Microscopy, Optical Spectroscopy, and Macroscopic Techniques

Transcription

1 Microscopy, Optical Spectroscopy, and Macroscopic Techniques

2 Methods in Molecular Biology John M, Walker, SERIES EDITOR 22. Microscopy, Optical Spectroscopy, and Macroscopic Techniques, edited by Christopher Jones, Barbara Mulloy, and Adrian H. Thomas, Protocols in Molecular Parasitology, edited by John E.Hyde, Protocols for Oligonucleotides and Analogs, edited by SudhirAgraival, Biomembrane Protocols: /. Isolation and Analysis, edited by John M. Graham and JoanA.Higgins, Transgenesis Techniques, edited by David Murphy and David A. Carter, Spectroscopic Methods and Analyses, edited by Christopher Jones, Barbara Mulloy, and Adrian H. Thomas, Enzymes of Molecular Biology, edited by Michael M. Burrell, PCR Protocols, edited by Bruce A. White, Glycoprotein Analysis in Biomedicine, edited by Elizabeth F. Hounsell, Protocols in Molecular Neurobiology, edited by Alan Longstaffand Patricia Revest, Pulsed-Field Gel Electrophoresis, edited by Margit Burmeister and Levy Ulanovsky, Practical Protein Chromatography, edited by Andrew Kenney and Susan Powell, Immunochemical Protocols, edited by Margaret M. Manson, Protocols in Human Molecular Genetics, edited by Christopher G. Mathew, Practical Molecular Virology, edited by Mary K. L. Collins, Gene Transfer and Expression Protocols, edited by Edward J. Murray, Plant Cell and Tissue Culture, edited by Jeffrey W. Pollard and John M. Walker, Animal Cell Cultvire, edited by Jeffrey W. Pollard and John M. Walker, New Nucleic Acid Techniques, edited by John M. Walker, New Protein Techniques, edited by John M. Walker, Nucleic Acids, edited by John M. Walker, Proteins, edited by John M. Walker, 1984

3 Methods in Molecular Biology 22 Microscopy, Optical Spectroscopy, and Macroscopic Techniques Edited by Christopher Jones, Barbara Mulloy, and Adrian H. Thomas National Institute for Biological Standards and Control, South Minims, Potters Bar, UK Humana Press ^j^ Totowa, New Jersey

4 1994 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey Allrightsreserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Photocopy Authorization Policy: Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by The Humana Press Inc., provided that the base fee of US $3.00 per copy, plus US $00.20 per page is paid directly to the Copyright Clearance Center at 27 Congress Street, Salem, MA For those organizations that have been granted a photocopy license from the CCC, a separate system of payment has been arranged and is acceptable to The Humana Press Inc. The fee code for users of the Transactional Reporting Service is: [ /93 $ $00.20]. Printed in the United States of America Library of Congress Cataloging in Publication Data Main entry under title: Methods in molecular biology. Microscopy, Optical spectroscopy, and macroscopic techniques / edited by Christopher Jones, Barbara Mulloy, and Adrian H. Thomas, p. cm. (Methods in molecular biology ; 22) Includes index. ISBN Biomolecules Spectra. 2. Electron microscopy. 3. Molecular biology methodology. I. Jones, Christopher, II. Mulloy, Barbara. III. Thomas, A. H. (Adrian H.) IV. Series: Methods in molecular biology (Totowa, NJ); 22. QP519.9.S6M '.45 dc CIP

5 Preface This is the second of three volumes of Methods in Molecular Biology that deal with Physical Methods of Analysis. The first of these, Spectroscopic Methods and Analyses dealt with NMR spectroscopy, mass spectrometry, and metalloprotein techniques, and the third will cover X-ray crystallographic methods. As with the first volume. Microscopy, Optical Spectroscopy, and Macroscopic Techniques is intended to provide a basic understanding for the biochemist or biologist who needs to collaborate with specialists in applying the techniques of modern physical chemistry to biological macromolecules. The methods treated in this book fall into four groups. Part One covers microscopy, which aims to visualize individual molecules or complexes of several molecules. Electron microscopy is the more familiar of these, while scanning tunneling microscopy is a new and rapidly developing tool. Methods for determining the shapes and sizes of molecules in solution are described in Part Two, which includes chapters on X-ray and neutron scattering, light scattering, and ultracentrifugation. Calorimetry, described in Part Three, provides the means to monitor processes involving thermodynamic changes, whether these are intramolecular, such as conformational transition, or the interactions between solutes or between a solute and its solvent. Part Four is concerned with optical and infrared spectroscopy and describes applications ranging from the measurement of protein concentration by UV absorbance to the analysis of secondary structure using circular dichroism and Fourier-transform infrared spectroscopy. As in our previous volume, authors were asked to emphasize those practical aspects of these techniques that reflect on their application, such as constraints on sample quantity, purity, and presenta-

