vot. 17 (1955) SHORT COMMUNICATIONS, PRELIMINARY NOTES I4I Studies on the structural basis of ribonuclease activity Since the fundamental studies of SUMNER and of NorTHROP AND Kuwnirtz on the protein nature of enzymes, most investigators in the field have tacitly assumed the relative inviolability of enzyme structure, both as regards the covalent linkages between amino acids in the peptide chains and also with relation to geometrical configurations imposed by non-covalent binding forces. Teleological reasoning has led to theories in which the protein structure as a whole was assumed to play a dominant role in catalytic action by transferring energy to the catalytic center through a resonance chain made up of regular hydrogen bonded structures?.?.?. It has, however, been known for some time that certain groupings (e.g. epsilon-amino groups on the lysine residues of pepsin‘) could be shown to be non-essential in catalytic activity and in more recent years it has been possible to modify, more drastically, certain biologically active proteins without apparent loss in activity. Thus, insulin®.*; ACTH?, TMV3, a-chymotrypsin®, and ribonuclease” have been found to exhibit their normal behavior even after removal with carboxypeptidase of one or more amino acid residues from the carboxyl-terminal ends of the peptide chains. In the case of the first three listed, such experiments are partly equivocal since activity tests (which might involve resynthesis) must necessarily be performed upon actively metabolizing tissues or upon intact organisms. The studies on a-chymotrypsin and ribonuclease, however, were performed using in vitro methods and are therefore of particular interest since they clearly suggest that the painstaking evolution of a unique structure for these enzymes (and by inference of other enzymes as well) may have been directed towards the fulfillment of more subtle biological requirements than catalytic activity alone. Previous studies have shown that a number of enzymes (e.g. trypsin, subtilisin!*, pepsin") maintain activity even when tested in strong urea solutions where, according to considerations by KauzMann"™ and ScHELLMAN45, secondary hydrogen-bonded structures are likely to be seriously distorted. However, the absence of adequate physical and chemical data on these proteins has precluded a proper appraisal of this rather surprising phenomenon. Recent investigations on ribonuclease structure! have suggested that this molecule exists in solution as a monodispersed!? unit consisting of a single peptide chain of 128 amino acids!®, cross- linked through four disulfide bonds. It has been found that restricted subtilisin digestion leads to a modified, fully active derivative® in which a new N-terminal end group can be demonstrated, the peptide chain which it terminates being attached to the main body of the molecule by disulfide bonding®*. Restricted pepsin digestion, on the other hand, appears to completely inactivate the enzyme, even after the cleavage of one or two peptide bonds®!, Complete loss of activity is also produced upon rupture of the disulfide linkages with performic acid'6. A series of experiments have now been conducted in this laboratory with the purpose of charac- terizing this enzyme under different conditions. They include (1) measurements of optical rotation and its dispersion (unpublished material, SCHELLMAN, compare”), (2) measurement of viscosity (un- published data of HaRRINGTON), (3) determinations of the rate of exchange of peptide bond hydrogen atoms (23.24, and unpublished data by Aa. Hvipt), and (4) measurements of enzymic activity against RNA and uridine-2’, 3’-phosphate. The results are summarized in Table I where a comparison is made between native RNase, RNase in 8 M urea or 2.5 M guanidine chloride, and oxidized RNase. TABLE I Native & M urea Oxidized on IN 60 ae Exchangeable . ‘ . . 70 rapidly ae : 40) peptide-bonded hydrogens exchangeable Allrapid” All rapid N (theoretical-127) *< 2 Intrinsic viscosity 0.033 0.089 0.116 “ in (g/100 ml)-+ \ [a]? 74.0 108 QL.I 1 Ac 2330 2170 2220 \ 5 “ Measurements made in 2.5 M guanidine chloride. = S Fig. 1. First order plot of change in viscosity of RNA with time of during RNase action in presence and absence of 8 M urea. RNA Hy N (2% in 8 Af urea, pH 5.0, 20°C) + RNase (2.6 y/ml 8 M urea, —3 pH 5.0): O. Same without urea @. 