Biochemical Spectroscopy

814 Pages·1995·13.968 MB·English

by  Abelson J.M., Simon M.I., Sauer K.

Preface
The use of spectroscopic methods to examine biomolecules has a long
and rich history. Such methods have the virtue of being largely noninva-
sive and capable of probing living materials as well as subcellular prepara-
tions and isolated biomolecules. The information gained is interpretable in
terms of structural parameters and intramolecular interactions. Using
time-resolved approaches, dynamics can be explored readily over a time
range from less than a picosecond to seconds and longer. This permits
ready exploration of intermolecular interactions and intramolecular mo-
tion relevant to biological processes.
Advances in technology and methodology in spectroscopy have
moved the field forward at a breathtaking pace in recent years. We have
come a long way from the era when cytochrome oxidation state changes
were monitored visually using a hand spectroscope or when absorption
spectrometry was done using photographic detection. In this volume the
reader can learn about instrumentation that uses diode array detectors to
monitor absorption or emission spectral properties with great precision at
hundreds of wavelengths simultaneously or Fourier transform methods
that provide a significant increase in the efficiency of collecting and ana-
lyzing spectroscopic information distributed over a wide wavelength
band. Mode-locked lasers and associated pulse-compression and contin-
uum-generation techniques allow pulse-probe measurements of fast (to l0
fsec = l0 4~- sec) absorption changes or fluorescence relaxation in the
picosecond regime. Single-photon counting methods have greatly im-
proved the signal-to-noise of optical detection systems for steady-state
spectroscopy and especially for time-resolved fluorescence measure-
ments. Pulsed lasers have advanced the application of time-resolved Ra-
man spectroscopy and the ability to discriminate between Raman and
fluorescence signals. In combination with the use of ultralow tempera-
tures, intense monochromatic laser sources can be used in hole-burning
experiments to probe chromophore local environments and the modes by
which the chromophores interact with their surroundings. Computation
intensive methods, such as Fourier transform infrared, electron spin echo
(ESE), pulsed electron nuclear doable resonance (ENDOR), and digital
imaging optical spectroscopy, have provided entirely new approaches to
data collection and processing. Major developments in radiation sources,
such as synchrotrons, have opened entirely new areas of investigation,
including X-ray absorption spectroscopy (XAS) and extended X-ray ab-
sorption fine structure (EXAFS). The reader will find descriptions and
examples of each of these new methodologies in the chapters that follow,
ix
xii ECAFERP
The audience for this volume includes the current generation of gradu-
ate students and professional scientists involved in biological or biochem-
ical studies seeking an introduction to modern spectroscopic methods and
instrumentation. To help the interested reader develop a deeper back-
ground and understanding of these methodologies and approaches, the
authors of the individual chapters have cited general references and pub-
lished reviews of the individual topics.
The chapters fall into major sections covering optical spectroscopy,
vibrational spectroscopy, electron paramagnetic resonance, and X-ray
spectroscopy. Areas such as nuclear magnetic resonance which have
been described extensively in recent volumes of this series have not been
included. A general overview of the contents of this volume and examples
of problems or situations to which the different approaches have been
applied are described in the first chapter.
I am indebted to many of my colleagues and students who have of-
fered suggestions, have read and provided valuable comments on many of
the contributed chapters, and have helped with the assembly of this vol-
ume. In particular, I wish to thank several Berkeley colleagues, including
Judith Klinman who was instrumental in initiating this project. Ignacio
Tinoco, Richard Mathies, and Melvin P. Klein have not only written
comprehensive overviews of the broader spectroscopic fields, but have
also reviewed the contributed chapters included in each section. Very
helpful insights for several chapters were provided by Joy Andrews, Mar-
tin Debreczeny, and Mary Talbot, based on their perspective as graduate
students in my research group. It is my hope that this volume will be
informative and beneficial to them and to their contemporaries and suc-
cessors in research laboratories throughout the scientific community.
