Denmark
J. K. N. JONES, Department of Organic Chemistry, Queen's University, Kingston,
Ontario, Canada
C. G. KING, St. Luke's Hospital Center, New York, New York
H. A. LARDY, Deparcmem of Biochemistry, University of Wisconsin, Madison
H. C. LICHSTEIN, Departmew of Microbiology, University of Cincinnati,
Cincinnati, Ohio
B. G. MALMSTRGM, Department of Biochemistry, University ofC&berg, Sweden
A. MEISTER, Department of Biochemistry, Tufts University School of Medicine,
Boston, Massachusetts
0. MICKELSEN, Department of Food and Nutrition, Michigan State University,
East Lansing, Michigan
J. ROCHE, Biochimie Gkdrale et Cornpar&, Coll2ge de France, Paris, France
A. TISELIUS, Institute of Biochemistry, University of Uppsala, Sweden
B. L. VALLEE, Biophysics Research Laboratory, Departmew of Biological Chemis-
try, H-d Medical School, Boston, Massachusetts
D. D. VAN SLYKE, Brwkhaven Nationul Laboratory, Upton, Long Island, New
York
METHODS OF
BIOCHEMICAL ANALYSIS
Edited by DAVID GLICK
Stanford University Medical school
Palo Alto, California
16
VOLUME
INTERSCIENCE PUBLISHERS adivision of
John Wiley & Sons, New London Sydney
York 0 Toronto
Copyright @ 1968, by John Wiley & Sons, Inc.
All rights reserved. No part of this book may be reproduced by any means,
nor transmitted, nor translated into a machine language without the written
permission of the publisher.
Library of Congress Catalog Card Number 547232
SBN 470 30750
PRINTED IN THE UNITED STATES OF AMERICA
METHODS OF BIOCHEMICAL ANALYSIS VOLUME 16
PREFACE TO SERIES
THE
Annual review volumes dealing with many different fields of science
have proved their value repeatedly and are now widely used and well
established. These reviews have been concerned primarily with
the results of the developing fields, rather than with the techniques
and methods employed, and they have served to keep the ever-
expanding scene within the view of the investigator, the applier, the
teacher, and the student.
It is particularly important that review services of this nature
should now be extended to cover methods and techniques,.because it
is becoming increasingly difficult to keep abreast of the manifold
experimental innovations and improvements which constitute the
limiting factor in many cases for the growth of the experimental
sciences. Concepts and vision of creative scientists far outrun that
which can actually be attained in present practice. Therefore an
emphasis on methodology and instrumentation is a fundamental
need in order for material achievement to keep in sight of the advance
of useful ideas,
The current volume is another in this series which is designed to try
to meet the need in the field of biochemical analysis. The topics to
be included are chemical, physical, microbiological, and if necessary,
animal assays, as well as basic techniques and instrumentation for
the determination of enzymes, vitamins, hormones, lipids, carbo-
hydrates, proteins and their products, minerals, antimetabolites, etc.
Certain chapters will deal with well-established methods or tech-
niques which have undergone sufficient improvement to merit
re-
capitulation, reappraisal, and new recommendations. Other chapters
will be concerned with essentially new approaches which bear promise
of great usefulness. Relatively few subjects can be included in any
single volume, but as they accumulate these volumes should comprise
a self-modernizing encyclopedia of methods of biochemical analysis.
By judicious selection of topics it is planned that most subjects of
current importance will receive treatment in these volumes.
V
vi PREFACE
The general plan followed in the organization of the individual
chapters is a discussion of the background and previous work, a
critical evaluation of the various approaches, and a presentation of
the procedural details of the method or methods recommended by
the author. The presentation of the experimental details is to be
given in a manner that will furnish the laboratory worker with the
complete information required to carry out the analyses.
Within this comprehensive scheme the reader may note that the
treatments vary widely with respect to taste, style, and point of view.
It is the Editor’s policy to encourage.individua1 expression in these
presentations because it is stifling to originality and justifiably annoy-
ing to many authors to submerge themselves in a standard mold.
Scientific writing need not be as dull and uniform as it too often is.
In certain technical details, a consistent pattern is followed for the
sake of convenience, in the form used for reference citations and
as
indexing.
The success of the treatment of any topic will depend primarily on
the experience, critical ability, and capacity to communicate of the
author. Those invited to prepare the respective chapters are scien-
tists who either have originated the methods they discuss or have had
intimate personal experience with them.
It is the wish of the Advisory Board and the Editor to make this
series of volumes useful as possible and to this end suggestions will
as
always be welcome.
DAVIDG LICK
May 1968
METHODS OF BIOCHEMICAL ANALYSIS VOLUME 16
CONTENTS
The Isotope Derivative Method in Biochemical Analysis. By
J. K. Whitehead and H. G. Dean, The Tobacco Research
Council Laboratories, Harrogate, Yorkshire, England. . 1
Bioluminescence Assay : Principles and Practice. By Bernard L.
Strehler, Department of Biological Sciences, University
of Southern California, Los Angeles, California, and
Aging Research Laboratory, Veterans Hospital,
Baltimore, Maryland. ......... ........... 99
Utilization of Automation for Studies of Enzyme Kinetics. By
Morton K. Schwartz and Oscar Bodansky, Sloan-
Kettering Institute for Cancer Research and Memorial
Hospital for Cancer and Allied Diseases, New York,
New York... .... .............. 183
Enzymatic Synthesis and Hydrolysis of Cholesterol Esters.
