MODERN PHYSICAL METHODS IN BIOCHEMISTRY, PART A New Comprehensive Biochemistry Volume 11A General Editors A NEUBERGER London L.L.M van DEENEN Utrecht ELSEVIER AMSTERDAMeNEW YORK*OXFORD Modern Physical Methods in Biochemistry Part A Editors A NEUBERGER and L.L.M VAN DEENEN London and Utrecht 1985 ELSEVIER AMSTERDAMeNEW YORK-OXFORD 1985, Elsevier Science Publishers B.V (Biomedical Division) All rights reserved N o 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, 1000 BM Amsterdam, The Netherlands 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 0-444-80649-0 (volume) ISBN 0-444-80303-3 (series) Puhlished hy: Elsevier Science Publishers B.V (Biomedical Division) P.O Box 21 I 1000 AE Amsterdam The Netherlands Sole distrihutors f o r the U S A and Canada: Elsevier Science Publishing Company, Inc 52 Vanderbilt Avenue New York, NY 10017 USA Library of Congress Cataloging in Publication Data Main entry under title: Modern physical methods in biochemistry (New comprehensive biochemistry; v 11) Bibliography: p Includes index Spectrum analysis Biological chemistry Technique I Neuberger, Albert 11 Deenen, Laurens L M van 111 Series QD415.N48 VOI 11 574.192 s [574.19'283] 85-4402 [QP5 19.9.S6] ISBN 0-444-80649-0 Printed in The Netherlands V Preface The great and, one might say without exaggerating, the amazing progress which has been made in the biological sciences, particularly in biochemistry, over the last 20 years has been caused to a large extent by the development of sophisticated physical methods and their application to biological problems Our knowledge of the structure and especially the conformation of protein and nucleic acids has been helped greatly by the use of mass spectrometry and a variety of optical methods, such as circular dichroism and the extension of optical rotary dispersion to low wavelengths The use of electron spin resonance has been of special use in our understanding of oxidation and reduction processes, and also has been helpful in other problems affecting the structure of important organic molecules The use of nuclear magnetic resonance has been another very important development in biological sciences It is even being used to an increasing extent in physiological investigations, and its application to clinical medicine is likely to be of considerable benefit The use of X-ray crystallography goes back to the 1930s, but in recent years the techniques have been refined so that resolution has been increased to a significant extent Therefore, it seems reasonable to describe the techniques used in a manner which is intelligible to the non-expert, and to describe at least some of the applications of these techniques to important biological problems The present book will be followed by a second dealing with a variety of other physical techniques It would be quite impossible to deal with all physical methods which will be used over the next or 10 years, but we hope to cover most of the major techniques which will be applied in solving important biological problems A, Neuberger L.L.M Van Deenen This Page Intentionally Left Blank vii Contents Preface V Chapter I Nuclear magnetic resonance spectroscopy in biochemistry, by J K M Roberts and Jardetzky 1 Introduction Theory (a) Nuclear spin (bj Nuclear precession (c) Nuclear magnetic resonance (i) In an isolated atomic nucleus (ii) In an assembly of identical nuclei (d) The free-induction decay and relaxation (e) The chemical shift (f) Spin-spin coupling (g) Spin-decoupling (h) Relaxation mechanisms (ij Cross-relaxation and the nuclear Overhauser effect (j)Chemical exchange (k) The spectrometer Biochemistry in vivo (a) Introduction (b) Experimental considerations (c) Observation and quantitation of metabolites (i) Assignment of resonances (ii) Quantitation of metabolites (d) Intracellular pH measurements (e) Compartmentation of metabolites ( f j Measurement of unidirectional reaction rates by saturation transfer (g) Tracing metabolic pathways by I3C- and "N-NMR Macromolecules in vitro (a) Introduction (b) Analysis of macromolecular spectra (i) Purely spectroscopic techniques (ii) Techniques dependent o n the knowledge of the crystal structure (iii) Combinations of chemical and spectroscopic methods independent of the knowledge of the crystal structure (c) The information content of macromolecular spectra (i) Chemical shift (ii) Coupling constants (iii) Relaxation parameters (iv) The problem of averaging 2 5 10 13 17 19 20 22 24 27 28 28 28 29 29 29 31 33 33 37 38 38 39 41 47 49 50 50 51 51 52 Vlll (d) Solution structure of proteins and nucleic acids (e) Dynamics of protein and nucleic acids (i) Hydrogen exchange between solvent and biopolymers (ii) Motion of aromatic side chains in proteins (iii) Information from relaxation data References 53 57 51 59 60 64 Chapter Electron spin resonance, b y R.C Sealy, J S Hyde and W.E Antholine 69 I Introduction (a) Classification with respect to technique (b) Classification with respect to order, motion and stability Nitroxide radical spin labels and spin probes (a) Labels and probes (b) Physical properties of spin labels (i) Intramolecular magnetic interactions (ii) Relaxation times (iii) Intramolecular motional modes (c) Spin-label information content (i) Intensity (ii) Lineshapes and rotational motions (iii) Spectral diffusion of saturation and rotational motions (iv) Translational diffusion (homospecies) and line broadening (v) Translational diffusion (heterospecies), line broadening, and saturation (vi) pH detection (vii) Polarity probes (viii) Distance determinations (fixed interaction distance) (ix) Distance determination (distribution of fixed interaction distances) (x) Concluding remarks Biological free radicals (a) Physical and chemical properties (b) Radicals from chemical oxidation/reduction (c) Radicals from enzymes, their substrates, and other macromolecular radicals (i) One-electron oxidation (ii) Rearrangement and related reactions (iii) One-electron reductions (iv) Mixed reaction mechanisms, redox equilibria (d) Radicals in drug metabolism (i) Oxidation reactions (ii) Reduction reactions Metal ions (a) General remarks (b) ESR of metalloproteins and metalloenzymes (c) Complementary probes (i) Isolated metal centers (ii) Coupled metal centers (d) Extensions of the standard ESR methods (i) S-band (ii) Spin echo spectroscopy (iii) ENDOR Instrumentation and methodology (a) The reference arm microwave bridge (b) Sensitivity 69 69 71 72 73 14 75 79 80 81 81 82 82 83 83 84 84 84 84 84 85 85 89 92 92 96 97 102 106 106 107 109 109 114 117 117 121 122 122 125 127 129 129 132 ix (c) Resonators (d) Field modulation (e) Accessories (f) ENDOR, ELDOR, time domain ESR and multifrequency ESR (g) ESR and computers References Chapter Mass spectroscopy, by J.C Tabet and M Fetizon I General (a) Peripheral techniques in mass spectrometry (b) Chemical ionization (CI) (i) Positive CI (i-a) Protonation reactions (and the formation of adducts) (i-b) Adduct ion formation reactions and their decompositions (i-c) Charge-exchange reactions (ii) Negative chemical ionization (c) Chemical ionization at atmospheric pressure (API) (d) Thermal desorption (i) Flash desorption (ii) Desorption by ‘electron (or ion) beam’ technique (iii) Formation and ionization of aerosols (e) Field ionization and desorption (i) Field ionization (FI) (ii) Field desorption (FD) (iii) Desorption by chemical ionization (DCI) (f) Other types of desorption (i) 25ZCfplasma desorption (PDMS) (ii) Laser-induced desorption (LDMS) (iii) Desorption by ionic bombardment (SIMS) Ion metastable studies and MS/MS methodology (a) Detections of metastable ions (i) Methods involving the variation of one field (i-a) Variation of accelerating voltage (HV scan or defocused metastable scanning) (i-b) Variation of the electric field (IKE technique) (i-c) MIKE (or DADI) technique (ii) Linked scan methods (ii-a) E Z I V linked scan (simulated MIKE) (ii-b) B/E linked scan method (daughter ml,, ions of ml) (ii-c) B / E linked scan method (precursors of ml: ions decomposing in the first FFR) (ii-d) B/E linked scan spectra (b) Collisionally activated fragmentations (c) Special case of negative ions (i) I K E spectra (ii) MIKE spectra and charge inversion reactions induced by collisions (d) Use of computers for processing unimolecular and collisional-induced decomposition spectra (e) New generation of mass spectrometers for MS/MS techniques (i) Magnet and electric analyzer instrument as tandems (ii) Triple quadrupole instruments (iii) Hybrid instruments (f) A new methodology for the study of mixtures: MS/MS Jw i35 136 137 138 139 140 149 149 149 151 151 151 151 154 155 157 157 157 158 159 160 160 160 161 163 163 164 165 167 167 168 169 171 172 175 176 177 179 181 184 190 190 190 192 193 193 194 195 196 414 204 205 206a 206b 207 208 209 210 21 212 213 214 215 216 217 218 219 220 22 222 223 224 225 226 227 228 229 230 23 232 233 '234 235 236 237 238 239 240 24 242 Branden, C.4 and Eklund, H (1980)in Dehydrogenases (Jeffry, J., ed.), pp &84, Birkhauser, Basle Remington, S., Wiegand, G and Huber, R (1982) J Mol Biol 158, 111-152 Rogers, N.K (1984) D.Phil Thesis, University of Oxford Pethig, R (1979) Dielectric and Electronic Properties of Biological Materials, John Wiley, New York Fink, A.L and Petsko, G.A (1981) Adv Enzymol 52, 177-246 Haas, D.J and Rossmann, M.G (1970) Acta Cryst B26,998-1004 Angell, C.A (1983) Annu Rev Phys Chem 34,593-630 Hobbs, P.J (1974) Ice Physics, Clarendon Press, Oxford Franks, F (1978) J Microsc 112, 3116 Costello, M.J and Corless, J.M (1978) J Microsc 112, 35-55 Douzou, P (1974) Methods Biochem Anal 22,401-512 Petsko, G.A (1975) J Mol Biol 96, 381-392 Wychoff, H.W., Doscher, M., Tsernoglou, D., Inagami, T., Johnson, L.N., Hardman, K.D., Allewell, N.M., Kelly, D.M and Richards, EM (1967) J Mol Biol 27, 563-578 Marsh, D.J and Petsko, G.A (1973) J Appl Cryst 6, 7680 