Contributors to Volume XLIX Article numbers are in parentheses following the names of contributors. Affiliations listed are current. LAWRENCE J. BERLINER (18), Department WILLIAM E. HULL (13), Bruker-Physik AG, of Physical Chemistry, University "fo Rheinstetten-Fo, West Germany Gronigen, Gronigen, ehT Netherlands JAMES S. HYDE (19), ehT Medical College qf J. F. BRANDTS (1), Department of Chemis- Wisconsin, Milwaukee Count)' Medical try, University of Massachusetts, Complex, Milwaukee, Wisconsin Amherst, Massachusetts AICIRTAP C. .lost (17), Institute of EZRA DANIEL (11), Department of Molecular Biology, and Department qf Biochemistry, ehT George S. Wise Center Chemistry, University of Oregon, Eugene. of Life Sciences, leT Aviv University, leT nogerO Aviv, Israel K. S. KRISHNAN (1), Department "q J. J. ENGLANDER (3), Department of Chemistry, University of Massachusetts, Biochemistry and Biophysics, University Amherst, Massachusetts of Pennsyh,ania School of Medicine, PAUL C. LEAVIS (10), Department o/" Mus- Philadelphia, Pennsylvania cle Research, Boston Biomedical Re- S. W. ENGLANDER (3), Department of search Institute, Boston, Massachusetts Biochemistry and Biophysics, University SHERWIN S. LEHRER (10), Department "/o of Pennsylvania School of Medicine, Muscle Research, Boston Biomedical Re- Philadelphia, Pennsylvania search Institute, and Department 1~ HAROLD P. ERICKSON (4), Department of Neurology, Harvard Medical School, Anatomy, Duke University Medical Cen- Boston, Massachusetts ter, Durham, North Carolina KEVIN LEONARD (4), European "uhuceloM JAMES A. FEE (20), Biophysics Research -iD Biology Laboratory, Heidelberg, West ,noisiv Department of Biological Chemis- Germany try, University of Michigan, Ann Arbor, ALBERT S. MILDVAN (15), Institute raJ. Michigan Cancer Research, Fox Chase Cancer Cen- O. HAYES GRIEFITH (17), Institute of ter, Philadelphia, Pennsylvania Molecular Biology, and Department of A. G. REDHELD (12, 16), Departments "/q Chemistry, University of Oregon, Eugene, Physics and Biochemistry, and ehT nogerO Rosensteil Bask" Medical Science Re- NHOJ J. GR|MALDI (14), Corporate search Center, Brandeis Universio,. Research and Development, General Waltham, Massachusetts Electric Company, Schenectady, New HAROLD A. SCHERAGA (5), Department qf kroY Chemistlw, Cornell University, Ithaca. RAJ K. GUPTA (15), lnstitutelbr Cancer Re- New kroY search, Fox Chase Cancer Center, MORDECHAI YKSVOLOKOS (11), Department Philadelphia, Pennsylvania of Biochemistry, ehT George S. Wise Cen- S. A. HAWLEY (2), Department of Physiol- ter of Lifo Sciences, leT A eiv University, ogy, School of Public Health, Harvard leT Aviv, Israel University, Boston, Massachusetts KAHCZ1 Z. STEINBERG (7), Chemical Physk's BARTON HOLMQUIST (6), Biophysics Re- Department, We&mann Institute of Sci- search Laboratory, Peter Bent Brigham ence, Rehovot, Israel Hospital, and Department of Biological EUGENE S. STEVENS (9), Department ff¢ Chemistry, Harvard Medical School, Bos- Chemistry, State University of New kroY ton, Massachusetts at Binghamton, Binghamton, New kroY vii viii CONTRIBUTORS TO VOLUME XLIX BRIAN D. SYKES (13, 14), Department of Chemistry, Harvard Medical School, Bos- Biochemistry, ehT University of Alberta, ton, Massachusetts Edmonton, Alberta, Canada HAROLD E. VAN WARr (5), Biophysics Re- DOUGLAS H. TURNER (8), Department of search Laboratory, Peter Bent Brigham Chemistry, College of Arts and Science, Hospital, and Department of Biological ehT University of Rochester, Rochester, Chemistry, Harvard Medical School, Bos- New kroY ton, Massachusetts BERT L. VALLEE (6), Biophysics Research WILLIAM A. VOTER (4), Department of Laboratory, Peter Bent Brigham Hospi- Anatomy, Duke University Medical Cen- tal, and Department of Biological ter, Durham, North Carolina Preface This is the second of two volumes of "Enzyme Structure" devoted to updating the treatment of physical methods (part F appeared recently). Although coverage of the various techniques is not exhaustive, it