4, = initial viscosity; Af = £h 20 4060 35° final viscosity. time, minutes [200] 142 SHORT COMMUNICATIONS, PRELIMINARY NOTES vor. 17 (1955) All the data indicate that the enzyme molecule denatured by urea or guanidine chloride approaches in shape and disorientation the molecule produced by performic acid oxidation. The activity of RNase toward RNA (Schwarz, dialyzed against 0.2 M acetate buffer, pH 5.02) was determined by measuring the change in viscosity with time. The results shown in Fig. 1 indicate that ribonuclease is fully active in 8 M urea solution (the enzyme having been incubated in 8 M urea for two days at 5° C prior to testing against RNA, also dissolved in 8 M urea). Determinations of non-precipitable!* and dialyzable e,,, absorbing material during the reaction and after maximum viscosity change also indicated that the hydrolysis of the substrate had proceeded unimpaired. The enzyme was further tested against the synthetic substrate, uridine-2’, 3’-phosphate® in the presence and absence of urea as above. The course of the reaction in this case was followed by a microspectrophotometric method developed by Dr. Frep M. Ricuarps. The hydrolysis of this substrate appears to be somewhat slower in urea than in its absence although an essentially linear production of uridylic acid with time was demonstrable by means of paper chromatographic analysis®* of the reaction mixture. The above data, considered together, suggest that only a relatively small part of the ribonuclease molecule is directly involved in catalytic activity and, that in the conversion from the native to the extended form, this part, the active center, may be protected from deleterious unfolding by restricting cross linkages. It seems impossible, at any rate, that an ordered secondary structure is responsible for its properties as a catalyst. The data further support the possibility that a considerable part of the enzyme structure may be superfluous from the catalytic standpoint, a possibility that is also suggested by the autodigestion experiments on pepsin previously published by PERLMANN?”’, The authors wish to thank Dr. L. Hepper for his generous gift of uridine-2’, 3’-phosphate. CHRISTIAN B. ANFINSEN* Carlsberg Laboratorium, Copenhagen (Denmark) W. F. Harrincton** Aa. Hvipt K. LINDERSTROM-LANG M. OTTESEN JouNn ScHELLMAN “* 1 A. SzENT-GyORGYI, Science, 93 (1941) 609. 2K. Z, Wirtz, Naturforsch., 2b (1948) 94; Chem. Zenir., 119 (1948) 657. 3 T, A. GEISSMAN, Quart. Rev. Biol., 24 (1949) 309. 4R. M. Herriott anp J. H. Norturop, J. Gen. Physiol., 18 (1934) 35. § J. Lens, Biochim. Biophys. Acta, 3 (1949) 367. 6 J. 1. Harris, J. Am. Chem. Soc., 74 (1952) 2944. 7 J. 1, Harris ann C. H. Li, J. Biol. Chem., in press. 8 J. I. Harris anp C. A. Knicut, Nature, 170 (1952) 613. ® I, A. GLADNER AND H. Neuratu, J. Biol. Chem., 206 (1954) 911. 10 G. KALNITSKY AND E, E. ANDERSON, Biochim. Biophys. Acta, 16 (1955) 302. 11 L. KorsGAARD-CHRISTENSEN, Compt. rend. trav. lab. Carlsberg, Sér. chim., 28, No. 2 (1952) 37. 12M. OTTESEN, unpublished results. 18 J, STEINHARDT, J. Biol. Chem.,123 (1938) 543. 14 W. Kauzmann, in Mechanism of Enzyme Action, edited by W. D. McELroy anp B. Grass, Johns Hopkin’s Press, Baltimore 1954. 15 J. SCHELLMAN, Compt. rend. trav. lab. Carlsberg, Sér. chim., 29, No. 15 (1955) 230. 16 C. B. ANFINSEN, R. R. REDFIELD, W. L. CHoate, J. PaGE anp W. R. Carroit, /. Biol. Chem., 207 (1954) 201. wD. W. Kupke, unpublished results. 18 C,H. W. Hirs, W. H. STEIN AND S. Moorz, J. Biol. Chem., 211 (1954) 941. WS. M. Katman, K. LinpErRsTROM-LANG, M. OTTESEN AND F. M. Ricuarps, Biochim. Biophys. Acta, 16 (1955) 297. 20 F, M. RICHARDS, unpublished results. 21 C. B. AnFInsEN, J. Biol. Chem., 196 (1952) 201. 22K, LINDERSTROM-LANG AND J. SCHELLMAN, Biochim. Biophys. Acta, 15 (1954) 156. 33 Aa. Hvipt anp K. LinpDERSTROM-LaNG, Biochim. Biophys. Acta, 16 (1955) 168. ™ Aa. Hvipt, G. JOHANSEN, K. LINDERSTROM-LANG AND F. VasLow, Compt. rend. trav. lab. Carlsberg, Sér. chim., 29, No. 9 (1954). % D. M. Brown, D. I. MaGRaTH AND A. R. Topp, J. Chem. Soc., (1952) 2708. 38 R. MARKHAM AND J. D. SmitH, Biochem. J., 52 (1952) 552. 37 G. E. PERLMANN, Nature, 173 (1954) 406. Received March 4th, 1955 “ Rockefeller Public Service Award, 1954-55. ** Post-doctoral fellow of the National Cancer Institute, USPHS. [201]