MODERN PHYSICAL METHODS IN BIOCHEMISTRY, PART B New Comprehensive Biochemistry Volume 11B General Editors A NEUBERGER London L.L.M van DEENEN Utrecht ELSEVIER AMSTERDAM NEW YORK OXFORD Modern Physical Methods in Biochemistry Part B Editors A NEUBERGER and L.L.M VAN DEENEN London and Utrecht 1988 ELSEVIER AMSTERDAM * NEW YORK OXFORD 1988, Elsevier Science Publishers B.V (Biomedical Division) All rights reserved No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the Publisher, Elsevier Science Publishers B.V (Biomedical Division), P.O Box 1527, loo0 BM Amsterdam, The Netherlands No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, neghgence or otherwise, or from any use or operation of any methods; products, instructions or ideas contained in the material herein Because of the rapid advances in the medical sciences, the Publisher recommends that independent verification of diagnoses and drug dosages should be made Special regulations for readers in the USA This publication has been registered with the Copyright Clearance Center, Inc (CCC), Salem, Massachusetts Information can be obtained from the CCC about conditions under which the photocopying of parts of this publication may be made in the USA All other copyright questions, including photocopying outside of the USA, should be referred to the Publisher ISBN 0-444-80968-6(volume) ISBN 0-444-80303-3 (series) Published by: Elsevier Science Publishers B.V (Biomedical Division) P.O Box 211 loo0 AE Amsterdam The Netherlands Sole distributors for the USA and Canada: Elsevier Science Publishing Company, Inc 52 Vanderbilt Avenue New York, NY 10017 USA Library of Congress Cataloging-in-PublicationData (Revised for volume 11 B) Modem physical methods in biochemistry (New comprehensive biochemistry; v 11 A, B) Includes bibliographies and index Spectrum analysis Biochemistry-Technique I Neuberger, Albert 11 Deenen, Laurens L.M van QD415.N48 vol 11 A, etc 574.19’2 s [574.19’283] 85-4402 [QP519.9S6] ISBN 0-444-80649-0 (v 11 A) 0-444-80968-6 (v 11 B) Acknowledgment Many illustrations and diagrams in this volume have been obtained from other publications In all cases reference is made to the original publication ThejuN source can be found in the reference list Permission for the reproduction of this material is gratefully acknowledged Printed in The Netherlands Preface In the former series of Comprehensive Biochemistry the contributions of physical methods to biochemistry were considered in volumes 1-4, a section which was devoted to the physicochemical and organic aspects of biochemistry In 1962 the series editors M Florkin and E.H Stotz emphasized the importance of these basic sciences for the future progress in the life sciences Since that time, the application of physical methods to biological problems has solved many questions and opened new avenues of research Volume 11,part A, of the present series contained chapters on protein crystallography, nuclear magnetic resonance spectroscopy, electron spin resonance, mass spectroscopy, circular dichroism and optical rotatory dispersion In this volume the range of spectroscopic techniques is extended to chapters on fluorescence and Raman spectroscopy One chapter deals extensively with neutron and X-ray solution scattering techniques, and a choice of rapid reaction methods is discussed in a further chapter The use of electron microscopy has been another very important development in the biological sciences and the results are illustrated by a chapter with emphasis on biomembranes The New Comprehensive Biochemistry series contains a volume (8) devoted to separation methods This area is now supplemented by a chapter in the present volume on high performance liquid chromatography of nucleic acids and a chapter on reversed phase HPLC of peptides and proteins The editors hope that the publication of this volume may serve the needs of many biochemists and thus contribute to further research in the biological sciences A Neuberger L.L.M van Deenen This Page Intentionally Left Blank Contents Preface V Chapter I Fluorescence spectroscopy; principles and application to biological macromolecules J.R Lakowicz (Baltimore, MD, USA) 1 The phenomenon of fluorescence Factors affecting the fluorescence emission 2.1 Solvent polarity and viscosity 2.2 Emission spectra of melittin 2.3 Quenching of fluorescence 2.4 Fluorescence energy transfer 2.5 Fluorescence anisotropy Time-resolved fluorescence spectroscopy 3.1 Resolution of the emission spectrum of liver alcohol dehydrogenase 3.2 Pulsed lasers for time-resolved fluorescence 3.3 Frequency-domain resolution of protein fluorescence 3.4 Anisotropy decays of protein fluorescence Harmonic-content frequency-domain fluorometry summary 4 11 13 15 18 19 21 23 25 Acknowledgements References 25 26 Chapter Raman and resonance Raman spectroscopy P.R Carey (Ottawa, Ont., Canada) 27 Introduction The units used in Raman spectroscopy A model for Raman scattering based on classical physics Raman and resonance Raman scattering: a quantum mechanical interpretation Polarisation properties of Raman scattering Basic experimental aspects Raman studies on biological materials 7.1 Proteins 7.1.1 Amide I and amide I11 features 7.1.2 Side chain contributions to the Raman spectrum 7.1.3 Applications 7.1.4 UV excited resonance Raman spectra of proteins 27 29 31 34 37 38 40 40 40 42 43 43 Vlll 7.2 Proteins containing a natural, visible chromophore 7.3 Resonance Raman labels 7.4 Nucleic acids 7.4.1 The purine and pyrimidine bases 7.4.2 Conformation of the (deoxy)ribose-phosphate backbone 7.4.3 Resonance Raman studies of nucleic acids 7.5 Viruses 7.6 Lipids and membranes 7.6.1 The C-C stretching region between 1050 and 1150 cm-' 7.6.2 The C-H stretching region between 2800 and 3000 cm-' 7.6.3 Deuterated lipids as selective probes 7.6.4 Lipid protein interactions and natural membranes 44 48 50 50 52 53 54 56 57 57 58 