Rudman, R (1976) Low Temperature X-ray Diffraction, Plenum Press, New York Douzou, P and Petsko, G.A (1984) Adv Prot Chem 36,245-361 Bishop, W.H and Richards, EM (1968) J Mol Biol 38, 315-328 Crank, J (1956) The Mathematics of Diffusion, Oxford University Press, Oxford Fink, A.L and Ahmed, A.I (1976) Nature 263, 294-297 Alber, T., Petsko, G.A and Tsernoglou, D (1976) Nature 263, 294-297 Alber, T., Gilbert, W.A., Ponzi, D.R and Petsko, G.A (1983) CIBA Symp 93, 4-24 Phillips, J.C., Wlodawer, A., Yevitz, M.M and Hodgson, K.O (1976) Proc Natl Acad Sci U.S.A 73, 128-132 Lemonier, M., Fourme, R., Rousseau, F and Kahn, R (1978) Nucl Inst Methods 152, 173-177 Kahn, R., Fourme, R., Gadet, A., Janin, J., Dumas, C and Andre, D (1982) J Appl Cryst 15, 330-337 Bartunik, H.D., Clout, P.N and Robrahm, B (1981) J Appl Cryst 14, 134-136 Bartunik, H.D., Fourme, R and Phillips, J.C (1982) in Uses of Synchrotron Radiation in Biology (Stuhrmann, H.B., ed.), Academic Press, New York Helliwell, J.R., Greenhough, T.J., Carr, P.D., Rule, S.A., Moore, R.R., Thompson, A.W and Worgan, J.S (1982) J Phys E Sci Inst 15, 1363-1372 Greenhough, T.J and Helliwell, J.R (1983) Prog Biophys Mol Biol 41, 67-124 Hendrix, J., Koch, M and Bordas, J (1979) J Appl Cryst 12, 467-472 McLaughlin, P.J., Stuart, D.I., Oihonomakos, N.G., Klein, H and Johnson, L.N (1984) Biochemistry 23, 5862-5873 Bartunik, H.D and Schubert, P (1982) J Appl Cryst 15, 227-231 Usha, R., Johnson, J.E., Moras, D., Thierry, J.C., Fourme, R and Kahn, R (1984) J Appl Cryst 17, 147-153 Huxley, H.E., Simmons, R.M., Faruqi, A.R., Kress, M., Bordas, J and Koch, M.H.J (1983) J Mol Biol 169, 469-506 Bartunik, H.D (1983) Nucl Inst Methods 208, 523-533 Sawyer, L and Helliwell, J (1984) Unpublished results Ford, G.C., Harrison, P.M., Rice, D.W., Smith, J.M and White, J.L (1984) Daresbury Lab Info Q 13, 15-16 Ramaseshan, S (1962) in Advanced Methods of Crystallography (Ramachandran, G.N., ed.), Academic Press, New York Phillips, J.C and Hodgson, K.O (1980) Acta Cryst A36, 8564364 Narayan, R and Ramaseshan, S (1981) Acta Cryst A37, 636-641 Schoenborn, B.P (1975) in Anomalous Scattering (Ramaseshan, S and Abrahams, S.C.,eds.), p 407, Monksgaard, Copenhagen 415 243a Templeton, D.H., Templeton, L.K., Phillips, J.C and Hodgson, K.O (1980) Acta Cryst A36, 435-442 243b Arndt, U.W., Greenhough, T.J., Helliwell, J.R., Howard, J.A.K., Rule, S.A and Thompson, A.W (1982) Nature 298, 835-838 244 Sakabe, N., Sakabe, K and Sasaki, K (1981) in Structural Studies on Molecules of Biological Interest (Dodson, G., Glusker, J.P and Sayre, D., eds.), pp 509-526, Clarendon Press, Oxford 245 Kossiakoff, A.A (1983) Annu Rev Biophys Bioeng 12, 159-182 246 Kossiakoff, A.A and Spencer, S.A (1981) Biochemistry 20, 6462-6484 247 Norvell, J.C., Nunes, A.C and Schoenborn, B.P (1975) Science 190, 568-569 248 Wlodawer, A and Hendrickson, W.A (1982) Acta Cryst A38, 239-247 249 Wlodawer, A., (1982) Prog Biophys Mol Biol 40, 115-159 250 Kossiakoff, A.A and Spencer, S.A (1980) Nature 288,414416 251 Mason, S.A., Bentley, G.A and McIntyre, G.J (1982) Brookhaven Symp Biol 32 252 Phillips, D.C (1967) Proc Natl Acad Sci U.S.A 57,493495 253 Wlodawer, A., Miller, M and Sjolin, L (1983) Proc Natl Acad Sci U.S.A 80, 3628-3631 254 Kossiakoff, A.A (1982) Nature 296,713-721 255 Hauptman, H and Karle, J (1953) The Solution of the Phase Problem, ACA Monograph No 3, Edwards Brothers, Ann Arbor 256 Karle, J and Karle, I.L (1966) Acta Cryst 21, 849-859 257 Germain, G and Wolfson, M.M (1968) Acta Cryst B24, 91-96 258 Gilmore, C.J (1982) in Computational Crystallography (Sayre, D., ed.), pp 126140, Clarendon Press, Oxford 259 Hull, S.E., Karlsson, R., Main, P., Woolfson, M.M and Dodson, E.J (1978) Nature 257, 206207 260 Jaynes, E.T (1957) Phys Rev 106, 62C631 261 Wilkins, S.W., Varghese, J.N and Lehmann, M.S (1983) Acta Cryst A39, 47-60 262 Wilkins, S.W., Varghese, J.N and Steenstrup, S (1984) in Indirect Imaging (Roberts, J.A., ed.), Cambridge University Press, Cambridge 263 Bricogne, G (1984) Acta Cryst A40, W 264 Bryan, R.K., Bansal, M., Folkhard, W., Nave, C and Marvin, D.A (1983) Proc Natl Acad Sci U.S.A 80,47284731 265 Diesenhoffer, J., Epp, O., Miki, K., Huber, R and Michel, M (1984) J Mol Biol 180, 385-398 266 Gilbert, W.A andPetsko, G A (1985), to be published Quoted in reference 218 267 Fink, A.L., Kav, D and Kolin, R (1985), to be published Quoted in reference 218 268 Deavin, A., Mathias, A.P and Rabin, B.R (1966) Biochem J 101, 14C 269 Kahn, R., Fourme, R., Bosshard, R., Wery, J.P., Dideberg, O., Rider, J.L., Brunie, S and Janin, J (1984) Symposium on New Methods in X-ray Absorption Scattering and Diffraction for Applications in Structural Biology, Bristol 270 Hails, J.E., Harding, M.M., Helliwell, J.R., Liddington, R and Papiz, M.Z (1984)Daresbury preprint DL/SCI/P428E, Daresbury Laboratory, Warrington This Page Intentionally Left Blank 417 Subject Index Absorption 275 fundamental aspects of 