is hoped that the intent of presenting a broad coverage of currently available methods has been reasonably fulfilled. As in the past, these volumes present not only techniques that are currently widely available but some which are only beginning to make an impact and some for which no commercial standard equipment is as yet available. In the latter cases, an attempt has been made to guide the reader in assembling his own equipment from individual components and to help him find the necessary information in the research literature. In the coverage of physical techniques, we have departed somewhat in scope from the traditional format of the series. Since, at the termination of an experiment, physical techniques frequently require much more in- terpretation than do organic ones, we consider that brief sections on the theoretical principles involved are highly desirable as are sections on theoretical and mathematical approaches to data evaluation and on as- sumptions and, consequently, limitations involved in the applications of the various methods. The organization of the material is the same as in Parts C and D. While Part F gave coverage mainly to the measurements of molecular weights and interactions, this volume is devoted to conformational analysis and optical and resonance spectroscopy. We wish to acknowledge with pleasure and gratitude the generous cooperation of the contributors to this volume. Their suggestions during its planning and preparation have been particularly valuable. Academic Press has provided inestimable help in the assembly of this volume. We thank them for their many courtesies. C. H. W. HIRS EGRES N. FFEHSAMIT ix METHODS IN ENZYMOLOGY EDITED BY Sidney P. and Colowick Nathan .O Kaplan VANDERBILT UNIVERSITY DEPARTMENT OF CHEMISTRY SCHOOL OF MEDICINE UNIVERSITY OF CALIFORNIA NASHVILLE, TENNESSEE AT SAN DIEGO AL JOLLA~ CALIFORNIA I. Preparation and Assay of Enzymes II. Preparation and Assay of Enzymes III. Preparation and Assay of Substrates IV. Special Techniques for the Enzymologist .V Preparation and Assay of Enzymes VI. Preparation and Assay of Enzymes )deunitnoC( Preparation and Assay of Substrates Special Techniques VII. Cumulative Subject Index ix METHODS IN ENZYMOLOGY FEIHC-NI-SROTIDE Sidney P. Colowick Nathan O. Kaplan EMULOV VIII. Coml)lex Carbohydrates Edited by HTEBAZILE F. DLEFUEN DNA ROTCIV ~(BUBSNI]( EMULOV IN. Carbohydrate Metabolism Edited !!b W~LLIS A. DOOW EMULOV X. Oxidation "tnd Phosl~horylation Edited bg DLANOR .V~" KOORBATbE DNA DRANYAI~ E. NAMLLUP EMULOV XI. Enzyme Structure Edited bg C. H. W. HIRS EMULOV XII. Nucleic Acids (Parts A and B) Edited !!b ECNERWAL NAM.SSOR3( KIVIE EVADLO[~ AND EMULOV XIII. Citric Acid Cycle Edited by ,l. M. NIETSNEWOL EMULOV XIV. Lipids Edited by ,l. M. NIETSNEWOL EI~.:~LOV XV. Steroids and Terpenoids Edited bg I~AYMOND B. CLAYTON EMULOV XVI. Fast Reactions Edited by KENNETH K,:STL~ Et~.VLOV XVII. Metabolism of Amino Acids and Amines (Parts A and B) Edited bg TREBREH ROBAT DNA CELIA WHITE ROBAT \'OH'ME XVIII. Vitamins and Coenzymes IParts A, B, and C) Edited by DLANOD B. KCIr~.ROCCM DNA LEMUEL D. WRIGHT E~.CLOV XIX. Proteolytic Enzymes Edited by EDURTREG E. NNAMLREP DNA OLZSAL DNAROL \'OLUME XX. Nucleic Acids and Protein Synthesis (Part C) Edited by KIVIE EVADLOI~ DNA EC',':ERWAL NAMSSORG xiii xiv METHODS IN ENZYMOLOGY EMULOV XXI. Nucleic Acids (Part )D Edited yb ECNERWAL NAMSSORG DNA KIVIE EVADLOM EMULOV XXII. Enzyme Purification and Related Techniques Edited yb WILLIAM B. YBOKAJ EMULOV XXIII. Photosynthesis (Part A) Edited yb YNOHTNA NAS ORTEIP EMULOV XXIV. Photosynthesis and Nitrogen Fixation (Part B) Edited yb YNOHTNA NAS ORTEIP EMULOV XXV. Enzyme Structure (Part B) Edited yb C. H. W. HIRS DNA EGRES N. FFEHSAMIT EMULOV XXVI. Enzyme Structure (Part C) Edited yb C. H. W. HIRS DNA EGRES N. FFEHSAMIT EMULOV XXVII. Enzyme Structure (Part D) Edited yb C. H. W. HIRS DNA EGRES N. FFEHSAMIT EMULOV XXVIII. Complex Carbohydrates (Part B) Edited yb ROTCIV GRUBSNIG EMULOV XXIX. Nucleic