HTENNEK REUAS
Contributors to Volume 246
elcitrA srebmun era ni parentheses gniwollof the seman of .srotubirtnoc
snoitailiffA listed era .tnerruc
ROBERT H. AUSTIN (7), Department of DANIEL R. GAMELIN (5), Department of
Physics, Princeton University, Princeton, Chemistry, Stanford University, Stan-
New Jersey 44580 ford, California 50349
GARY W. BRUDVlG (22), Department of ELLEN GOLDMAN (29), Curagen Corpora-
Chemistry, Yale University, New Haven, tion, Branford, Connecticut 50460
Connecticut 11560
DONALD M. GRAY (3), Department of Mo-
PETER S. BRZOVI~ (8), Department of Bio- lecular and Cell Biology, University of
chemistry, University of Washington, Se-
Texas at Dallas, Richardson, Texas 38057
attle, Washington 59189
BRIAN M. HOFFMANN (23), Department of
THERESE M. COTTON (28), Department of
Chemistry, Northwestern University,
Chemistry, Iowa State University, Ames,
Evanston, Illinois 80206
Iowa 11005
ROMAN S. CZERNUSZEWlCZ (18), Depart- ALFRED R. HOLZWARTH (14), Max-Planck-
Institut for Radiation Chemistry, 07454-D
ment of Chemistry, University of Hous-
Miilheim/Ruhr, Germany
ton, Houston, Texas 40277
VICTORIA J. DEROSE (23), Department of Su-Hwl HUNG (3), Department of Molecu-
Chemistry, Northwestern University, lar and Cell Biology, University of Texas
Evanston, Illinois 80206 at Dallas, Richardson, Texas 38057
SIMON DELAGRAVE (29), Department of Bi- DAVID M. JAMESON (12), Department of
ology, Brandeis University, Waltham, Biochemistry and Biophysics, University
Massachusetts 43220 of Hawaii, John A. Burns School of Medi-
SHAOJUN DONG (28), Laboratory of Electro- cine, Honolulu, Hawaii 22869
analytical Chemistry, Changchun Insti- KENNETH H. JOHNSON (3), Baylor College
tute of Applied Chemistry, Chinese Acad- of Medicine, Center for Biotechnology,
emy of Sciences, Changchun ,220031 The Woodlands, Texas 18377
China
JAMES R. KINCAID (19), Department of
MICHAEL F. DUNN (8), Department of -DiB
Chemistry, Marquette University, Mil-
chemistry, University of California, Riv-
waukee, Wisconsin 33235
erside, Riverside, California 12529
MARTIN L. KIRK (5), Department of Chem-
SHYAMSUNDER ERRAMILLI (7), Department
istry, Stanford University, Stanford, Cali-
of Physics, Princeton University, Prince-
fornia 50349
ton, New Jersey 44580
WAYNE R. FIORI (24), Department of Chem- MELVIN P. KLEIN (21), Structural Biology
istry, Sinsheimer Laboratories, Univer- Division, Lawrence Berkeley Laboratory,
sity of California, Santa Cruz, Santa University of California, Berkeley, Berke-
Cruz, California 46059 ley, California 02749
JOSEF FRIEDRICH (10), Physikalisches Insti- SUE LEURGANS (27), Department of Preven-
rut, Universitdt Bayreuth, D-95440 Bay- tive Medicine, Rush Medical College,
reuth, Germany Chicago, Illinois 21606
ix
X CONTRIBUTORS TO VOLUME 246
AUGUST H. MAKI (25), Department of Radiation Biology, University of Frei-
Chemistry, University of California, burg, D-7800 Freiburg, Germany
Davis, Davis, California 61659 DRAWDE .1 NOMOLOS (5), Department of
DRAHCIR A. MATHIES (16), Department of Chemistry, Stanford University, Stan-
Chemistry, University of California, ford, California 50349
Berkeley, Berkeley, California 94720 SAMOHT G. SPIRO (18), Department of
NAHBOIS M. MIICK (24), Department of Chemistry, Princeton University, Prince-
Chemistry, Sinsheimer Laboratories, ton, New Jersey 08544
University of California, Santa Cruz, WALTER S. STRUVE (11), Ames Labora-
Santa Cruz, California 95064 tory-United States Department of En-
GLENN L. MILLHAUSER (24), Department of ergy, and Department of Chemistry, Iowa
Chemistry, Sinsheimer Laboratories, State University, Ames, Iowa 11005
University of California, Santa Cruz, JOHN C. SUTHERLAND (6), Department of
Santa Cruz, California 95064 Biology, Brookhaven National Labora-
JIANJUN NIU (28), Laboratory of Electroan- tory, Upton, New York 37911
alytical Chemistry, Changchun Institute OICANGI TINOCO, JR. (2), Department of
of Applied Chemistry, Chinese Academy Chemistry and Chemical Biodynamics
of Sciences, Changchun 130022, China Laboratory, University of California,
RENRAW L. SALOCITEP (17), Department of Berkeley, Berkeley, California 02749
Chemistry, University of Oregon, TREBREH NAV NEGNOREMA (9, 11), Depart-
Eugene, Oregon 30479 ment of Physics and Astronomy, Free