276 Absorption curve 341 Absorption edges 367 Absorption flattening 308,334 quotient 330 Absorption obscuring 336 Acid-base reactions 151 Active site geometry stereo diagram of 383 Active site mobility of 393 Acyl CoA-dehydrogenase 100 ADP 31 Adenine mass spectrum of 164 Adenosine mass spectra of 159 Adenosine dinucleotides 275 Adenylyl (3’,5’)cytidine260 Adrenal 206 Adriamycin 107 Alanine 21 Alkaloids analysis of 246 identification of 248 Allosteric proteins 349 Amide exchange 58 Amino acid 160 derivatives 219 Amino acid analysis 235 MS/MS contribution to 235 Aminocyclitol-aminoglucoside aintitiiotics 238 Androstane derivatives 209 Androstanediols 212 B/E spectra of 213 a-Angelicalactone 256 Anhydronucleotides 253 1,5-Anhydroribofuranose 239 1.5-Anhydroxyfuranose 239 Anisotropic electronic shielding 21 harmonic oscillator 300 thermal ellipsoids 392 Anis6tropy 306 metastable transitions 219 Anomalous dispersion 289 interactions 290 Anomalous scattering 366, 367 effects of heavy atom 368 Antibiotics 160, 161,240 Antibiotic compounds analysis of 236 Antiparallel alignment 283 Antiparallel-P-pleated sheet CD pattern 309 Apolipoprotein B 228 Aromatic side chains 59 Arrhenius equation 395 Artifact identification 233 signals 224 widths 192 Artificial intelligence 150 Ascorbate ESR spectra of radicals from 105 radical ion 104 Asparagine 219 Atmospheric pressure ionization 157 Atomic scattering factors 367 Auto oxidation 89 Averaging 52 Avian pancreatic polypeptide atomic positions in 392 Babinet’s principle 381 Bacteriorhodopsin 339 Bap 259 2-OH-Bap 259 Bap diol epoxide 257 E/E/CAD linked scan methods 215 B/E/CAD and MIKEjCAD spectra comparison of 261 B / E constant parent spectrum 168 418 B / E constant daughter spectrum 168 ( B / E )(I - E/E,)1’2 constant neutral fragment spectrum 168 B/E linked scan method 177 B / E linked scan method 179 B/E scan techniques determining peptide structures 221 Beefheart mitochondria CD spectra of 325 Beer’s law 276, 280 1,2-Benzanthracene 179 2,3-Benzanthracene 179 Benzo(a)pyrene257 Biogenic amines 236 Biological free radicals 85 Biomembranes CD data on 325 CD spectra of 328 Biotin 180 Biotine methyl ester 174 E / E linked scan spectra of 178 B / E linked scan spectra of 180 EI mass spectrum of 175 MIKElCAD analyses of 197 MIKE spectrum of 175 Bleomycin (Blm) 110, 124 complexes with metal ions 110 Cu(l1) complex of 126 ESR spectra of 112 structure of 11 X-band ENDOR spectrum of 128 Bloch equations 34 Blue shift 280,282 Biological molecules 160 Birefringence 291 Boltzmann equation expression 23 BPTI 53,58,388 ‘H-NMR spectrum of 46 BIQIQ 195 Bragg’s law 350 Broad-band proton decoupling 20 p-Bromophenol 183 Brownian rotational diffusion 82 t-Butyldimethylsilylethers of isomeric androstanediols 213 Caffeine 199 Calcium-binding protein 388 Calusterone molecular ion 207 Carbohydrates 160 determination of 151 Carbon tetrachloride 109 Carboxypeptidase 347,387 Carcinogenic diols 260 Cardenolides 160 Cascaroside A 163 Cationization reaction 160 Cationized molecular ions collisional (MIKE/CAD) spectra 244 Cationized molecular peak 243 Cellobiose 241 MIKEjCAD spectra of anomers 242 Cerane 170 Charge-exchange reactions 154 Charge localization 160 Charge stripping of MH’ 249 Charge-transfer reactions 154 Chemical ionization (CI) 155 of inorganic ions 90 Chemical modification 50 Chemical shift 13, 50 Chirality 291,292 quantitization of 300 p-Chlorobenzoic acid MIKE/CAD spectra of 191 p-Chlorophenol 183 Chlorpromazine 106 Cholestane 170 Cholestane skeletons 207 Cholest-I-ene-3-one 207 Cholesterol 203 Chromophore 275,283,296 Chrysene 179 Chymotrypsin 347,383, 387 CIDEP 72 Cinerubine A 241 structure and main decompositions of 241 Cinnamoylcocaine 248 distribution in E coca Lam 249 Circular birefringence 292 Circular dichroism 275,291,292,293, 302 spectra 302 reciprocity 299 Circularly polarized light 292 cisltrans distinction 209 Coacervate 13 Coal liquid mass spectrum, 189 Cocaine 198, 199,248 distribution in E coca Lam 249 molecular ion 198 Coca leaves 198 419 Collagen CD and absorption spectra 309 Collision-activated dissociation (CAD) 184 Collision-activation (CA) 167, 184 Collisional charge inversion 191 Collisional-induced decomposition 206, 224 of MH' ions 217 Collisionally activated decompositions (CAD) 167 Collision cell 184, 195, 234 Complementary probes 17 Computer protein crystallography and 406 ESR and 139 Concanavalin A 386 Condon expression 295 Conformational flexibility 394 Coniine 248 Coplanar alignment of chromophores 282 Copper proteins 113 Cosine law 366 COSY 44 Cotton effects 290, 295 Coupled metal centers 121 Coupled copper pairs 121 Coupling constants 51 Cowpea mosaic virus 402 Crambin 405 o-Cresol 183 Cross-peaks 45 Cross-relaxation 22, 62 Cryoprotectant 396 Crystal activity 386 Crystal field theory 109 Crystallisation 355 and data collection 360 and protein gross structure 383 cessation of growth and 358 crystal growth and 358 