59 References 61 Chapter Rapid reaction methods in biochemistry Quentin H Gibson (Ithaca, NY, USA) 65 Introduction Continuous flow Stopped flow 3.1 Miscellaneous stopped-flow devices 3.2 Relaxation methods 3.2.1 Flash sources 3.2.2 Observation light sources 3.2.3 Light detectors Combinations of flash photolysis with other techniques Temperature jump Miscellaneous methods 6.1 Time-resolved resonance Raman spectroscopy 6.2 Competitive methods Data reduction 65 65 69 71 72 73 75 75 76 76 77 77 78 78 References 83 Chapter High performance liquid chromatography of nucleic acids M Colpan and D Riesner (Dusseldorf, FRG) 85 Introduction Techniques 2.1 Size exclusion chromatography 2.2 Anion-exchange chromatography 2.3 Reversed phase and hydrophobic interaction chromatography 2.4 RPC-5 and other mixed mode chromatography 2.5 Sample preparation and recovery Applications 3.1 Oligonucleotides 3.2 Natural RNA 3.3 DNA fragments 3.4 Plasmids Concluding remarks 85 86 86 88 91 94 95 96 96 98 99 101 102 Acknowledgements References Chapter Reversed phase high per,,rmance liquid chromatography of peptides and proteins M.T.W Hearn and M.I Aguilar (Clayton, Vic., Australia) 103 103 107 Introduction Retention relationships of peptides in RP-HPLC The relationship between peptide retention behaviour and hydrophobicity coefficients Bandwidtb relationships of peptides in RP-HPLC Dynamic models for interconverting systems Conclusion 107 111 120 126 131 139 Acknowledgements References 139 140 Chapter X-ray and neutron solution scattering S.J Perkins (London, UK) 143 Introduction Part A: Theoretical and Practical Aspects Theory of X-ray and neutron scattering 2.1 Scattering phenomena and their angular ranges 2.1.1 X-ray scattering 2.1.2 Neutron scattering 2.1.3 Scattering angles, vectors and resolution 2.2 The scattering event and the Debye equation 2.3 Scattering densities and allowance for solvent 2.3.1 Concept of scattering densities 2.3.2 Scattering densities and volumes 2.3.3 The contrast difference A p 2.3.4 Mean macromolecular scattering densities p 2.3.5 Scattering density fluctuations pF(r) 2.4 The Guinier plot: Z ( ) and R , 2.4.1 The innermost scattering curve 2.4.2 Cross-sectional and thickness Guinier analyses 2.5 Analyses of I ( ) values 2.6 Analyses of R, values 2.7 Non-uniform scattering densities and contrast variation 2.7.1 The Stuhrmann plot 2.7.2 Solvent penetration and exchange effects 2.7.3 Isomorphous replacement 2.7.4 Matchpoints of multicomponent systems 2.8 Label triangulation 2.9 Wide-angle scattering and modelling strategies 2.9.1 Spheres and ellipsoids 2.9.2 Scattering curves at large Q 2.9.3 Independent parameters from scattering 2.9.4 Debye curve simulations 2.9.5 Interparticle interference 2.10 Distance distribution functions 143 144 144 144 144 145 146 147 149 149 150 152 154 154 160 160 162 163 165 167 167 170 172 173 173 175 175 177 178 178 180 180 294 Fig 17 Cell surface distribution of EGF receptors in A431 cells as visualised by label-fracturing showing 12 nm gold particles in projection with the high-resolution image of the exoplasmic fracture face antibody-antigen interactions Another important feature in the efficiency of immunogold labelling concerns the accessibility of the antigenic sites and the penetration characteristics of the marker system In principle, all antigenic sites on the surface of the cryosection are fully accessible to the antibody However, the penetration of the antibody into the cryosection might be differently hindered by cytoplasmic structures and might be decreased on the cell surface in a cryosection due to the embedding material, usually gelatin, as discussed above From surface labelling studies on the pore protein PhoE of E coli K-12 it has been shown that steric hindrance can cause a drastic reduction of surface label [174] and therefore putting in doubt the results obtained from biochemical binding studies Using freeze-etch labelling only a small percentage of the cell population is heavily labelled, similar to results obtained by immunofluorescence and whole mount labelling In contrast, however, a uniform, dense labelling of all cells was observed using cryo-ultramicrotomy By the use of mutants it was concluded that the antigenic sites of the PhoE pore protein were not accessible in normal cells, due to steric hindrance caused by the lipopolysaccharide carbohydrate chains Such a phenomenon may occur in various systems and thus emphasize the necessity of simultaneously applying several, basically different approaches as cross-references to study the presence and localization of antigens on the cell surface and intracellularly 295 Another parameter in label efficiency concerns the penetration phenomena of the probe in relation to size of the gold label and the use of protein A or antibody-conjugated gold It has been demonstrated that the efficiency of labelling decreased with increasing particle size [151,178-1801 In particular a reduced labelling efficiency was found for gold probes with a diameter more than nm [151] In addition, the gold-associated protein appears to influence the penetration capacity of the complex Preliminary experiments have indicated that gold particles directly coupled to goat anti-rabbit antibodies gwe a better penetration into cryosections than protein A-gold conjugates Conclusions With the development of low temperature techmques in combination with immunolabelling methods the period of molecular electron microscopy has started In this rapidly developing field information has been obtained, which was not possible before, due to this unique combination of high technology In addition, electron microscopy of frozen hydrated biological material appears to be a promising line in electron microscopy [181] Acknowledgements We are very grateful to Johannes Boonstra, Gerd Knoll, Jan Leunissen, Jan Andries Post and Jose Leunissen-Bijvelt for their