Methods of Biochemical Analysis, Volume 27

543 Pages·1981·13.854 MB·English

METHODS OF BIOCHEMICAL ANALYSIS
Volume 27
Advisoy Board
N. G. ANDERSON, Division uf Biological and Medical Research, Argonne National
Laboratories, Illinois
TH. BUCHER, Institute of Physaologzcal Chemistry, a d Physical Biochaistry and Cell
Biology, University of Munich, West Germany
W.E . COHN, Oak Rzdge National Laboratory, Trnnessee
P. DOUZOU, Institute of Physico-Chemical Biology, Edmond de Rothschild Foundation, Paris,
France
R. W. ESTABROOK, Department of Biochemistry, Southwestern Medical School, Dallas, Texas
S. GATT, Department of Biochemistry, Hebrew University-Hadmmh Medical School, Jem-
salem, Israel
I. C. GUNSALUS, Department of Biochemistry, University of Illinois, Urbana, Illinois
H. A. 0. HILL, Department of Inorganic Chemistry, University of Oxford, England
J. H. R. KAGI, Biochemical Institute, University of Zurich, Switzerland
B. G. MALMSTROM, Department of Biochemistry and Biophysics, Chalmers University of Tech-
nology and University of Goteborg, Sweden
A. MEISTER, Department ofBiochemist6, Cornell Medical College, New York,N ew York
R. S. MELVILLE, Bureau o f Medical Seruices, Food and Drug Administration, Silver Spring,
Maryland
M. OTTESEN, Carlsberg Laboratory, Copenhagen, Valby, Denmark
J. E. SCOTT,D epartment o f Medical Biochemistry, University o f Manchester, England
E. C. SLATER, Laboratory of Biochemistry, B. C. P. Jansen Institute, University of Amsterdam,
The Netherlands
B. L. VALLEE, Biophysics Research Laboratory, Department of Biolopcal Chemistry, Haruard
Medical School, Boston, Massachusetts
P. VENETIANER, Institute of Biochemistry, Hungarian Academy of Sciences, Szeged,
Hungary
K. YA GI, Institute o f Biochemistry, University of Nagoya Medical School, Japan
METHODS OF
BIOCHEMICAL ANALYSIS
Edited by DAVID GLICK
Cancer Biology Research Laboratory
Stanford University Medical Center
Stanford, Cal$orniu
VOLUME
27
An Interscience@P ublication
J 0 H N W I L & S 0 N S ,N ew York. Chichester- Brisbane Toronto
E Y
An Interscience@P ublication
Copyright @ 1981 by John Wiley & Sons, Inc.
All rights reserved. Published simultaneously in Canada.
Reproduction or translation of any part of this work
beyond that permitted by Sections 107 or 108 of the
1976 United States (hpyright Art without the permission
of rhe copyright owner is unlawful. Requests for
Fermission or further information should be addressed
to the Permissions Department,J ohn Wiley & Sons, Inc.
Library of Congress Catalogue Card Number: 54-7232
ISBN 0-47 I-06503-X
Printed in the United States of America
I 0 9 8 7 6 5 4 3 2 1
METHODS OF BIOCHEMICAL ANALYSIS Volume 27
PREFACE
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, not only with the results
in the developing fields, but also with the techniques and methods
employed, and they have served to keep the ever-expanding scene within
the view of the investigator, applier, the teacher, and the student.
It is particularly important that review services of this nature should
have included the area of methods and techniques, because it is becom-
ing increasingly difficult to keep abreast of the manifold experimental in-
novations 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 volumes in this series are 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, carbohydrates, proteins and their products,
minerals, antimetabolites, etc.
Certain chapters will deal with well-established methods or techniques
which have undergone sufficient improvement to merit recapitulation,
reappraisal, and new recommendations. Other chapters will be con-
cerned 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 judi-
cious selection of topics it is planned that most subjects of current
importance will receive treatment in these volumes.