error treatment in 369 heavy atom derivatives 363 nucleation and 357 of membrane proteins 359 practical techniques 359 seeding and 357 Crystallographic techniques 387 and NMR evidence 387 Crystallography 355 atom displacement and 391 low temperature and 395 neutron diffraction in 404 Crystal structure 47, 382 CTPO 77 ESR spectra of 78 spin label of 80 Cu-Cu dimer ESR spectra of 116 CuKTSM, ESR spectra of 118 Cu-monomer ESR spectra of 116 Curie Point 161 CW experiments 70 Cyclic conformation 13 correlate 313 1,4-Cyclohexane 185 Cyclohexane oxide 185 Cyclohexanone 185 MIKE/CAD spectra of 187 Cyclopentadecapeptide stereo pair plot of molecular structure 314 Cyclopeptides fragmentation of 234 structure of 234 Cytidylyl(3',5')adenosine 260 Cytochrome b, reductase 100 Cytochrome c 383,384,388, 389,402 Cytochrome c oxidase ENDOR spectrum of 94 Cytochrome c peroxidase 93 ESR spectrum of 94 Cytochrome P-450 reductase 99 DAP I/IV digestion 230 Data collection 360 Debye-Waller factor 355 Decomposition maps 208 Defocused metastable scanning (HV scan) 169 Dehydrocephalosporin 250 Dehydroepiandrosterone 17 Deoxyadenine 257 Deoxycytidine 257 Deoxyguanosine 257 Derivative peptides 222 Desorption 158 by ionic bombardment (SIMS) 165 1,2-DiacyI glycerylphosphatidylcholine236 Diamond, synthetic 134 ESR signals of 134 Diastereoisomers 153 Diastereomeric diols 152, 157 Diastereomeric dipeptides 157 2,5'-Dichloro-3-methoxybiphenyl177 Diels-Adler retrogression 247 of isoquinolein derivative 247 420 Dieth ylenetriamine Cu(I1) complex of 126 Difference Fourier synthesis 378 Differential absorption flattening 334 Differential absorption obscuring 334 Differential light scattering 335,336 Differentiated 158 Diffractometer 361 Diffusion times 398 5a-6-Dihydroergosterol207 3a-17~-Dihydroxy-5~-androstane-l1,16a 206 Diketo steroids 212 H transfer 212 N,N-Dimethylacetamide 'H-spectra of 25 Dinucleotides 160,323 Di-OTBDMS groups 212 Dioxin (dimethoxane) 156 Dipolar Hamiltonian 84 Dipole-dipole interactions 21,60 Dipole interaction potential 281 moment 276 strength 277 Direct analysis of daughter ions (DADI) 172 Dispersion force interactions 275,284,286,290, 329 Dissymmetric 291 Distearoyl 236 Distinguishing ring junction geometries 212 DNA 161,284 DNA proteolyse products conventional mass spectra of 256 n-Dotriacontane 192 Double charged ions 160 Double refraction (see Birefringence) 291 Double resonance technique 19 Drug metabolism 106 radicals in 106 Drugs 218 Drude equation 294 E,/2 188 spectra 189 Edman degradation 218 EI/FD source 233 Elastase 383,399,400 Elastin 8-spiral of polypentapeptide 312 ELDOR 71,79,83,127,138 Electric transition dipole moment 277 Electromagnetic radiation Electron distribution 279 Electron density equation 353 Electron density map 373 computer graphics and 371 of glycogen phosphorylase b 372 of insulin 354 interpretation of 371 Electronic area detectors 361 magnetic moment 13 Electron impact ionization (EI) 149 Electron spin resonance classification of 69 Ellipticity 292,296,318,326,334,337 distortion quotient 334 END mechanism 79 MIKE spectra 247 ENDOR 71,86,127,138 8-Endorphin 163 Energy release 185 Enrichment 235 Enzyme activity 393 Erythromycin 163 Escherichia coli CAP protein 348 31P-NMR spectra of 35 ESR methods extensions of 122 ESR sample classes 133 ESR spectrometer accessories of 137 field modulation 136 reference arm microwave bridge 129 resonators of 135 sensitivity of 132 Estradiol mixture with estrone, estriol 202 Estriol mixture with estrone, estradiol 202 Estrone derivatives 203 Ethylhydrindanones 173 p-Ethyl phenol 183 E / V linked scan 176 Excitation resonance interaction 275,281,306 Excitons 286 External magnetic field 2,5 Eyring theory 300,310 one-electron theory of optical rotation 310 FAB 178 Family tree constructing 167 fragment ions 206 Fast-Fourier least-squares 376 Feher's formula 133 42 Ferriheme undecapeptide 288 Ferritin 402 Ferroheme undecapeptide CD of Soret (y) band 321 FFID 165 FFR first 184 second 184 Field desorption (FD) 160 Field ionization (FI) 160 Figure of merit 370 Fingerprint(s) 161, 167 Flattening quotients 333 Flavin methyl protons in 98 semiquinones 98 Flavin adenine dinucleotide (FAD) 298 CD spectra 324 isoalloxazine band 324 Flavoprotein ESR spectra of 99 ENDOR spectra of 99 Radicals 97 Fomblin oil 178 Four field ( E / B / E / B )tandem instrument 193 Free-induction decay 10 Free radical chemistry 86 Freeze-quenching 72,87 Frequency of radiation o-Fructose 161 FD spectrum of 161 Functional group 150 Furfuryl alcohol 257 Gasoline 199 GC/MIKE 196 GC/MS/HR 196 GCIMSIMS 196 Gentamicines 238 Gentibiose 241 MIKEjCAD spectra of anomers 244 Glucagon 388,389 Glucose 245 D-Glucose FD spectrum of 161 Glucose 6-phosphate titration curve of 32 Glutamine 219 Glyceraldehyde 3-phosphate dehydrogenase 383, 387 Glycine 219 Glycogen phosphorylase b 387, 399 electron density map of 372 Gorgosterol 217 Gramicidin A 31 CD and absorption spectra 