Acids and Protein Synthesis (Part )E Edited yb ECNERWAL NAMSSORG DNA KIVlE EVADLOM EMULOV XXX. Nucleic Acids and Protein Synthesis (Part F) Edited yb KIVIE EVADLOM DNA ECNERWAL NAMSSORG EMULOV XXXI. Biomembranes (Part A) Edited yb YENDIS REHCSIELF DNA LESTER REKCAP EMULOV XXXII. Biomembranes (Part B) Edited yb YENDIS REHCSIELF DNA LESTER REKCAP EMULOV XXXIII. Cumulative Subject Index Volumes I-XXX Edited yb AHTRAM G. DENNIS DNA EDWARD A. SINNED EMULOV XXXIV. Affinity Techniques (Enzyme Purification: Part B) Edited yb WILLIAM B. YBOKAJ DNA MEIR KEHCLIW METHODS IN ENZYMOLOGY XV EMULOV XXXV. Lipids (Part B) Edited by JOHN M. NIETSNEWOL EMULOV XXXVI. Hormone Action (Part A: Steroid Hormones) Edited by W. BERT O'MALLEY DNA JOEL G. NAMDRAH EMULOV XXXVII. Hormone Action (Part :B Peptide Hormones) Edited by W. BERT O'MALLEY DNA JOEL G. NAMDRAH EMULOV XXXVIII. Hormone Action (Part C: Cyclic Nucleotides) Edited by JOEL G. NAMDRAH DNA BERT W. O'MALLEY EMULOV XXXIX. Hormone Action (Part D: Isolated Cells, Tissues, and Organ Systems) Edited by JOEL G. NAMDRAH DNA BERT .W O'MALLEY EMULOV XL. Hormone Action (Part E: Nuclear Structure and Function) Edited by BERT W. O'MALLEY DNA JOEL G. NAMDRAH EMULOV XLI. Carbohydrate Metabolism (Part B) Edited by W. A. DOOW EMULOV XLII. Carbohydrate Metabolism (Part C) Edited by .W A. DOOW EMULOV XLIII. Antibiotics Edited by JOHN H. HSAH EMULOV XLIV. Immobilized Enzymes Edited by KLAUS HCABSOM EMULOV XLV. Proteolytic Enzymes (Part B) Edited by LASZLO DNAROL EMULOV XLVI. Affinity Labeling Edited by WILLIAM B. YBOKAJ DNA MEIR W|LCHEK EMULOV XLVII. Enzyme Structure (Part E) Edited by C. H. W. HIRS DNA EGRES N. EFEHSAMIT xvi METHODS IN ENZYMOLOGY VOLUME XLVIII. Enzyme Structure (Part F) Edited yb C. H. W. HIRS DNA SERGE N. TIMASHEFF VOLUME XLIX. Enzyme Structure (Part G) Edited yb C. H. W. HIRS DNA SERGE N. TIMASHEFF VOLUME L. Complex Carbohydrates (Part C) (in preparation) Edited yb VICTOR GRUBSN1G EMULOV LI. Purine and Pyrimidine Nucleotide Metabolism (in preparation) Edited yb PATRICIA A. HOFEEE DNA MARY ELLEN JONES VOLUME LII. Biomembranes (Part C) (in preparation) Edited yb SIDNEY FLEISCHER DNA LESTER REKCAP VOLUME LIII. Biomembranes (Part D) (in preparation) Edited yb SIDNEY ELEISCHER DNA LESTER REKCAP VOLUME LIV. Biomembranes (Part E) (in preparation) Edited yb SIDNEY FLEISCHER DNA LESTER REKCAP 1 SCANNING CALORIMETRY 3 1 Scanning Calorimetry yB K. S. KmSHNAN and J. F. BRANDTS An interesting property of biological macromolecules is their ability to undergo structural changes with temperature. Lipids in aqueous environ- ments undergo gel-to-liquid-crystal transitions, proteins undergo unfold- ing or denaturation transitions, and base-paired nucleic acids unwind. These transitions have proved to be highly cooperative in nature and hence are sharply defined. They may be reversible or irreversible and are strongly influenced by solvent. Calorimetry suggests itself as an ideal technique for measurement of thermodynamic parameters associated with such phenomena. The technique of calorimetry has been the subject of an extensive review in an earlier volume, ~ and that review included a fairly complete discussion of differential heat capacity calorimetry. Hence, the objectives of the present chapter are fairly limited in scope. It will be devoted primar- ily to some of the newer areas of research where differential heat-capacity calorimetry'" appears to be a powerful tool. The frontier areas of modern biochemical research are more suscepti- ble to meaningful calorimeteric investigation than has ever been the case in the past. This is due to the complicated nature of the systems now being investigated. These would include "superstructures," such as mem- branes, ribosomes, viruses, multisubunit proteins, and chromatin, to name a few. With many of these systems, certain of the physical methods which have proved to be the most powerful in the elucidation of the structural features of simpler systems in the past are much less