ENIBAS PULVER (5), Department of Chemis- University of Amsterdam, 1081 HV Am-
try, Stanford University, Stanford, Cali- sterdam, The Netherlands
fornia 50349 RIENK NAV ELLEDNORG (9), Department of
TREBOR T. ROSS (27), Department of -DiB Physics and Astronomy, Free University
chemistry, Ohio State University, Colum- of Amsterdam, 1081 HV Amsterdam, The
bus, Ohio 43210 Netherlands
KENNETH SAUER (1), Department of Chem- ALAN RENOGGAW (15), Department of Bio-
istry, and Structural Biology Division, La- logical Sciences, and Center for Light Mi-
wrence Berkeley Laboratory, University croscope Imaging and Biotechnology,
of California, Berkeley, Berkeley, Califor- Carnegie Mellon University, Pittsburgh,
nia 02749 Pennsylvania 31251
MAILLIW H. SAWYER (12), Russell TREBOR W. YDOOW (4), Department of Bio-
Grimwade School of Biochemistry, Uni- chemistry and Molecular Biology, Colo-
versity of Melbourne, Parkville, Victoria rado State University, Fort Collins, Colo-
3052, Australia rado 80523
HUGO SCHEER (30), Botanisches Institut der VITTAL K. ARDNAHCAY (26), Structural Bi-
Universitiit Miinchen, D-80683 Miinchen, ology Division, Lawrence Berkeley Labo-
Germany ratory, University of California, Berkeley,
PAUL R. SELVIN (13), StructuralBiology Di- Berkeley, California 94720
vision, Calvin Laboratory, Lawrence MARY M. YANG (29), KAIROS Inc., San
Berkeley Laboratory, and Department of Jose, California 63159
Chemistry, University of California, SALGUOD C. YOUVAN (29), PaiD Alto Insti-
Berkeley, Berkeley, California 94720 tute of Molecular Medicine, Mountain
F. SIEBERT (20), Institute of Biophysics and View, California 34049
1 NOITCUDORTNI 1
1 Why Spectroscopy? Which Spectroscopy?
yB KENNETH SAUER
Biochemists and scientists in related fields have a "need to know"
about information covering an enormous range of disciplines. They are
interested in the involvement and fates of very small molecules like 02,
CO2, n20, and N 2, as well as those of the largest proteins and nucleic
acids. Scientists also want to know about the interactions of the small
molecules with the large ones: not only to view the needle in the hay-
stack, but to determine whether "it is threaded and ready to use." These
studies may involve "simple" reactions like the interchange between
dissolved CO2 gas and bicarbonate ion in aqueous solution, as well as the
role of enzymes that can speed up such processes by orders of magni-
tude.
The context in which biochemical processes occur is essential to defin-
ing mechanisms and functions. The component proteins and nucleic acids
exhibit dramatic changes in properties when they are incorporated into
ribosomes or chromosomes or functional membranes. Even then, it is not
sufficient to determine a static or average structure of the components
in such complex assemblies, because the function typically depends on
essential structural fluctuations or driven conformational changes. In this
way time enters as a significant variable. Short-term events (picosecond
time scale and shorter) can involve vibrational motion to dissipate heat
energy or to initiate conformational events that trigger vision processes
or start the electron transfer reactions of photosynthesis. Diffusion of
small substrate molecules is a few orders of magnitude slower. The motion
of major portions of proteins may require only microseconds, whereas
the complex decision-making required for enzymes to function selectively
may require times in the range of seconds.
Such an enormous range of conditions and interests places enormous
demands on the panoply of experimental approaches needed to address
the related issues. In a recent single issue of the American Chemical
Society journal yrtsimehcoiB there were 25 articles, of which all but 4
explicitly involved some form of spectroscopy among the methods that
were cited. In many cases two or three spectroscopic methods were used
in the same study, and none of these entailed simply using absorbance
at a particular wavelength to monitor the concentration of a particular
component. Even the other 4 articles, which included one study involving
X-ray crystallography and another that followed the course of a radioiso-
Copyright © 1995 by Academic Press, Inc.