contributions to the work presented in this review, Dick Smit for graphical support and Peter Thomas for correcting the English This work was supported by The Netherlands Foundation for Chemical Research (SON) with financial aid from The Netherlands Organization for the Advancement of Pure Research (ZWO) References Haschemyer, R.H and Meyers, R.J (1972) In: Principles and Techniques of Electron Microscopy (M.A Hayet, ed.) Vol 2, Ch 3, pp 101-143, Van Nostrand Reinhold Company, New York Femandez-Moran, H (1962) Circulation 26, 1039-1065 Racker, E., Tyler, D.D., Estabrook, R.W., Conover, T.E., Parsons, D.F and Change, B (1965) In: Oxidases and Related Redox Systems (T.E King, H.S Mason and M Morrison, eds.) pp 1077-1101, John Wiley, New York Crowther, R.A and Klug, A (1975) Annu Rev Biochem 44, 161-182 Caspar, D.L.D., Goodenough, D.A., Makowsky, L and Phdlips, W.C (1977) J Cell Biol 74, 605-628 Bangham, A.D (1963) Adv Lipid Res 1, 65-104 Kleinschmidt, A.K and Zahn, R.K (1959) Z Naturforsch, 14B, 770-779 Bos, J.L., Heyting, C., Borst, P., Amberg, A.C and Van Bruggen, E.F.J (1978) Nature (London) 275, 336-338 Shotton, D.M., Burke, B.E and Branton, D (1979) J Mol Biol 131, 303-329 296 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 Crowther, R.A and Pearse, B.M.F (1981) J Cell Biol 91, 790-797 Ungewickell, E and Branton, D (1981) Nature (London) 289, 420-422 Ungewickell, E (1983) EMBO J 2, 140-1408 Fernandez-Moran, H and Finean, J.B (1957) J Biophys Biochem Cytol 3, 725 Gorter, E and Grendel, F (1925) J Exp Med 41, 439-443 Hackenbrock, C.R (1968) Proc Natl Acad Sci USA 61, 598-605 Hackenbrock, C.R (1968) Biochemistry 61, 598-605 Knoll, G and Brdiczka, D (1983) Biochim Biophys Acta 733, 102-110 Weakley, B.S (1981) In: A Beginners Handbook in Biological Electron Microscopy, 2nd ed Churchill Livingstone, New York Fernandez-Moran, H (1964) J R Microsc Soc 83, 183-195 Van Harreveld, A,, Crowell, J and Malhotra, S.K (1965) J Cell Biol 25, 117-137 Van Harreveld, A and Crowell, J (1964) Anat Rec 149, 381 Heuser, J and Salpeter, R (1979) J Cell Biol 82, 150-173 Bachmann, L and Schmidt, W.W (1971) Natunvissenschaft 58, 217-218 Schwabe, K.G and Terrachio, L (1980) Cryobiology 17, 571-584 Moor, H., Kistler, J and Miiller, M (1976) Experienta 32, 805 Pscheid, P., Schudt, C and Plattner, H (1981) J Microsc 121, 149-167 Sitte, H., Neumann, K and Edelmann, L (1986) In: The Science of Biological Specimen Preparation (M Miiller, R.P Becker, A Boyde and J.J Wolosewick, eds.) p 103, AMF OHare, SEM Inc., Chicago, Illinois Plattner, H and Bachmann, L (1982) Int Rev Cytol 79, 237-304 Escaig, J (1982) J Microsc 126, 221-229 Jones, G.J (1984) J Microsc 136, 349-360 Heuser, J.E., Reese, T.S., Dennis, M.J., Jan, Y , Jan, L and Evans, L (1979) J Cell Biol 81, 27 5-300 Van Harreveld, A and Trubatch, J (1979) J Microsc 115, 243-256 Bald, W.B (1983) J Microsc 131, 11-23 Kopstad, G and Elgsaeter, A (1982) Biophys J 40, 163-170 Sowers, A.J and Hackenbrock, C.R (1981) Proc Natl Acad Sci USA 78, 6246-6250 Kleemann, W., McConnell, H.M (1974) Biochm Biophys Acta 345, 220-230 Ververgaert, P.H.J.T., Verkleij, A.J., Verhoeven, J.J and Elbers, P.F (1973) Biochm Biophys Acta 311, 651-654 Verkleij, A.J and Ververgaert, P.H.J.T (1975) Annu Rev Phys Chem 26, 101-122 Van Venetie, R., Hage, W.J., Bluemink, J.G and Verkleij, A.J (1981) J Microsc 123, 287-292 Gulik-Krzywicki, T and Costello, M.J (1978) J Microsc 112, 103-113 Melchior, D.L., Bruggemann, E.P and Steim, J.M (1982) Biochim Biophys Acta 690, 81-88 Chandler, D.E and Heuser, J (1979) J Cell Biol 83, 91-108 Pinto da Silva, P and Kachar, B (1980) Cell Biol Int Rep 4, 625-640 Moor, H., Miihlethaler, K., Waldner, H and Frey-Wessling, A (1961) J Biophys Biochem Cytol 10, 1-13 Steere, R.L (1957) J Biophys Biochem Cytol 3, 45-60 Miihlethaler, K (1971) Int Rev Cytol 31, 1-19 Branton, D (1971) Phil Trans R SOC.London B 261, 133-138 Moor, H (1971) Phil Trans R SOC.London B 261, 121-131 Zingsheim, H.P (1972) Biochim Biophys Acta 265, 339-366 Bullivant, S (1974) Phd Trans R SOC.London B 268, 5-14 Zingsheim, H.P and Plattner, H (1976) In: Methods in Membrane Biology (E.D Korn, ed.) Vol 7, Ch 1, pp 1-146, Plenum Press, New York Sleyter, U.B and Roberts, A.W (1977) J Microsc 110, 1-25 Deamer, D.W and Branton, D (1967) Science 158, 655-657 Pinto da Silva, P and Branton, D (1970) J Cell Biol 457, 598-605 Branton, D., Bullivant, S., Gilula, N.B., Kamovsky, N.J., Moor, H., Miihlethaler, K., North, D.H., Parker, L., Sarir, B., Speth, V., Staehelin, J.A., Steere, R.L and Weinstein, R.S (1975) Science 190, 54-56 297 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 Gross, H., Bass, E and Moore, H (1978) J Cell Biol 76, 712-728 Reimer, L and Schulte, C (1966) Naturwissenschaften 19, 489-494 Branton, D (1966) Proc Natl Acad Sci USA 55, 1048-1056 Verkleij, A.J and Ververgaert, P.H.J.T (1978) Biochim Biophys Acta 515, 303-327 Pinto da Silva, P and Nicolson, G.L (1974) Biochim Biophys Acta 363, 311-319 Yu, J and Branton, D (1976) Proc Natl Acad Sci USA 73, 3891-3895 Barratt, D.G., Sharom, F.J., Thede, A.E and Grant, C.W.M (1977) Biochim Biophys Acta 465, 191-197 Gerritsen, W., Verkleij, A.J., Zwaal, R.F.A and Van Deenen, L.L.M (1978) Eur J Biochem 85, 255-261 Murphy, C.R and Swift, J.G (1983) Acta Anat 116, 174-179 Blaurock, A.E and Stoeckenius, W (1971) Nature New Biol 233, 152-153 Chen, Y.S and Hubbell, W.L (1973) Exp Eye Res 17, 517-532 Verkleij, A.J., Lugtenberg, E.J.J and Ververgaert, P.H.J.T (1976) Biochim Biophys Acta 426, 581-586 Verkleij, A.J., Van Alphen, L., Bijvelt, J and Lugtenberg, E.J.J (1977) Biochim Biophys Acta 466, 269-282 Verkleij, A.J., Mombers, C., Leunissen-Bijvelt, J and Ververgaert, P.H.J.T (1979) Nature (London) 279, 162-163 Verkleij, A.J (1980) In: Electron Microscopy at Molecular Dimensions (W Baumeister and W Vogell, eds.) pp 328-337, Springer-Verlag, Berlin/Heidelberg/New York