317 stereo pair plots transmembrane channel 316 transmembrane channel 315 Gramicidin S 406 Grandparent ion 212 Guanosine 165 Haemoglobin 56,81, 287, 383, 385, 387 saturation recovery signal of 81 Haemaglutinin 348 Hahhzcterium halobium 276 Hedomycin 163 8-Helices 304, 315, 317 a-Helix 284, 326, 328, 342 polypeptide conformations 304 Helix-helix interactions 328 Heme chromophore 285 Heme-heme association 285 Heme-heme interactions 319,322 CD spectra 320 Heme moieties 319 Heme octapeptide 285, 321 absorption and C D spectra 322 Heme peptides 276 Heme proteins 276, 299 ESR of 115 Hemerythrin 348 Heme SBret band 288 Hemochromagen 319 Henderson-Hasseibalch pattern 54 Heterocycles 246 analysis of 246 n-Hexadecane 179 Hexokinase 384,386, 390 High-molecular-mass molecules 161 High-resolution 174 analyses 193 High voltage scan (HV) 168 Homologous proteins 49 Hordenine 246 structure of 246 Horseradish peroxidase 92 ESR spectra of 95 HR/MS 193 Humic acid 199 HV scan 168, 169 Hybrid mass spectrometer instruments 195 H ydrindanones MIKE spectra of 174 Hydrogen atom migrations 211 Hydrogen bonds 152 422 Hydrogen exchange mechanisms of 57 Hydroquinone 102 22/l-Hydroxycholesterol acetate 207 17-Hydroxy-19-nor-17a-pregn-4-en-20-yne-3-one 205 Hydroxyphenyl-2-butanone 199 Hydroxysterone 203 Hyperchromism 280,321 Hypochromism 276,280,305,321 Identical nuclei ensemble of IKE 168 IKE spectra 190,236 sophorose, kogibiose, etc 237 Ile-Ile] ions identification of 226 Imidazol 32 Cu(I1)complex of 126 Immobilization 87 Immobilized semiquinones 101 Immune response system 304 In-beam CI 158 In-beam EI 158 Incident power 70 Indole alkaloids 248 Information theory 407 Inhibitors 363 Insulin 383, 385 electron density map of 354 Internal energies 204 motional freedom 56 Ionization methods comparison between 162 Ion kinetic energy (IKE) 168, 171 Ion metastable studies 167 Ion-molecule reactions 151, 154 Ion precursor search 169 Isobaric ion 186 Isoleucine 158,219 Isomeric linkages identification MS/MS methodology 227 Isomeric monosaccharides 161 Isomeric peptides distinguishing 221 Isomeric peracetates of gentiobiose 236 kojibiose 236 laminaribiose 236 maltose 236 melibiose 236 sophorose 236 trehalose 236 Isomers differentiation of 235 Isoprene 188 Isoquinolein derivative Diels-Adler retrogression 247 Isotope "CI 191 exchange 49 Isotopic labelling 149 substitution 49 Joint probability distribution 378 J-spectroscopy 45 + Kaqiplus curve 51, 52 Keto and diketo steroids conventional mass spectra of 210 22-Ketocholesterol acetate 207 16-Keto estradiol 203 Kirkwood coupled oscillator mechanism 296 Konnert-Hendrickson method 375 Kuhn-Thomas sum rule 287 Lactate dehydrogenase 348 P-Lactoglobulin 386, 399 Lagrange method 373 Lanosterol 217 Larmor equation frequency Laser-induced desorption 164 Laser radiation 232 Laue photographs 403 LC/MS 150 Leigh effect 84 Leu-Leu] + ions identification of 226 Leucine 158,219 Librational entropy mechanism 314 Ligand binding 54 Light scattering 335 distortions and 308 Linear conformational correlate 313 Linked scan 175 techniques 183 Lipoprotein 228 Liquid phase magnetic parameters 76 Localization of D 200 of methyl group in indolic ring 249 423 Long-range NOE 48 Loop-gap resonator 134, 135 Lorentz correction factor 295 Lorentz factor 337 Lorentzian line 11 Low internal energy 160 Low temperature applications 400 devices 397 Lysozyme 347, 348,383, 384,386,387, 388,392, 405 Macromolecular spectra 50 Macromolecules 38 Magnetic moment 2,279 quantization of Magnetic transition dipole moment 278 Magnetogyric ratio Maleimide spin label effect of solvent of 77 Q-band spectra of 76 Maltose MIKE/CAD spectra of anomers 242,244 Maltotriose peracetate 243 Manganese 119 Marcellomycin 241 Marine sterols 215 Mass analyzed ion kinetic energy 172 Mass spectrometry artifacts in 208 Melanin ESR spectra of 103 Metabolic pathways tracing of 37 Metabolites compartmentation of 33 observation of 29 quantitation of 29 Metal centers 117 ions 109 Metalloenzymes ESR of 114 Metalloproteins ESR or 114 Metastable decompositions 209 ion method 200 Metastable peaks widths of 192 Metastable transitions 219 amino acid derivatives 219 Methionine 219 1-Methyladenine 254,255 2-Methyladenine 255 N,-Methyladenine 254, 255 5-Methyldeoxycytidine 5'-monophosphoric acid 254 Methyl hexapyranoside stereomers identification of 238 Methyl-thiohydantoin 21 derivatives N-Methyl tyramine 246 structure of 246 Methyloxime 215 Methyl trinor-5-cholestane-3-one-24-oate 207 Mexaline 199 Microcrystallinity 355 Mie scattering theory 329,338, 339 MIKE and MIKEjCAD spectra peptides 226 MIKE/CAD and B/E/CAD spectra comparison of 261 MIKEjCAD spectra 184, 185 analysis steroid mixtures 206 computers and 192 penicillin of 252 MIKE/CAD linked scan methods 215 MIKE scan techniques 221 determining peptide structures 221 MIKE spectra 190 fragments ions 223 M+ products from peptide isomers 223 Miscellaneous perturbations 48 Mitochondria1 