METHODS IN ENZYMOLOGY, VOL. 246 All rights of reproduction in any form reserved.
2 INTRODUCTION 1
tope, contained procedures that implied the application of spectroscopic
methods in the preparation and purification of materials.
Numerous, wide-ranging spectroscopic techniques will be presented
in this volume, with the exception of nuclear magnetic resonance 1,)RMN(
which was the subject of Volumes 176, 177, and 932 of Methods ni Enzy-
mology, and mass spectrometry,: which was the subject of Volume .391
Examples of techniques from each of three major areas, ultraviolet/visible
spectroscopy, vibrational spectroscopy, and electron or electron/nuclear
magnetic resonance, are presented in this volume. Also included are
special topics like rapid-scan diode-array spectroscopy, terbium labeling
ofchromopeptides, and deconvolution of complex spectra that are covered
in chapters in Section IV of this volume.
Why Spectroscopy?
The range of areas of scientific interest represented in the Biochemistry
issue mentioned above was extremely broad. A sampling includes the
following: the interaction with DNA of a cobalt probe that resulted in
shifts in the electron paramagnetic resonance (EPR) spectrum as well as
changes in the circular dichroism, a study of protein oligomerization,
premelting flexibility of a synthetic oligonucleotide, the formation of cross-
links between DNA and protein using platinum and ruthenium compounds,
enzyme-substrate interactions, the structure and dynamics of channel-
forming membrane proteins, the dynamics of unfolding and refolding of
a redoxactive protein, and the use of a fluorescent amino acid reporter
to send signals from the channel of a transporter protein. Spectroscopic
methods can be used to examine the behavior of cell organelles or macro-
scopic assemblies that are not yet amenable to interpretation at the molecu-
lar level.
One of the virtues of spectroscopic approaches is that they are com-
monly nondestructive and noninvasive, although some procedures involve
the attachment of labels chemically at specific sites. The former ap-
proaches are particularly valuable for ni uivo studies. In laboratory experi-
ments, the amounts of materials required range from the femtomole (I0-,5
tool) level for some particularly sensitive fluorescence approaches to milli-
grams or more for typical EPR, infrared, or X-ray spectroscopy. Ongoing
requirements in instrumentation and techniques are continually whittling
away at the limits to the amount of material required.
t N. J. Oppenheimer and T. L. James (eds.), this series, Vols. 176, 177, and 239: Nuclear
Magnetic Resonance.
2 j. A. McCloskey (ed.), this series, Vol. 193: Mass Spectrometry.
1 NOITCUDORTNI 3
Which Spectroscopy?
One of the most important questions facing an experimentalist at the
outset of a research project is the decision about which of the available
(or potentially available) spectroscopic methods is most suitable to a par-
ticular problem or investigation. The decision is complex, in that it in-
volves consideration of the nature of the biological or chemical materials
being investigated, the particular goals of the research, the availability of
suitable spectroscopic tools, and the ability to interpret the results in a
meaningful way. The latter requirement implies the need for at least a
vestige of a theoretical framework in which to formulate the interpretation
and, in many cases, access to an "expert" who is knowledgeable about
the relation between the experimentally observed quantities and their
origin at the molecular or electronic level. One important source of such
expertise is a suitable text or reference book that describes the underlying
principles in terms that are both informative and understandable. Although
the relation between underlying principles and spectroscopic measure-
ments properly involves subjects such as quantum mechanics, statistical
mechanics, and molecular dynamics, one does not necessarily need to be
an authority in these fields to be sufficiently conversant to make intelligent
use of the spectroscopic observables.