Singer, S.J and Nicolson, G.L (1972) Science 75, 720-731 Hargreaves, W.R., Giedd, K.N., Verkleij, A and Branton, D (1980) J Biol Chem 255,11965-11972 Goldstein, J.L., Anderson, R.G.W and Brown, M.S (1979) Nature (London) 279, 679-685 Henderson, R and Unwin, P.N.T (1975) Nature (London) 257, 28-32 Malhotra, S.K and Van Harreveld, A (1965) J Ultrastruct Res 12, 473-487 Van Venetie, R and Verkleij, A.J (1982) Biochim Biophys Acta 692, 379-405 Heuser, J (1981) Trends Biochem Sci 6, 64-68 Heuser, J.E and Kirschner, M.W (1980) J Cell Biol 86, 212-234 Hirokawa, N., Tilney, L.G., Fujiwara, K and Heuser, J.E (1982) J Cell Biol 94, 425-443 Hirokawa, N and Tilney, L.G (1982) J Cell Biol 95, 249-261 Heuser, J and Evans, L (1980) J Cell Biol 84, 560-583 Espevik, T and Elgsaeter, A (1984) J Microsc 134, 203-211 Hirokawa, N., Cheney, R.E and Willard, M (1983) Cell 32, 953-965 Lawson, D (1984) J Cell Biol 99, 1451-1460 Luzzati, V and Tardieu, A (1974) Annu Rev Phys Chem 25, 79-95 Deamer, D.W., Leonard, R., Tardieu, A and Branton, D (1970) Biochim Biophys Acta 219,47-60 Bangham, A.D and Home, R.W (1964) J Mol Biol 8, 660-668 Szoka, F.C and Papahadjopoulos, D (1980) Annu Rev Biophys Bioeng 9, 467-508 Van Venetie, R., Leunissen-Bijvelt, J., Verkleij, A.J and Ververgaert, P.H.J.T (1980) J Microsc 118, 401-408 Chapman, D (1968) In: Biological Membranes (D Chapman ed.) Vol 1, Ch 4, pp 125-199, Academic Press, London Luzzatti, V., Gulik-Krzywiclu, T and Tardieu, A (1968) Nature (London) 218, 1031-1034 Ladbrooke, B.D and Chapman, D (1969) Chem Phys Lipids 3, 304-347 Steim, J.H., Tourtelotte, M.E., Reinert, J.C., McElhaney, R.N and Rader, R.L (1969) Proc Natl Acad Sci USA 63, 104-107 Verkleij, A.J., Ververgaert, P.H.J.T., Van Deenen, L.L.M and Elbers, P.F (1972) Biochim Biophys Acta 288, 326-332 Ververgaert, P.H.J.T., Verkleij, A.J., Elbers, P.F and Van Deenen, L.L.M (1973) Biochim Biophys Acta 311, 320-329 Ververgaert, P.H.J.T., Verkleij, A.J., Verhoeven, J.J and Elbers, P.F (1973) Biochim Biophys Acta 311, 651-654 Verkleij, A.J and De Gier, J (1981) In: Research Monographs in Cell and Tissue Physiology (C.G Knight, ed.) Vol 7, Ch 4, pp 83-103 Elsevier/North-Holland Biomedical Press, Amsterdam 298 98 99 100 101 102 Janiak, M.J., Small, D.M and Shipley, G.G (1976) Biochemistry 15, 4575-4580 Luna, E.J and McConnell, H.M (1977) Biochim Biophys Acta 466, 381-392 Shimshick, E.J and McConnell, H.M (1973) Biochemistry 12, 2351-2360 James, R and Branton, D (1973) Biochim Biophys Acta 291, 621-628 Haest, C.W.M., Verkleij, A.J., De Gier, J., Scheek, R, Ververgaert, P.H.J.T and Van Deenen, L.L.M (1974) Biochim Biophys Acta 356, 17-26 103 Kleeman, W and McConnell, H.M (1974) Biochim Biophys Acta 345, 220-230 104 Shechter, E., Letelier, L and Gulik-Krzywicki, T (1974) Eur J Biochem 49, 61-76 105 Speth, V and Wunderlich, F (1973) Biochim Biophys Acta 291, 621-628 106 Wunderlich, F., Speth, V., Batz, W and Kleinig, H (1973) Biochm Biophys Acta 293, 39-49 107 Tsien, H.C and Higgins, M.L (1974) J Bactenol 118, 725-734 108 Verkleij, A.J., Ververgaert, P.H.J.T., Prins, R.A and Van Golde, L.M.G (1975) J Bactenol 124, 1522-1528 109 Trauble, H and Eibl, H (1974) Proc Natl Acad Sci USA 71, 214-218 110 Verkleij, A.J., De Kruijff, B., Ververgaert, P.H.J.T., Tocanne, J.F and Van Deenen, L.L.M (1974) Biochim Biophys Acta 339, 432-437 111 Kimelberg, H.K and Papahadjopoulos, D (1974) J Biol Chem 249, 1071-1080 112 Papahadjopoulos, D., Vail, W.J., Jacobson, D and Poste, G (1975) Biochim Biophys Acta 394, 483-491 113 Post, J.A., Leunissen-Bijvelt, J., Ruigrok, T.J.C and Verkleij, A.J (1985) Biochim Biophys Acta 845, 119-123 114 Rand, R.P., Tinker, D.O and Fast, P.G (1971) Chem Phys Lipids 6, 333-342 115 Cullis, P.R and De Kruijff, B (1978) Biochim Biophys Acta 513, 31-42 116 Cullis, P.R and De Kruijff, B (1979) Biochim Biophys Acta 559, 399-420 117 Wieslander, A,, Ulmius, J., Lindblom, G and Fontell, K (1978) Biochim Biophys Acta 554, 340-357 118 Shipley, G.G (1973) In: Biological Membranes (D Chapman and D.F.H Wallach, eds.) Vol 2, Ch 1, pp 1-89, Academic Press, New York 119 Cullis, P.R., De Kruijff, B., Hope, M.J., Verkleij, A.J Nayar, R., Farren, S.B., Tilcock, C., Madden, T.D and Bally, M.B (1983) In: Membrane Fluidity in Biology (R.C Aloya, ed.) Vol I, Ch 2, pp 39-81, Academic Press, New York 120 De Kruijff, B., Verkleij, A.J., Van Echteld, C.J.A., Genitsen, W.J., Mornbers, C., Noordam, P.C and de Gier, J (1979) Biochim Biophys Acta 555, 200-209 121 Verkleij, A.J., Mombers, C., Gerritsen, W.J., Leunissen-Bijvelt, J and Cullis, P.R (1979) Biochim Biophys Acta 555, 358-361 122 Verkleij, A.J., Van Echteld, C.J.A., Gerritsen, W.J., Cullis, P.R and De Kruijff, B (1980) Biochim Biophys Acta 600, 620-624 123 Lucy, J.A (1970) Nature (London) 277, 814-817 124 Poste, G and Allison, A.J (1973) Biochm Biophys Acta 300, 421-465 125 Papahadjopoulos, D., Poste, G and Vail, W.J (1979) Methods Membr Biol 10, 1-121 126 Verkleij, A.J (1984) Biochim Biophys Acta 779, 43-63 127 Wilschut, J., Holsappel, M and Jansen, R (1982) Biochim Biophys Acta 690, 297-301 128 Bearer, E.L., Duzgunez, N., Friend, D.S and Papahadjopoulos, D (1982) Biochim Biophys Acta 693, 93-102 129 Verkleij, A.J., Van Venetie, R., Leunissen-Bijvelt, J., De Kruijff, B., Hope, M.J and Cullis, P.R (1983) In: Physical Methods on Biological Membranes and Their Model Systems (F Conti, W.E Blumberg, J De Gier, and F Pochiari, eds.) pp 179-192, Plenum Press, New York 130 Schmidt, W., Patzak, A., Ling, G., Winkler, H and Plattner, H (1983) Eur J Cell Biol 32, 31-37 131 Coons, A.H., Creech, H.J and Jones, R.N (1941) Proc SOC.Exp Biol 47, 200-202 132 Singer, S.J (1959) Nature (London) 183, 1523-1524 133 Plattner, H and Zingsheim, H.P (1983) In: Subcellular Biochemistry (D.B Roodyn, ed.) Vol 9, Ch 1, pp 1-194, Plenum Press, New York 134 Moeremans, M., Daneels, G and De Mey, J (1985) Anal Biochem 145, 315-321 135 