6 vi Preface tion, the level of time and expense involved, the problems the technique is best suited to solve, and how the results may be interpreted. We hope that each of these volumes will encourage effective and productive multidisciplinary approaches to problems in molecular biology and biochemistry. We would particularly like to thank Robin Wait for his continuing help in the preparation of this book. Christopher Jones Barbara Mulloy Adrian H. Thomas

7 Contents Preface Contributors v ix PART I. MICROSCOPY CH. 1. Electron Microscopy of Protein-Nucleic Acid Complexes: Enhanced High-Resolution Shadowing, Carta W. Gray 1 CH. 2. Visualization of Unshadowed DNA by Electron Microscopy: Adsorption to a Bacitracin Film, Carta W.Gray 13 CH. 3. Biological Applications of Scanning Tunneling Microscopy, Phillip M. Williams, Mano S. Cheema, Martyn C. Davies, DavidE. Jackson, andsaulj. B. Tendler 25 PART II. SCATTERING AND SEDIMENTATION CH. 4. High-Flux X-Ray and Neutron Solution Scattering, Stephen J. Perkins 39 CH. 5. DeterminationofMacromolecular Homogeneity, Shape, and Interactions Using Sedimentation Velocity Analytical Ultracentrifugation, Stephen E. Harding 61 CH. 6. Determination of Absolute Molecular Weights Using Sedimentation Equilibrium Analytical Ultracentrifugation, Stephen E. Harding 75 CH. 7. Classical Light Scattering for the Determination of Absolute Molecular Weights and Gross Conformation of Biological Macromolecules, Stephen E. Harding 85 CH. 8. Determination of Diffusion Coefficients of Biological Macromolecules by Dynamic Light Scattering, Stephen E. Harding 97 PART III. CALORIMETRIC METHODS CH. 9. Introduction to Microcalorimetry and Biomolecular Energetics, Alan Cooper and Christopher M. Johnson 109 CH. 10. Differential Scanning Calorimetry, Alan Cooper and Christopher M. Johnson 125 vii

8 via Contents CH. 11. Isothermal Titration Microcalorimetry, Alan Cooper and Christopher M. Johnson 137 PART IV. OPTICAL SPECTROSCOPY CH. 12. Optical Spectroscopy: Principles and Instrumentation, Alex F. Drake 151 CH. 13. The Measurement of Electronic Absorption Spectra in the Ultraviolet and Visible, Alex F. Drake 173 CH. 14. Analysis of Polypeptide and Protein Structures Using Fourier Transform Infrared Spectroscopy, Parvez I. Maris and Dennis Chapman 183 CH. 15. Fluorescence Spectroscopy, Paul G. Varley 203 CH. 16. Circular Dichroism, Alex F. Drake 219 Index 245

9 Contributors DENNIS CHAPMAN Department of Protein and Molecular Biology, Royal Free Hospital School of Medicine, University of London, UK MANO S. CHEEMA Department of Pharmaceutical Sciences, ALAN COOPER Department of Chemistry, Glasgow University, Glasgow, Scotland, UK MARTYN C. DAVIES Department of Pharmaceutical Sciences, ALEX F. DRAKE Department of Chemistry, Birkbeck College, London, UK CARLA W. GRAY Program in Molecular and Cell Biology, The University of Texas at Dallas, Richardson, TX STEPHEN E. HARDING Department of Applied Biochemistry and Food Science, PARVEZ I. HARIS Department of Protein and Molecular Biology, Royal Free Hospital School of Medicine, University of London, UK DAVID E. JACKSON Department of Pharmaceutical Sciences, CHRISTOPHER M. JOHNSON Department of Chemistry, Glasgow University, Glasgow, Scotland, UK STEPHEN J. PERKINS Departments of Biochemistry and Chemistry, and of Protein and Molecular Biology, Royal Free Hospital School of Medicine, London, UK SAUL J. B. TENDLER Department of Pharmaceutical Sciences, PAUL G. VARLEY National Institute for Biological Standards and Control, South Mimms, Potters Bar, UK PHILLIP M. WILLIAMS Department of Pharmaceutical Sciences, IX