Each of the individual chapters in this volume presents references to
authoritative monographs or compendia of relevant information on the
main topic covered. There are, in addition, introductory books that de-
scribe a broad range of relevant methods; these books are particularly
helpful in making the initial decision as to which spectroscopic methods
to choose for a particular problem. 4,3 Sections that involve spectroscopy
are included in a variety of texts in biochemistry, 5 physical chemistry for
biological scientists, 6-1° or biophysics. At a somewhat more advanced
3 ,R E. Hester dna .R .B Stirling, "Spectroscopy of lacigoloiB Molecules." CRC Press,
Boca Raton, Florida, .1991
4 I. .D Campbell and .R .A Dwek, "Biological Spectroscopy." ,sgnimmuC/nimajneB Menlo
Park, California, .4891
L. Stryer, "Biochemistry," dr3 Ed. Freeman, naS Francisco, California, .8891
6 I. Tinoco, Jr., K. Sauer, and J. C. Wang, "Physical Chemistry--Principles dna -acilppA
tions ni lacigoloiB Sciences," dr3 Ed. Prentice-Hall, Englewood Cliffs, New Jersey, .5991
7 .D Eisenberg and .D Crothers, "Physical Chemistry, with Applications to the Life Sci-
ences." ,sgnimmuC/nimajneB Menlo Park, California, ,9791
8 .A G. Marshall, "Biophysical Chemistry--Principles, Techniques dna Applications."
Wiley, weN York, .8791
9 .D Freifelder, "Physical Biochemistry--Applications to Biochemistry dna Molecular -loiB
ogy," dn2 Ed. Freeman, naS Francisco, California, .2891
0~ K. E. naV Holde, "Physical Biochemistry," dn2 Ed. Prentice-Hall, Englewood ,sffilC
weN Jersey, .5891
4 NOITCUDORTNI ll
level, the monograph by Cantor and Schimme111 is a particularly valuable
resource for this purpose. Greater detail in the theoretical underpinnings
of many of the types of spectroscopy used by biochemists is covered in
an excellent book by Struve. 21 Nevertheless, it is inevitable that some of
the topics covered in the present volume are not to be found or are only
briefly mentioned in the broader surveys. This occurs either because the
fields, or the applications to biological problems, are relatively new X-ray
spectroscopy, hole burning, Fourier transform infrared spectroscopy
(FTIR) or because until recently the applications to biology were con-
strued to be highly specialized electron nuclear double resonance spec-
troscopy (ENDOR), spin labels, digital imaging spectroscopy. Several of
these methods have sufficient potential that they should be considered
to be a part of the reserve toolkit of experimental biochemists and biolo-
gists.
Spectrometric methods rely on the availability of reference data or
relevant properties of well-examined biochemical materials. Such data
are abundant and widespread in the literature, but compilations of such
spectrometric data are not nearly so available as are those in the areas
of chemistry or physics. Even the few compilations that exist 4a31 are not
particularly recent or up-to-date. This is a shortcoming that the leaders
of the discipline should endeavor to remedy.
To aid the reader in the process of choosing suitable tools from the
toolkit, the following paragraphs focus on a variety of objectives that are
typical of biochemical and biological investigations and how each of the
objectives may be productively illuminated by the methods described in
detail in the subsequent chapters.
Structure of Biomolecules
The distinction between monomer, duplex, and triplex helices of DNA
or RNA can be established by a comparison of absorption and circular
dichroism (CD) spectroscopy. A clear difference in the CD is seen that
distinguishes triplexes of RNA from those of DNA. 6A51 The CD spectra
II C. R. Cantor and P. R. Schimmel, "Biophysical Chemistry--Part II: Techniques for the
Study of Biological Structure and Function." Freeman, San Francisco, California, 1980.
21 W. S. Struve, "Fundamentals of Molecular Spectroscopy." Wiley (Interscience), New
York, 1989.
31 G. D. Fasman (ed.), "Handbook of Biochemistry and Molecular Biology, Vol. :1 Physical
and Chemical Data," 3rd Ed. CRC Press, Cleveland, Ohio, 1976.
4i G. D. Fasman (ed.), "Practical Handbook of Biochemistry and Molecular Biology." CRC
Press, Boca Raton, Florida, 1989.