By George V.V ahouny and C. R. Treadwell, Department
of Biochemistry, The George Washington University
School of Medicine, Washington,D . C.. ........ 219
Determination of Histidine Decarboxylase Activity. ByR ichard
W.S chayer, Research Center, Rockland State Hospital,
Orangeburg, New York.. ..... 273
+
The Estimation of Total (Free Conju
and Some Catecholamine Metabolites in Human
Urine. By H. Weil-Malherbe, Division of Special Mental
Health Research Programs, National Institute of
Mental Health, St. Elizabeths Hospital, Washington,
D. C.. . .................................. 293
The Automated Analysis of Absorbent and Fluorescent Sub-
stances Separated on Paper Strips. By Alan A.
Boulton, Psychiatric Reseach Unit, University Hospital,
Saskatoon, Saskatchewan, Canada.. ........ 327
Author Index. .......................................... 395
Subject Index. .......................................... 409
Cumulative Index. ........................ ....... 429
vii
Methods of Biochemical Analysis, Volume 16
Edited by David Glick
Copyright © 1968 John Wiley & Sons, Inc.
METHODS OF BIOCHEMICAL ANALYSIS VOLUME 16
The Isotope Derivative Method
in Biochemical Analysis
J. K. WHITEHEADAN D H. G. DEAN*T he Tobacco Research Council
Laboratories, Harrogate, Yorkshire, England
I. Introduction .................................................. 2
1. General.. . . . . . . . . . . . . . 2
11. Methods and Materials.. . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1. Introduction.. . . . . . . . . . . . . . . . . . . . 5
2. General Principles.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
5
6
7
3. Factors Affecting Accuracy.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
A. Purity of the Materials. . . . . . . . . . . . 11
B. Complete Equilibration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
C. Purity of Final Samples.. . . . . . . 12
D. Unstable Substances. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
E. Addition of Tracer Compound in Double Isotope Deriva-
tive Techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
4. Counting Methods.. . . . . . . . . . . . . . 13
................................. 13
13
14
16
16
16
................................... 16
19
28
31
.......................... 31
B. Chromatography.. . . . . . . . 32
34
D. Isotope FractionLtion. . . . . 36
37
1. Amino Acids, Proteins, and Peptides. . . . . . . . . . . . . . . . . . . . . . . . 37
* Department of Pharmacology, School of Medicine, Led,Y orkshire, England.
I
2 J. WHITEHEAD AND H. 0. DEAN
K.
A. Amino Acid Composition Analysis. . 37
B. End Group Sequence Analysis of Pro 44
2. Steroids .................................................. 52
A. Introduction.. .. ...................... 52
54
59
D. Determinations Using Thiosemicarba ........... 61
E. Determinations Using Other Labeled 83
3. Other Compounds.. ....................................... 86
A. Introduction. .............. 86
charides. ................... 87
88
I). Alkaloids .......................................... 90
E. Biochemical Intermediates. ..........................
90
F. Pesticides.. ........................................ 91
IV. Conclusion. . .... .. 93
References .................................................... 93
I. INTRODUCTION
1. General
Isotopes are now common tools in biochemistry, and their use has
greatly facilitated the recent advances in the understanding of
biochemical mechanisms and metabolic pathways. Analytical tech-
niques have made a similar advance, but this has been due to the
further development of physiochemical methods rather than isotopic
techniques. Thus the sensitivity of determination techniques in-
creased from milligram to microgram amounts, whereas the most
recently developed, isotopic analytical methods have a sensitivity in
the nanogram range.
Physiochemical analytical techniques have many disadvantages,
especially with material of biological origin. These arise mainly
from the difficulty in isolating a material which has to be determined
in a pure state, since the accuracy of the technique usually depends
on the purity of the sample. Advances in chromatographic purifi-
cation techniques during the past decade have greatly simplified
these purifications, but they are still laborious and time consuming.
The greatest disadvantage associated with purification of material
for analysis by physiochemical methods is the necessity for a quanti-
tative, or accurately reproducible, recovery. This cannot be satis-
ISOTOPE DERIVATIVE METHOD BIOCHEMICAL ANALYSIS 3
IN
factorily overcome in any analytical technique of this kind, with the
possible exception of gas-liquid chromatography, where internal
standards can be used. Even in this case the compound used is not
the same as the unknown, although it may be very similar chemically.
Thus the method assumes that the internal standard behaves in
exactly the same way the unknown throughout the initial isolation
as
and purification or that the ratio of recovery of internal standard to
recovery of unknown does not vary with the concentration of either.
Such losses can only be overcome in a reliable, reproducible, and
accurate way by the use of isotopic tracer compounds. It is the use
of such tracers which gives isotopic techniques, and particularly
isotope-derivative techniques, their advantages. The simple use of
tracers still does not overcome the necessity for the isolation of a
chemically pure sample for assay by some physiochemical method.
The application of a double isotope derivative technique, where one
isotope gives a measure of the amount of the unknown while the
second allows correction for any losses during the analytical pro-
cedure can overcome this, since such a method requires only the
determination of radioactivity. Thus the criterion of chemical
purity is replaced by one of radiochemical purity. The only labeled
compound in the final sample assay must be the one of interest,
although other unlabeled compounds may be present, providing that
they do not interfere with the counting procedure. For this reason
it is possible to add milligram amounts of carrier to a nanogram
sample of doubly labeled derivative in order to facilitate the radio-
chemical purification without altering the accuracy or sensitivity of
the method in any way.
Double isotope techniques have a further advantage over other
analytical methods, because their sensitivity depends on the specific
activity of the labeled compound available. Recent advances in the
techniques of preparation, purification, and storage of these labeled
materials have made them readily available at high specific activity.
The sensitivity of analytical techniques has, therefore, been increased
to the nanogram level which is now necessary in the advancing field of
biochemical and physiological research. The study of hormones is a
field in which this technique has found particular application.
Isotopes available from atomic reactors shortly after World War I1
were soon utilized in biochemical studies, and in the succeeding 20
years a large number of analytical methods were developed. These