valuable. For example, nuclear magnetic resonance (NMR) and circular dichroism (CD) both suffer fl-om problems associated with the size and heterogeneity of many of these superstructures. Also, because of their complexity, less detailed answers to structural questions constitute more meaningful prog- ress than would be true with monomolecular systems. Thus, techniques with rather low information content, like calorimetry, can sometimes be more wfluable than the very sophisticated techniques. Differential heat-capacity calorimetry has the potential for resolving all the structural transitions that a system undergoes as it is perturbed by systematic temperature variation. Even for a monomolecular system, there may be multiple transitions due to the fact that the molecule is ' J. M. Sturtevant. this series Vol. 26, p. 227. " In this chapter, we use the terms 'scanning calorimetry" and "'differential heat-capacity calorimetry" interchangeably. 4 CONFORMATION AND TRANSITIONS 1 folded into more than one "cooperative unit." Thus, it appears that bovine serum albumin consists of three distinct structural units that un- fold nearly independently. ~:.'-' Immunoglobulins also have at least three semi-independent regions? On the other hand, some of the small proteins, such as ribonuclease, chymotrypsinogen, myoglobin, and cytochrome c appear to possess only one major cooperative unit? ~" We will refer to these independent or semi-independent structural re- gions as domains. The most important feature of heat-capacity calorimetry which makes it a powerful tool for modem biochemical re- search is that it often permits one to look individually at some or all of the different domains in a complicated multidomain system. In the human erythrocyte membrane, as an example, at least six distinct structural transitions can be seen with a sensitive heat-capacity calorimeter over the temperature range from 40 ° to 80°. 7 ': These arise from different domains on the native membrane. Two of the domains appear to consist predomi- nantly of proteins, while the others most likely involve phospholipid in addition to protein. Calorimetric studies of this type can be important over and above the specific information that might be obtained as to the nature of the structural transition that gives rise to each endotherm. For example, at least one of the recognizable domains of the erythrocyte membrane can be shown by calorimetry to be intimately involved in anion transport, since the transition of this domain interacts strongly with specific inhibitors of anion transport when these are present in function- ally effective concentrations. In such a way, calorimetry can be used to elucidate the various interactions in which a domain might be involved, and thereby identify its function. This seems to be the real value of calorimetry in biochemical research involving multidomain systems. Thus, the calorimetric "'spectrum'" (heat absorption versus thermal en- ergy) can be used in much the same way as spectra from more con- ventional forms of spectroscopy such as NMR or infrared (IR). If the different transitions of a system can be separately resolved on the temper- ature axis, then one is able to study each domain independently. Any '- R. V. Decker and J. F. Foster. Biochemistry 5, 1242 (1966). ~: V. R. Zurawski, W. J. Kohr, and J. F. Foster, Biochemistry 14, 5579 (1975). L G. M. Edelman, B. A. Cunningham, W. E. Gall, E D. Gottlief, V. Rutishauser, and M. J. Waxdal, Proc. Natl. Acad. Sci. U.S.A. 63, 78 (1969). '; W. M. Jackson and J. F. Brandts, Biochemistry 9, 2294 (1970). ;' P. L. Privalov and N. N. Khechinashvili, J. Mol. Biol. 86, 665 (1974). ~ W.M. Jackson, J. Kostyla, J. Nordim and J. F. Brandts, Biochemistry 12, 3602 (1973J. J. F. Brandts, K. Lysko, T. A. Schwartz, L. Erickson, R. B. Carlson, J. Vincentelli, and R. D. Taverna, Colloq. Int. C. N. R. S. 246, 169 (1976). 0 j. F. Brandts, L. Erickson, K. Lysko, T. A. Schwartz, and R. D. Taverna, Biochemistry Biochemistry 16, 3450 (1977).