Moeremans, M., Daneels, G., Van Dijk, A., Langanger, G and De Mey, J (1984) J Immunol Methods 74, 353-360 299 136 Guesdon, J.L., Ternynck, T and Avrameas, S (1979) J Histochem Cytochem 27, 1131-1139 137 Hsu, S.M., Raine, L and Fanger, H (1981) J Histochem Cytochem 29, 577-580 138 Bonnard, C., Papermaster, D.S and Kraehenbuhl, J.P (1984) In: Immunolabelling for Electron Microscopy (J.M Polak and I.M Varndell, eds.) Ch 8, pp 95-111, Elsevier, Amsterdam 139 Bendayan, M (1984) J Electr Micr Techn 1, 263-270 140 Amzel, L.M and Poljak, R.J (1979) Annu Rev Biochem 48, 961-997 141 Fauk, W.P and Taylor, G.M (1971) Immuno-cytochemistry 8, 1081-1083 142 Romano, E.L., Stolinsky, C and Hughes-Jones, N.C (1974) Immuno-cytochemistry 11, 521-522 143 Geoghegan, W.D., Scillian, J.J and Ackerman, G.A (1978) Immunol Commun 7, 1-12 144 Geoghegan, W.D and Ackerman, G.A (1977) J Histochem Cytochem 25, 1187-1200 145 Goodman, S.L., Hodges, G.M., Trejdo, J., Siewicz, L and Livingstone, D.C (1981) J Microsc 123, 201-203 146 De Mey, J., Moeremans, M., De Waele, M., Geuens, G and De Brabander, M (1981) In: Protides of the Biological Fluid (H Peeters, ed.) Vol 29, pp 943-947, Pergamon Press, London 147 Brandon, C (1985) J Histochem Cytochem 33, 715-719 148 Horisberger, M (1979) Biol Cellul, 36, 253-258 149 Horisberger, M (1981) In: Scanning Electron Microscopy I1 (0.Johari, ed.) pp 9-31, AMF OHare, SEM Inc., Chicago, Illinois 150 Goodman, S.L., Hodges, G.M and Livingston, D.C (1981) In: Scanning Electron Microscopy I1 (0 Johari, ed.) pp 133-145, AMF O’Hare, SEM Inc., Chicago, Illinois 151 Van Bergen en Henegouwen, P.M.P and Leunissen, J.L.M (1986) Histochem J 85, 81-86 152 Bendayan, M (1982) J Histochem Cytochem 30, 81-85 153 Slot, J.W and Geuze, H.J (1985) Eur J Cell Biol 38, 87-93 154 Romano, E.L and Romano, M (1977) Imrnunochemistry 14, 711-715 155 Horisberger, M and Clerc, M.F (1985) Histochemistry 82, 219-223 156 Tokuyasu, K.T (1973) J Cell Biol 57, 551-565 157 Tokuyasu, K.T (1978) J Ultrastruct Res 62, 287-307 158 Boonstra, J., Van Mourik P., Defize, L.H.K., De Laat, S.W., Leunissen, J.L.M and Verkleij, A.J (1985) Eur J Cell Biol 36, 209-216 159 Boonstra, J., Van Belzen, N., Van Maurik, P., Hage, W.J., Blok, F.J., Wiegant, F.A.C and Verkleij, A.J (1985) J Microsc 140, 119-129 160 Tokuyasu, K.T and Singer, J.S (1976) J Cell Biol 71, 894-906 161 Tokuyasu, K.T (1983) J Histochem Cytochem 31, 164-167 162 Tokuyasu, K.T., Maher, P.A and Singer, S.J (1984) J Cell Biol 98, 1961-1972 163 Tokuyasu, K.T., Maher, P.A and Singer, S.J (1985) J Cell Biol 100, 1157-1166 164 Geuze, H.J., Slot, J.W., Strous, G.J.A.M and Schwartz, A.L (1983) Eur J Cell Biol 32, 38-44 165 Geuze, H.J., Slot, J.W., Strous, G.J.A.M., Peppard, J., Von Figura, K., Hasilik, A and Schwartz, A.L (1984) Cell 37, 195-204 166 Tommassen, J., Leunissen, J., Van Damme-Jongsten, M and Overduin, P (1985) EMBO J 4, 1041- 1047 167 Haigler, H., Ash, J.F., Singer, S.J and Cohen, S (1978) Proc Natl Acad Sci USA 75, 3317-3321 168 Haigler, H., McCanna, J.A and Cohen, S (1979) J Cell Biol 81, 382-395 169 McCanna, J.A., Haigler, H.T and Cohen, S (1979) Proc Natl Acad Sci USA 76, 5689-5693 170 Willingham, M.C., Haigler, H.T., Fitzgerald, D.J.P., Callo, M.G., Rutherford, A.V and Pastan, T.H (1983) Exp Cell Res 146, 163-175 171 Pinto da Silva, P., Moss, P.S and Fudenberg, H.H (1973) Exp Cell Res 81, 127-138 172 Pinto da Silva, P., Kachar, B., Torrisi, M.R., Brown, C and Parkinson, C (1981) Science 213, 230-233 173 Pinto da Silva, P and Kan, F.W.K (1984) J Cell Biol 99, 1156-1161 174 Voorhout, W.F., Leunissen-Bijvelt, J.J.M., Leunissen, J.L.M and Verkleij, A.J (1986) J Microsc 141, 303-310 175 Hohenberg, H., Bohn, W., Rutter, G and Mannweiler, K (1986) In: The Science of Biological Specimen Preparation (M Miiller, R.P Becker, A Boyde, J.J Wolesewick, eds.) p 235, AMF OHare, SEM Inc., Chicago, Illinois 300 176 Tillack, T.W., Allietta, M., Moran, R.W and Young, W.W (1983) Biochim Biophys Acta 733, 15-24 177 Bullock, G.R (1984) J Microsc Oxford 133, 1-15 178 Vernooi-Gerritsen, M., Leunissen, J.L.M., Veldink, G.A and Vliegenthart, J.F.G (1984) Plant Physiol 76, 1070-1079 179 Horisberger, M (1983) Trends Biochem Sci 7, 395-397 180 De Mey, J (1983) In: Immunochemistry Practical Application in Pathology and Biology (S Polak and S Van Noorden, eds.) pp 82-112, Wright-PSG, London 181 Stewart, M and Vigers, G (1986) Nature (London) 319, 631-636 301 Subject Index A/D conversion 70 Accelerated-flow apparatus 67 Accessibility of antigenic sites 293 Acetonitrile 93 Acetylcholine receptor 224, 226, 227 Acid group in Raman spectrum 43, 55 Actin 211 Adenosine triphosphatase 197 ADP/ATP carrier protein 224 Adsorption 86 Agarose 87 Aggregation 167, 183, 202 Alfalfa mosaic virus 249 Algae 46 Alkyl-ammonium salt 93 Allostericism 198 Amide I Raman bands 40-43, 54 Amide I1 Raman bands 43 Amide 111 Raman bands 40, 41, 43, 54 Aminegroup 89 Amphpathic helix 127, 130 Amphotericin B 60 Amplifier 67, 79 Analog data plot 79 Anaphylatoxin fragments 109 Andrenodoxin 45 Anion-exchange chromatography (IEC) 86-102 Anisotropy decay 21-25 Anisotropy fluorescence 11-13 Anode X-ray sources I87 Anomalous X-ray scattering 205 Antibody-hapten complexes resonance Raman labels 49 Antigenicity 286 Anti-Stokes Raman scattering 33, 35, 36 Apolipoprotein C-111 221 L-Arabinose binding protein 197 Arginine kinase 197 Aspartate aminotransferase 196 Aspartate transcarbamylase (ATCase) 199, 202 ATPase 211, 222, 224 ATPase inhibitor 211 Atrial natriuretic peptide Autoradiography 286 109 Babinet’s theorem 150, 173 Bacteriophage T4 gene 43 protein tqptic peptides 108 Bacteriophages 245, 249 Bacteriorhodopsin 45 Band-broadening 111, 127 Bandspreading model 128 Bandwidth behaviour 129, 130 Base-pairing 51, 52 Base-stacking 51, 52 Basic nuclear protein phosphopeptides 109 Basic scattering functions 237 Beam divergence 179 Beeman camera 190 Bilayer phase lipid 