The general plan followed in the organizatioa 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 pro-
cedural details of the method or methods recommended by the author.
V
vi PREFACE
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 analysis.
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 individual expression in these pre-
sentations because it is stifling to originality and justifiably annoying 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 conveni-
ence, as in the form used for reference citations and indexing.
The success ofthe 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 scientists 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 as useful as possible and to this end suggestions will be always
welcome.
DAVIDG LICK
~~~ ~~ ~ ~~
METHODS OF BIOCHEMICAL ANALYSIS Volume 27
CONTENTS
Recent Developments in Amino Acid Analysis by Gas-Liquid
Chromatography. By S.L. MacKenzie, P r a i ~R egional
Laboratory, National Research Council of Canada, Saskatoon,
Saskatchewan, Canada ............................................................... 1
Hydrophobic Interaction Chromatography of Proteins, Nu,cleic
Acids, Viruses, and Cells on Noncharged Amphiphilic
Gels. By Stellan Hjmtin, Institute of BiochemisQ, University of
Uppsala, Biomedical Center, Uppsala, Sweden ............................. 89
Immunological Techniques for Studies on the Biogenesis of
Mitochondria1 Membrane Proteins. By Sigurd Werner
and Walter Sebald, Institut fur Physiologzsche Chemie, Physika-
lkhe Biochemie und Zellbiolop der Universitiit Munchen und
Gesellschaft fur Biotechnologische Forschung mbH, Braun-
schweig-Stockheim, Germany ...................................................... 109
Thermodynamic Flow Methods in Biochemistry: Calorimetry,
Densimetry, and Dilatometry. By Camel Jolicoeur,
Department of Chemistry, Universiti de Sherbrooke,
Sherbrooke, Qutbec, Canada ..................................................... 171
Ion Binding in Biological Systems Measured by Nuclear
Magnetic Resonance Spectroscopy. By Sture Forsin and
Bjorn Lindman, Department of Physical Chemistry, Chemical
Centre, Lund University, Lund, Sweden ...................................... 289
Author Index ..................................................................................... 487
Subject Index ..................................................................................... 507
Cumulative Author Index, Volumes 1-27 and Supplemental
Volume .................................................................................... 513
Cumulative Subject Index, Volumes 1-27 and Supplemental
Volume .................................................................................... 525
vii
Methods of Biochemical Analysis, Volume 27
Edited by David Glick
Copyright © 1981 John Wiley & Sons, Inc.
METHODS OF BIOCHEMICAL ANALYSIS VOLUME 27
Recent Developments in Amino Acid
Analysis by Gas-Liquid
Chromatography
s.
L. MACKENZIEP,r airie Regional Laboratwy, National Research Council o f Canada,
Saskatoon, Saskatchewan, Canada
I. Introduction ......... ..... 2
..... 3
1. Protein Amino Acids ...................... ... ..... 3
..... 3
B. Multiple Derivatizations ........................... .__6_ .
a. N-TFA Methyl Esters ............................... __._6 .