membrane 100, 325 Modified nucleosides 258 Mole fractions 318 Molecular replacement 368, 369 vibrations 393 Mononucleotides 160 Monosaccharides 161 Morphine 199 Mouse liver "C-NMR spectra of 38 MS/MS 196,262 analysis of amino acids 235 applications of 201 comparisons with GC/MS 198 isomeric linkages identification 227 isotopic assay 200 methodology 167 peptide sequence determination with 227 techniques 193 Multifrequency ESR 70, 139 Multiheme proteins 322 424 Multiple ion detection (MID) 150, 215 Multiwavelength method in synchroton radiation 403 Myoglobin 347, 383, 393, 405 displacement values for 394 myosin 'H-NMR spectra of 40 Na,ATP 165 Naringin 163 Natural metal complexes 161 Negative chemical ionization 155 Negative ions 190 Neuraminidase from influenza virus 348 Neuropeptides sequence determination of 230 Neurotoxin 388 New ionization methods 150 NICI 156 NI(I1) complexes 113 Nicotinamide-adenine dinucleotide 298, 324 8-NAD CD spectra Nigerose 241 Nitro-anion free radicals 108 ESR spectra of 108 Nitrobenzoyl derivatives 235 Nitrogen hyperfine interaction 75 Nitro-radical anions 107 Nitroxide spin label 71, 72 Nitroxides 74, 79 intramolecular magnetic interactions 74 intramolecular motional mode 75 relaxation times of 74 NMR chemical exchange 24 in vitro 38 in vivo 28 line widths 25 NOE 22 reference line 16 solid-state 55 solution 55 spectrometer 27 NOE 22,48,50,51,56, 31 NOESY 44 Non-superimposable mirror images 291 Non-volatile nucleic acids 165 Norethynodrel 205 Norgetrol 205 N-terminal sequences identification of 229 Nuclear dipole Nuclear magnetization vector Nuclear moments Nuclear Overhauser enhancement Nuclear Overhauser effect (NOE) 22,48, 50,51, 56,311 Nuclear precession 2,4 spin quantum number Nucleophilic substitution reaction 153 Obscuring quotient 335 Octacosane 178 Octant rule 300 Oleonafic acid 184 Oligonucleotide 164 One-electron oxidation 92 reduction 97 One electron theory of optical rotation Eyring theory 300, 310 Optical activity 291 density (OD) 276 Optical rotation 291 fundamental aspects of 276 reciprocal relations 298 Optical rotatory dispersion 275, 291,294 Oscillation camera 361 photograph 362 Oxidation reactions 106 Papain 387 Parallel stacking of chromophores 282 Paramagnetic perturbations 48 probe 118 proteins 54 Paramagnetism 13 Partial molar rotatory powers 300, 302 Penicillin 250 MIKEjCAD spectra of 252 Pentapeptides 160 z-Pentapeptide 158 Peptide compounds analysis of 18 Peptide isomers MIKE spectra of M + 223 Peptides 160 determination of 151 distinguishing of isomers 221 MIKE and MIKE/CAD spectra of 226 425 Peptide sequence 157 Peptide sequences determination MS/MS methodology 227 Peptide structures 218 Peptide structures determining MIKE or B/E linked scan techniques 221 Peracetylated disaccharides 236 Permanent magnetic moment 13 Permethylated polypeptide 229 Permethylated tetrapeptide PPNICI spectrum of 228 Perturbing group 300 PGA absorption spectra of 327 C D spectra of 327 differential scatter of 331 Phase calculation 364 Phase probability function 369 Phenols 182 constant neutral fragment spectra of 182 Phenacetin 199 Phenobarbitol 199 Phenylalanine 60, 219 PhenyL(D5)propene oxide 185 Phenylthiohydantoin 218 Phosphate dinucleotides 262 Phosphocreatine ATP formation of 36 Phosphoglycerate kinase 383 Phosphoryl exchange 34 Phosphotriester 164 3-Phenylpropene oxide MIKE spectrum of 185 Photooxidation 90 Photoreduction 90 Plane polarized light 291 8-Pleated sheet 284 polypeptide conformations 304 P-NMR pH measurements by 32 Pollution studies 156 Poly-~-alanine305 absorption curve 305 CD spectra and 8-pleated sheet 309 CD spectrum 305 Polychlorinated biphenyl (PCB) 200 Polyethylene glycol (PEG) 356 Poly-L-glutamic acid (PGA) 326 refractive index of 336 Polymethyl- glutam am ate 309 Polypeptide conformations 304 Polysaccharides 161 analysis of 236 ’ Poly-L-serine 307 CD and absorption spectra 307 8-pleated sheet conformations 307 Poole’s treatise 125 Positive CI 151 ‘Potato’ phenomenon 137 Precession camera 361 Primary reagent radical 91 Probability distribution 370 Proline 219 n-Propylbenzene 199 Prosthetic group 275 Protease A 383,393 of Streptomyces griseus 393,399 Protein crystallography 347 direct methods in 406 dynamics of 390 flexibility of 390 maximum entropy in 406 methods in 350 synchroton radiation 401 Protein crystals 399 diffusion times in 399 Protein gross structure 383 Proteins solubility of 356 Protein structures X-ray crystallography and 348 Proton affinity 151 noise 20 Protonated molecule 151 Pseudoreference state 337, 341 approach 339 Pulse-chase 37 Pulsed ESR 70,87 Purple membrane 276,341 CD spectra of 340, 342 of Halobacterium halobium 276, 339 Pyrimidine Cu(l1) complex of 126 Pyrimidine bases SIMS spectra of 166 ~-Pyrrolid-2-ones301 Quadrant rule 300 Quadrupolar coupling 21 Quadrupoles 150 Quarter wave retarder 292 Quinone 102 Radicals 89 from enzymes 92 426 macromolecular 92 substrate-derived 93 