279-286 Bile pigments 46 Biliverdin dimethylester 46 Bimodal dependence of In k ’ 123 Binding capacity 90 Binding region 217 Biological macromolecules 1, Biosil 94 Biotin-avidin method 288 Biotin-streptavidin method 288 Blue copper 45 Boltzmann distribution 36 Bondapack 94 Bovine serum albumin 202, 205, 21 8Br-adenosine, resonance Raman spectrum 54 Bragg’s law 146 Bromegrass mosaic virus 249 Buffer subtraction 184 C1 220 Clq 220 Clr,Cls, 220 c 220 C4b binding protein 220 Cadmium in X-ray scattering 186, 193 302 Caerulein 109 Calmodulin 108,197 Capsid proteins 108 Carbohydrate scattering densities 156 y-Carboxyglutamic acid protein 109 Carotenoids resonance Raman spectra 46,60 Casein 210 p-Casein tryptic peptides 108 Catalase 205,208,209 Cell-dye interactions resonance Raman labels 49,60 Cell fractionation studies 290,291 Cell membranes Cemulsol 224,227 Chironomur ihummi ihummi 47 Chlorophylls 46 Chloroplasts 46,276 Cholera toxin 109 Chromatin 233,236,239 Chromatographic bandwidth 127 conditions 88-108 residence time 128 resin 85 selectivity 107 Chromatography 85-103,107-120,139 Chromophore 44 Chromosomes 239 Chymotrypsinogen 196 Clathrin triskelions 270,271 Cleared lysate 92 Co-crystallization 280 Coherent neutron scattering 145 Coherent X-ray scattering 144 Colipase 224,227 Collagen 185,186 Collisional quenching 7-9 Colloidal gold 286 Column configuration in RP-HPLC 110 Complement glycoprotein 219,220 Computerized data acquisition system 70 Conductivity measurements 71 Con formational equilibria 111 interconversion 132,136 reorientation 128 Conformationally rigid species 127 Contact sites 270 Continuous flow 65 Continuous flow apparatus 67 149-152, 167, 168, 182, 204-212,217,226 Core particles 233,236,237,238 Correlation length 178 Contrast variation Cowpea chlorotic mottle virus Raman spectrum 54 Cross-contamination 90 Cross-sectional Guinier plot 162,231,234,238, 249 Cryofixation 272,286 Cryo-fractures 290 Cryo-ultramicrotomy 268,286,288,290 Crystallins 203 Cucumber mosaic virus 99 CURL 290 Cyclic AMP (CAMP) dependent kinase I1 109 Cylindrical viruses 246 Cysteine proteases 49 Cytochrome b, 222 Cytochrome be, subunit 226,227 Cytochrome c 44-46 Cytochrome oxidase 45 Cytochrome P-450 45 Cytochrome reductase 224,226,227 Cytoskeleton 276,279 D-amino acid oxidase 45 Data recording 69 Data reduction 78,193 Data storage 69 DDAO 224 Debye equation 144,147-149,160 Debye sphere 178 Deoxyadenine-5’-monophosphate, RR spectrum 53,54 (Deoxy)ribose-phosphate backbone, conformation of 52 Depolarisation ratio 37,38 Desmearing 179 Detector response 185,186,193 Detergent scattering densities 159 Detergents 224 Deuterated lipids, Raman spectra 58 Deuteration 172,173,186,211-213,224,230, 234,240-244 Diarachidoyl phosphatidylcholine 57,58 Diatomic molecules Raman scattering from 32-34 Diethylamino group 89 Dimyristoyl phosphatidylcholine 56,59,60 1,2-Dimyristoyl-sn-glycero-3-phosphocholines 58 Dipaimitoyl phosphatidylcholine 58,59 303 Distance distribution function 180, 212 Distribution coefficient 86 Disulfide group vibrations 42 DNA 85, 86, 92, 95, 96, 99-103 A, B, and Z forms 52 electron microscopy 270 Raman spectra 51, 52, 54 supercoiling 231 X-ray studies 230, 234 DNA dependent RNA polymerase 196, 200, 201,211 DNA-drug complexes resonance Raman labels 49 Doubled frequency 74, 75 Dye laser 74 First-order rate constant 79 Fixatives on antigens 293 Flash photolysis 72, 75, 76, 78 Flash sources 73, 74 Flavoproteins 45 Flavovirus membrane proteins 108 Floppy disk 70 Flow-quench methods 67 Flow rate 91 Fluctuations scattering density 149, 154, 205, 236 Fluorescence 1-25, 35, 71, 75, 77 frequency-domain data 14, 15, 19-24 polarization 71 spectroscopy 1-25 time-domain data 14, 15, 19, 20, 22, 23 Fluorometry 15, 23 Fluorophore 2, 3, 11 Fourier transform 19, 20, 23 Freeze-etching 268, 274, 275, 290-292 Freeze-fracturing 268, 273-284, 287 Freeze-substitution 268, 273 Frictional ratio 165 EGF-receptor 289 Egg-white flavoprotein 45 Electric polarisability 32-34 Electron density 204, 205 Electron microscopy 267-272, 295 Electron paramagnetic resonance spectroscopy 68 Electronic absorption 29 Electronic transitions 34 Elongation factor EF-TU 236 Elongation ratio 165, 195, 196, 239 Elution volume 86 Encephalin analogues 109 P-Endorphin 108-109, 116, 128, 130, 131 Endothelial cell growth factors 109 Energy transfer fluorescence 1, 9-11, 14 Enzyme 67, 79 Enzyme-substrate complexes 67 resonance Raman labels 49, 50 Erythrocyte 276, 292 Escherichia coli 276, 277, 280, 290-294 Ethanol precipitation 96 Exchange loss factor 171, 172 Excimer laser 75 Excitation profile 37 Excited state Eye lens proteins Raman spectroscopy of 43, 45 Gap junction 268 Gaussian function 180 Gel permeation chromatography (GPC) 86 Gene protein 233, 234 Gene technology 85 Glass filter 73 Clatter indirect transform 181 Glutamate dehydrogenase 201, 202 Glutathion transferase 109 Glyceraldehyde 3-phosphate dehydrogenase 198 Glycophorin 59 Glycoproteins Stuhrmann plot of 214, 216, 225 Gonadotropin-releasing hormone 109 Gradient elution 112 Gradient slope 91 Growth hormone 107, 108 Guanine ring-breathing 51 Guinier plot 160, 161, 234, 238, 249 Fab fragments 288 Fast freezing 267, 274, 278, 280, 282 Fermi resonance 42 Femdoxin 45 Ferritin 205, 206, 209, 210 Ferrocytochrome c 47 Fe(II1)-tyrosinate proteins 45 Fibrinogen 217 'H-'H exchange 150,152,163,165,170,171, 184 Haemocyanins 199, 200 Haemoglobin 198, 203, 208, 211 Haernoglobin tryptic peptides 108 Halobacterium halobium 278 Halorhodopsin 45 Hankel transformation 249 304 Hartridge 65, 66, 69, 72 spectroscope 68 Heavy metal label 173, 186, 207, 220 a-Helix 130 amide I and 111 characteristics 41 Heme group 83 Heme proteins 44, 45 marker bands 44 resonance Raman spectra 44-46 Soret region 44 X-ray crystallography 46, 48 Hemerythrin 45 Hemocyanin 45, 67 Hemoglobin 44, 45, 65, 66, 76, 77 Heparin-binding growth factors 