b. N-TFA n-Butyl Esters ........... ..... 7
..... 9
... .... .I2
e. N-Acetyl n-Propyl Esters ............................ .... .12
.... .15
.... .18
h. N-HFB Isopropyl Esters .............. ... .... .19
... .... .I9
2. Nonprotein Amino Acids .................................... .... .20
3. Analysis of Specific Amino Acids ....... ................ .... .29
A. Tryptophan and its Metabolites ............. .... .30
B. Histidine and Related Compounds .............................. .32
C. Glutamine and Asparagine ....................
.34
D. Hydroxyproline and Hydro
E. Methionine ........ ...................... .36
F. Cysteine and Cystine ................... ............. .39
A. Primary N- and C-Terminal Analysis ...............
B. Analysis of Protein Sequencing Products ......................... .41
a. Thiohydantoins .................... .............. .41
5. Ultramicro Amino Acid Analysis .............. .............. .45
111. Resolution of Optical Isomers ............................
1. Resolution of Diastereomers .................................
A. Resolution Using Optically Active Esterification Reagents .......... .53
B. Resolution Using Optically Active Acylating Reagents ............. .55
1
2 S. L. MACKENZIE
1. Sample Preparation ................................................ .72
2. Derivarization ...................... ........ ..... .74
A. Equipment .................... ...................... .74
B. Reagents ...................................................... .75
C. Esterification ...... ......... ............
D. Acylation ..................................................... .78
3. Chromatography .............................................
V. Conclusions ............................................................. .81
References ............................................................. .81
I. INTRODUCTION
In the period since amino acid analysis by gas-liquid chromatography
(GLC) was last discussed in this series (Weinstein, 1966), there have
been substantial developments in all aspects of volatile derivative for-
mation and chromatography. Progress in the 20 years following the
first GLC analysis of amino acids was thoroughly reviewed by Husek
and Macek (1975). The subject has also been covered in other reviews
(Coulter and Hann, 1971; Darbre, 1974; Kolb, 1974; Vitt et al., 1976).
This chapter, therefore, deals primarily with developments in the past
decade. Much of the earlier work is omitted because it has already been
adequately covered elsewhere and only selected earlier papers leading
directly to the more recent developments will be cited.
Studies on the analysis of amino acids by GLC have concentrated on
the search for derivatives and column systems suitable for the separa-
tion and quantitation of all the protein amino acids in one analysis.
Since accurate quantitation, regardless of the separation, presupposes
quantitative derivatization, simultaneous studies on this aspect have
usually, but not always, been conducted in parallel with the search for
suitable derivatives. These developments are discussed in chronological
sequence in Section 11.1.
The difficult resolution problems encountered in the analysis of
nonprotein amino acids (Section 11.2) have almost invariably been
addressed using methods first developed for protein amino acids.
In addition to those methods used to obtain an overall profile of
amino acids, methods have also been devised to analyze one or only a
few amino acids. Although the data may sometimes be required in the
AMINO ACID ANALYSES BY GAS-LIQUID CHROMATOGRAPHY 3
context of a complete protein or biological fluid analysis, these methods
cannot usually be used for other amino acids and are therefore de-
scribed separately in Section 11.3.
Sequencing of proteins does not fall within the scope of this review.
Nevertheless, there have been significant developments in the use of
GLC for the identification and quantitation of amino acid methyl- or
phenylthiohydantoins produced by the Edman or related degradations.
Such developments are discussed in Section 11.4.
Ultramicroanalysis presents special problems; these are also discussed
separately (Section 11.5) although the basic methods have already been
described in Section 11.1.
Along with the development of methods for analyzing amino acid
mixtures, there have been continuing efforts not only to separate the
amino acids but also simultaneously to resolve their optical isomers.
This has required specialized techniques which are discussed in Section
111.
Although treatises on gas chromatographic techniques are readily
available, a section on techniques is included to emphasize some aspects
considered essential for reproductible quantitation of amino acid de-
rivatives (Section IV). Likewise, this section includes a discussion of
derivatization, emphasizing procedural details common to many of the
methods described more generally in Section 11.1.
11. ANALYSIS OF PROTEIN AND NONPROTEIN AMINO
ACIDS
1. Protein Amino Acids
This section deals primarily with the methods that have been used
for the routine analysis of most, if not all, the amino acids in protein
hydrolysates. Amino acids are not stable at elevated temperatures and
thus require conversion to stable, volatile derivatives before they can be
analyzed by GLC. The most common derivatizations are based on the
two-step formation of N-acyl amino acid alkyl esters. Several single-step
derivatization procedures have also been studied but most of these have
not been generally applied.
A. SINGLE DERIVATIZATION
The ideal derivatization procedure to render amino acids volatile
should consist of only one chemical reaction. To this end, there have
been several promising developments which unfortunately have not