Raffinose 163 Rearrangement 96 Reciprocal relations 276,297,324 Red cells NMR spectra of 30 Redox equilibria 102 Red shift 280, 282, 308, 313, 325, 327 Reference arm bridges 131 ESR bridge 130 Reference beam 280 Refinement 373 constrained-restrained 376 fast-Fourier least-squares 376 simultaneous energy 377 Refraction 275 Refractive index 288, 294,330, 336 Relaxation 10 data 60 mechanisms 20 parameters 51 time 79 Resolution enhancement 63,348 Resonance frequencies 18 Restrained least-squares 374 Retrosine 253 Reversed geometry identification 233 RF radiation absorption of Rhodes equation 284 Riboflavin 163 Ribonuclease 40,347,383,384,387,399,400,405 Ribonucleotide reductase 97 ESR spectra of tyrosyl radical of 97 Richard‘s Box 371 Riddelliine 251,253 Ring conformation 21 Ring current shift 47, 53 Rotational motion 82 strength 292, 293, 294, 295,296, 297, 301 Reversed geometry instruments 173 Salmon DNA MIKE/CAD spectra of 254 Salmon sperm DNA MIKEfCAD spectra of 255 pyrolysis of 254 Salting in 356 Salting out 356 Sample beam 280 Saturation recovery 70 Saturation transfer spectroscopy 83 S-band 122, 123 Scan methods MIKE/CAD or E/E/CAD linked 215 Scattering theory 338 Schiffs base 153 Screw sense 29 Segmental flexibility 64 Selected ion monitoring (SIM) 150, 199 Semiquinones 89,98, 102 ESR spectra of 96 Senecionine 253 Seneciphylline 253 Serine proteinase family 383 Shift correlation spectroscopy 45 Sickle-cell hemoglobin (HcS) 228 Side-chain decoupling 41 Sigmoid oxygen-binding curve of hemoglobin 322 Signal intensity 133 Sim’s derivation 378 Simulated MIKE 168 Smoluchowski equation 83 Solution absorption spectrum 328 structure 382 Solvent structure 380 Sophorose 241 Soret band 288,319 Spectral density function 60 Spectroscopic techniques 41 Spin-decoupling 19 Spin diffusion 62 echo 70 spectroscopy 125 Spin-immunoassay 81 Spin labels information 81 physical properties of 74 Spin-lattice relaxation 12 Spin-probe-spin-label method 121 Spin resonance data 14 Spin-rotation 21 Spin-spin coupling 17 constants 19 Spin-spin relaxation 10 Spin-stabilization 88 fi-Spiral 304,312 polypentapeptide of elastin 312 Split-beam spectrophotometer 280 SRC I1 refined coal 194 427 1-Stearoyl-2-oleo1236 Sterane 170 Stereochemical effects 155 Stereochemistry of functional groups 209 Steroid compounds analysis 201 Steroid skeletons 208 Stone theory 80 Strawberry flavour 199 Strong electric dipole moment 296 Structure factor 353,364,365,373 amplitude of 366 Studies of mixtures 197 Subtilisin 383 Succinate dehydrogenase 100 Sucrose FD and E/E/FD spectra of 240 Supersaturation 356 Sussman method 376 Suspension absorption spectrum 328 Synchroton 401 Tandems 193 TBDMS ether 215 Temperature dependence 393 Temperature factors 391 Terpane 170 Testerone 217 Testosterone 217 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) 193,199 Tetramethylthiazolodine 250,251 TFA group 239 Thenoyl trifluoroacetone 101 Thermal equilibrium vibrations of atoms 391 Thermo-labile 157 biological molecules 165 Thiophene 199 Threonine 219 Time domain ESR 70,139 Tissue radicals 104 TOF instruments 263 Torque Tortoise eggwhite lysozyme electron density map of 381 Transition dipole moment 285 Transition probabilities 61 Translational diffusion 83,84 Transmembrane channel 305 Transverse relaxation 10 Trehalose 163,241 Trierucin 162 Trimyristin 162 Triolein 162 Triose phosphate isomerase 348,386 Tripeptide mixture OH-/NICI mass spectrum of 231 Tripeptides 160 Triphenylene 179 Triple quadrupole instruments 194 Trypsin 383,388,405,406 neutron diffraction studies on 405 Trypsin inhibitor (BPTI) 388 Trypsinogen 386,393 Tryptopan 219 P-Turn conformational feature 310 type I310 type I1 310,313 type I1 CD pattern 312 Twist conformation 212 Two-dimensional Fourier transform 43 NMR spectroscopy 44 Tyrosine 60 Ubine hydrochloride MIKE spectra of 247 Ubisemiquinone ESR spectra of 100 Unimolecular decompositions under electron impact 214 Urine 217 glucose detection in 245 samples 198 Valine 219 Vanadyl 120 Vector model of excitation splitting 282 Vestal interface 159 Vitamin C 309 Vitamins 160,163 Wiggler beam lines 403 Xanthine oxidase 102, 109 X-band 122 X-ray crystallography 53 X-ray determination information theory for 407 X-ray diffraction 350,352 schematic diagram of 352 Zeeman interaction 75 This Page Intentionally Left Blank ... Classification with respect to order, motion and stability Nitroxide radical spin labels and spin probes (a) Labels and probes (b) Physical properties of spin labels (i) Intramolecular magnetic interactions... Relaxation times (iii) Intramolecular motional modes (c) Spin-label information content (i) Intensity (ii) Lineshapes and rotational motions (iii) Spectral diffusion of saturation and rotational... so-called spin-spin coupling Figure 11 shows the two possible orientations of nuclear and valence electron spins in a covalent bond Note that whereas nuclear spins can be parallel or antiparallel