108 Hexagonal I1 phase lipid 283-286 Hexokinase 197 High density lipoproteins (HDL) 226, 228 High speed rotor 71 High voltage system 74, 77 High performance columns 87, 94 High performance liquid chromatography (HPLC) 85, 86, 96-103 Histidine decarboxylase 109, 208 Histidine vibrations 43 HLA-I1 108 HMG-CoA synthase 109 HMG proteins 108 Homoribopolynucleotides 93 Horseradish peroxidase 45, 288, 289 HPLC (see high performance liquid chromatography) Hybrid protein 290, 291 Hydration effect 151, 152, 163, 170 Hydration shell 205, 210 Hydrophilic acrylic polymer 89 Hydrophilic surface 87 Hydrophlicity 87 Hydrophobic interaction chromatography (HIC) 86, 91-98 Hydrophobic surface 93, 118, 127, 130 Hydrophobicity coefficient 119- 124, 130 Hydroxyethylmethacrylate 87 Hydroxylated polyether 89 Hypersil 95 IGF inhibitor 108 IgG subclasses 218 Immunoblotting technique 287 Immunocytochemistry 287-290 Immunoglobulins 218 Immunogold labelling 287-293 Immunolabelling 267, 289, 291, 295 Incoherent neutron scattering 144-146, 164, 186 Incoherent X-ray scattering 144-146 Independent parameters 178 Induced dipole moment 31 Influenza virus 248, 249 Infrared absorption spectroscopy 33 Instrumental calibration 185 Insulin 44, 108, 109 Interacting surface 89 Interconverting systems 131-135 Interfacing 70 Interference function 174 Interferon 108 Interleukin 108, 118 Interparticle interference 149, 180, 203 Interstitial volume 86 Intramembranous particles (IMPS) 276, 281, 292 Invariant 177 Ion-pair chromatography 93 formation 93 Ionic additives 111 interactions 86 Isocratic elution 112 Isometric viruses 245, 246 Isopropanol 93, 96 Isotope effect 48 Jablonski diagram Jet-freezing 273 4, Kinetic effect 136 measurement 78, 83 Kratky camera 191, 200 Kratky’s equation 163 Label efficiency 293 fracture 292 triangulation 173, 207, 211-213, 242, 243 Lac repressor 234 a-Lactalbumin 44, 196 LAPAO 224 Laser 74, 75 pulse 18, 19 Lateral diffusion 273 LiChrospher 88, 94 Light detectors 75 Linear plasmid DNA 102 Linearizing data 79 305 Link protein 109, 217 Lipid bilayer 270, 284 Lipid-cholesterol interactions 59 Lipid-metal ion interactions 59 Lipid phase transition 59 Lipid polymorphism 279 Lipid-protein interactions 59 Lipid-protein interface 59 Lipid scattering densities 158, 159 C-H stretching vibrations 56, 57 C-C stretching vibrations 56, 57 gel-to-liquid crystalline transitions 57 intermolecular chain disorder 57 intermolecular chain packing 56 phase segregation 273 phase transition 273, 274, 279, 280 Raman spectra 56, 57 Lipid vesicles 221 Lipoproteins 223, 225, 226 Liquid-liquid chromatography 95 Liver alcohol dehydrogenase emission spectrum 15-18 Lobster shells 46 Log k’ value 112-116, 118-120 Low density lipoproteins (LDL) 226, 229, 230 Low temperature techniques 267, 272, 295 Luminescence Lysozyme 196, 208, 209 a,-Macroglobulin 217 Magnetic effect 74 Malate dehydrogenase 21 Malate synthase 197, 201, 202 Matchpoint 163, 173, 204, 210-212, 245, 247 Melittin emission spectra 6-13, 20-23 Membrane potential 60 Membranes Raman spectra 56, 59 resonance Raman probes 60 Messenger RNA (mRNA) 99, 103 Metal mirror freezing 273 Metal shadowing 269, 270 Metalloproteins 45 Micelles 210, 224 Microcomputer 70 Minicomputer 69 Mirror galvanometer 66 Mitochondria 268, 270, 276, 278, 279 Mixed mode chromatography 94 Mixing chamber 66 Mo-Fe nitrogenase 211 Mobile phase 86 Mobile phase flow rate in RP-HPLC 110 Molecular weight calculations 163, 164, 165 Mono-Q 89, 99 Monoclonal antibody 287 Monodispersity 149, 183 Moore indirect transform 181 Mouse salivary gland glycoprotein 109 Multimer-monomer dissociation 132 Multiple mixer 71 Multiple zones 133 Multisite interaction 111 Myelin 276 Myelin basic protein 108 Myoglobin 44,45, 66, 196, 205, 208, 209 NADPH-cytochrome P-450 reductase 45 Near equilibrium assumption 136 Negative staining 268, 270 Neodymium glass laser 74 Neutron camera 191, 192 Neutron scattering densities 154, 159 Neutron solution scattering 143 Non-ionic interactions 89 Nucleic acid scattering densities 154, 155, 157 Nucleic acids fractionation 85-89 Raman spectra 50 resonance Raman studies 53 Nucleogen 89-92, 97-99, 102 Nucleoproteins 108 Nucleosil 88, 94 Nucleosomes 233, 236, 238 Observation light sources 75 Oestrogen synthetase 108 Ohmic heating 76, 77 Old yellow enzyme 45 Oligonucleotides 96-98 Optimization procedures 80-82 Oxygen-myoglobin reaction 67 Oxyhemoglobin 47 Parallel axes theorem 166, 170, 212, 213, 219, 220, 236, 239 Parathyroid hormone 108 Partial specific volume 152, 165 Partisil SAX 89 Parvalbumin 208 Patterson function 180 PDGF-receptor fragments 109 Peak capacity (PC) 126-129 PEI-silica 89 306 Penetration, label efficiency 293 PEP-RP1 94 Peptide folding 118 Perrin equation 12 pH-dependent ionisation equilibria 111 Phage + X 174 100 PhageMS2 99 Phenylalanine Phosphatidylcholines 280, 281 Phosphofructokinase 198 Phosphoglycerate kinase 197 Phospholipids 221, 274, 280, 284 Phosphorescence Photodiodes 71 Photographic flash 73 Photomultiplier 71, 74, 75 Photon-molecule interaction 35 Photosynthetic membrane 46, 60, 222 Photovoltaic cells 66 Phytochrome 46 Plant seed globulins 199, 200 Plaskon 95 Plasma glycoproteins 213 Plasmids 85, 92, 101 Platelet factor 211 Point collimation camera 191 Polarisability tensor 32-34, 36 Polarization 11, 66 Poliovirus protein 108 Poly(rA)poly(rU) 51 Poly(rC)poly(rG) 52 Polyanions 87, 88 Polyclonal antibody 287 Polydispersity 209, 221, 222, 229, 248 Polyene 46 Polyether 87, 94 Polyethyleneglycol 96 Polyethyleneimine 89 (Po1y)peptides RP-HPLC 107-133 Polysaccharides 213, 217 Porcine liver fatty acyl-CoA dehydrogenase 45 Porod’s law 177 Preresonance Raman conditions 36 Pretransition 280 Pro RPC 94 Protein A 288, 295 Protein-detergent complex 224, 225, 227 Protein fluorescence 2, 5, 6, 14-16, 19, 21 Protein-lipid vesicles 221, 223 Protein-nucleic acid complexes 233 Protein-protein aggregation 