51 D. M. Gray, S.-H. Hung, and K. H. Johnson, this volume 3.
61 R. W. Woody, this volume 4.
ll NOITCUDORTNI 5
of proteins and polypeptides are also useful in determining the content of
zc helix,/3 sheet, and/3 turns.~6 The coordination geometry and spin state
of transition metal centers in biomolecules can be characterized using CD
and magnetic circular dichroism (MCD) measurements.17 The conforma-
tion of macromolecules in solution is sensitively measured by excitation
transfer between fluorescence donor-acceptor molecular pairs that are
covalently or noncovalently attached. 9~,8~ Detailed information about the
backbone conformation of proteins or nucleic acid helices can be read
from Raman °2 or infrared ~2 spectra. Resonance Raman spectra are useful
in documenting the subtle distinction between the a- and/3-subunit hemes
in hemoglobin. 22 Changes in the oxidation state of iron- or copper-contain-
ing proteins allow ENDOR spectroscopy to elucidate the coordination
environment of the redox-active cofactors. 32 Rotational diffusion con-
stants that are sensitive to molecular extension can be determined using
spin-labeled macromolecules. The difference between a peptide in the a
helix or 0~3 helix configuration is readily seen in the EPR spectra of doubly
spin-labeled molecules. 42
Changes in Structure or Conformation
The transformation of polyproline from a right-handed to a left-handed
3-fold helix can be followed using CD.~6 MCD spectra are sensitive to the
conformation of the heme-imidazole ligand interaction in cytochromes. 52
Hole-burning measurements provide sensitive tools for detecting differ-
ences in structure produced on thermal cycling or as a consequence of
increasing pressure on horseradish peroxidase solutions. 62 Attachment of
a fluorescence label 72 provides a useful indicator of the loss of activity of
human immunodeficiency virus (HIV) protease, 8~ and lifetime measure-
ments can be used to determine the binding of ligands, of drugs to proteins,
or the association of proteins with membranes. 91 Similarly, the decreasing
content of free sulfhydryl groups that accompanies aging is detectable by
Raman spectroscopy using deuterium exchange methods.2° Raman spectra
7I E. I. Solomon, .M L. Kirk, .D .R Gamelin, and .S Pulver, this volume .5
81 p. .R Selvin, this volume .31
91 .A .R Holzwarth, this volume .41
02 .W L. Peticolas, this volume .71
i2 F. Siebert, this volume .02
22 j. .R Kincaid, this volume .91
32 .V J. DeRose and .B .M Hoffman, this volume .32
42 .G L. Millhauser, .W .R Fiori, dna .S .M Miick, this volume .42
52 j. .C Sutherland, this volume .6
62 j. Friedrich, this volume .01
72 .A Waggoner, this volume .51
6 NOITCUDORTNI 1
also can be used to follow the course of DNA or RNA melting, °z The
involvement of aspartate in the photocycling of bacteriorhodopsin can
be followed using difference infrared studies of isotopically substituted
molecules. 12 Electron spin-labeled proteins exhibit striking changes in
EPR spectra on undergoing structural transformations, 4z
noitisopmoC of sexelpmocoiB
The stoichiometry of RNA in duplex or DNA in triplex complexes is
discernible from CD spectra, as are hybrid complexes between RNA and
DNA. 51 The amino acid content of proteins and polypeptides is reflected
in the UV absorption and CD spectra, as is the presence of cofactors
such as heme or flavin. The CD spectrum is also sensitive to interactions
between proteins and nucleic acids. 61 In general, cofactors that involve
transition metals with incomplete d electron shells exhibit characteristic
features in absorption, CD, and MCD 52,71.artceps The presence of trypto-
phan gives rise to a distinctive band in the MCD spectrum of any protein.25
The binding of substrate analogs like e-AMP to carbamoyl-phosphate
synthase can be detected by the influence on the anisotropy of the fluores-
cence. Protein-DNA interactions have been monitored using the anisot-
ropy of fluorescence of Trp of the Ada protein and the cal promoter with
cyclic AMP in the presence of receptor protein, 8z Added fluorescent dyes
can be used to tag DNA molecules, even during electrophoresis.~8 FTIR
spectra reflect the interaction between certain drugs and DNA, and they
characterize caged Ca +z and caged ATP as well. ~2 Stacking interactions in
protein-DNA complexes have been investigated using optically detected
magnetic resonance (ODMR). 92 Photosynthetic complexes that contain
antenna complexes and reaction centers exhibit complex fluorescence
relaxation that reflects the spectroscopic heterogeneity of the complexes. 9~
The identity and integrity of photosynthetic reaction center complexes
are readily determined using chromatography with a diode-array detector,
as is the degradation of isolated photosynthetic pigments. °3
noitatneirO of Component Structures
The orientation of structural components in proteins can be character-
ized using polarized absorption or emission spectroscopy. Where single
crystals are not available or convenient, anisotropic macromolecules may
be oriented using gel compression, electric or magnetic fields, or stretched
82 D. M. Jameson and W. H. Sawyer, this volume 12.
92 A. H. Maki, this volume 25.
o3 H. Scheer, this volume 30.