132 Protein Raman spectra 40, 43, 54 Protein re-orientation, in RP-HPLC 132 Protein scattering densities 154, 155 Protein secondary structure 40,41, 42, 130 Proteins conformation of 131 RP-HPLC 107-117, 130-133 UV excited resonance Raman spectra 43 Proteoglycans 213, 217 Prothrombin 217 Purines, Raman spectra 50 Pyrimidines, Raman spectra 50 Pyruvate kinase 109 Quarternary diethylamino group Quenching 78 of fluorescence 7-9 Quinaldine red resonance Raman label 60 89 Radio frequency interference 74 Radius of gyration 165-167, 194, 204 Raman depolarisation symmetry dependence 38 Raman effect 27, 35, 40 Raman hyperchromism 51 Raman scattering 28, 31, 34, 36, 37 polarisation of 37 Raman spectrometer 28, 39, 40 Raman spectroscopy 27-60, 77 Raman spectrum detection of 28, 39, 40 Rapid reaction 65 Rayleigh scattering 33-35, 143 RBC membrane proteins 108 Reaction center protein 222, 224 Reactor neutron sources 189 Relaxation methods 72, 73 Relaxation spectrum 76 Relaxed plasmid DNA 102 Resolution in chromatography 90 in scattering experiments 147 Resonance Raman (RR) effect 28, 29, 37, 40 labels 48, 60 scattering 34 spectroscopy 77 spectrum 28, 29, 36, 37, 43-50, 53, 54, 60 Restriction fragments 92 Retinal 45 Reversed phase chromatagraphy (RPC) 91-97 Reversed phase high performance liquid chromatography 107-120, 126, 128, 132-134, 139 Reversion spectroscope 72 307 Rhodopsin 45, 222, 224 Ribo-oligonucleotides 90 Riboflavin-binding protein 197 Ribonuclease 196, 211 Ribonucleotide reductase 45 Ribosomal proteins 235, 241 Ribosomal RNA (rRNA) 88,98 Ribosomal RNA (rRNA) X-ray and neutron studies 232, 235, 241, 242 Ribosomes 225, 239 Ribulose biphosphate carboxylate 197, 211 RNA 85-91, 98, 99 RNA-DNA in electron microscopy 269, 270 Raman spectra 50-52, 54, 55 Rotational diffusion Roughton 65-69, 72 RP-HPLC 107-120, 126, 128, 132-134, 139 RPC-5 chromatography 86, 94, 95, 98-102 Rubredoxin 45 Ruby laser 74 S value 112-116 S, nuclease emission spectra 20-23 Sample assays X-ray and neutron experiments 183 Sample concentration X-ray and neutron experiments 183 Sample holders X-ray and neutron experiments 184 Sample preparation in HPLC 96 Sample recovery in HPLC 96 Sample size in RP-HPLC 110 Scanning spectra 71 Scattering curve Debye sphere 178, 182, 200, 220, 237, 238 ellipsoid 174, 200 hollow sphere 173, 182 sphere 173, 182 Scattering density 150, 151, 153, 154, 167, 172, 205, 206 Scattering length 145, 146 Scattering tensor 38 Secondary minima 231 Selectivity-functional group dependency in RP-HPLC 113 Sendai virus protein 108 P-Sheet 130 amide I and 111 characteristics 40, 41, 54 Silica 87-89, 94, 97, 98, 110, 112, 116 Size exclusion chromatography (SEC) 86-88, 94, 98-101 Slow kinetics 136 Sol-gel equilibria 111 Solute aggregation 111 Solute-solvation equilibria 110 Solvophobic-silanophilic interactions 123 Southern bean mosaic virus 247, 248 Space charge 76 Spallation neutron sources 190 Specific surface 178 Specific tRNA 98 Spectrin 211 Spectrophotometer 66 Spectrophotometry split-beam 71 Spherical harmonics 179, 208, 248 Spheron 87 Spray-freezer 272 Stationary phase 86 Steric hindrance label efficiency 293 Stern-Volmer plot Stoichiometry 236 Stokes Raman scattering 33, 35 Stokes spectrum 36 Stopped-flow procedure 69, 71, 76 Storage oscilloscope 70 Structure-retention relationship 139 Stuhrmann plot 167, 172, 207-246 Stuhrmann a; p; R, 168, 169, 170, 204-246 Sulfide group vibrations 42 Supercoiled plasmid DNA 92, 101, 102 Superose 87 Surface amino acid residues 118 Surface located antigens 291 Surface topographical mapping 139 SynChropack 88, 89, 94 Synchroton X-ray sources 187-189 Synthetases 213, 235 Synthetic oligonucleotides 85 Teflon 95 Temperature in RP-HPLC 110 Temperature jump 71, 76, 77 Tetrapyrrole 46 Theoretical curves scattering 175 Thermal method 68 Thermopile 68 Thermotropic phase transition 274 Thickness Guinier plot 162, 163, 221, 222 308 Thiolase subunits 109 Thionoester hydrolysis 49 4-Thiouridine resonance Raman spectrum 54 Thomson’s constant 145 Thomson’s equation 145 Three-dimensional image reconstruction 268 Time resolution 273 Time-resolved fluorescence 13-21 Time-resolved studies 202 Tobacco bushy stunt virus 248 Tobacco mosaic virus 249 Transfer RNA (tRNA) 85, 93, 95, 98, 99 X-ray and neutron studies 231-235 Transferrins 45 Transmission measurements 183 Trigger transformer 74 Triton X-100 224, 227 Tropomyosin tryptic peptides 109 Tryptic peptides 107 Tryptophan 2, 9-19, 78 Tryptophan synthase 200, 201, 212 Tryptophan vibrations 43 TSK 87, 89, 94, 98-100 Tubulin 201, 202 Tungsten-halogen lamp 75 P-Turn 40 42 Tylorrhynchus sp haemoglobin 109 Type IV collagen fragments 109 Tyrosinase 45 Tyrosine Tyrosine doublet 54 Tyrosine vibrations 42 Unordered protein amide I and I11 characteristics Urea 92 41 Vacuum photocell 67 Vasoactive intestinal peptide 109 Vibrational transitions 34, 36 Viral DNA 231 Viral membrane glycoprotein fragments Viral RNA 85, 99 X-ray and neutron studies 232 Viroid infected plants 91 Viroid RNA 85 Viruses Raman spectroscopy of 43, 54 Visual pigments 45 Void volume 86 Vydac RP 94 Wavelength spread 179 Wheat proteins 108 X-ray camera 190 X-ray crystallography 42, 46, 48 X-ray scattering 143 Yeast fatty acyl-CoA flavin oxidase 45 glutathione reductase 45 Zorbax 87 108 ... Cataloging -in- PublicationData (Revised for volume 11 B) Modem physical methods in biochemistry (New comprehensive biochemistry; v 11 A, B) Includes bibliographies and index Spectrum analysis Biochemistry- Technique.. .MODERN PHYSICAL METHODS IN BIOCHEMISTRY, PART B New Comprehensive Biochemistry Volume 1 1B General Editors A NEUBERGER London L.L.M van DEENEN Utrecht ELSEVIER AMSTERDAM NEW YORK OXFORD Modern. .. questions, including photocopying outside of the USA, should be referred to the Publisher ISBN 0-444-80968-6(volume) ISBN 0-444-80303-3 (series) Published by: Elsevier Science Publishers B. V (Biomedical