Reprinted from Tue JouRNAL oF Bro.ocgica, CHEemIstry Vol. 207, No. 1, March, 1954 STUDIES ON THE GROSS STRUCTURE, CROSS-LINKAGES, AND TERMINAL SEQUENCES IN RIBONUCLEASE By CHRISTIAN B. ANFINSEN, ROBERT R. REDFIELD, WARREN L. CHOATE, JUANITA PAGE, anp WILLIAM R. CARROLL (From the Laboratory of Cellular Physiology, National Heart Institute, and the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, United States Department of Health, Education, and Welfare, Bethesda, Maryland) (Received for publication, October 2, 1953) Previous studies (1, 2) have shown an asymmetric labeling of amino acids derived from different points along the chains of radioactive proteins. These results have suggested, as one major possibility, that proteins are * synthesized by an assembly mechanism involving the condensation of preformed peptide fragments not in ready equilibrium with the pools of free amino acid. An attractive corollary of this general hypothesis is that such fragments may serve as common building blocks for a number of proteins. The studies of Sanger and his collaborators (3, 4) have elucidated the amino acid sequences in the insulin molecule and have made available methodology (8, 5) for similar studies on other proteins. The present report deals with the progress to date of our investigations on the fine and gross structures of ribonuclease, particularly in regard to N and C terminal sequences and the nature of the cross-linkages in this single chain protein. It is hoped that, as comparison of the fine structure of these two pancreatic proteins becomes possible, a more rational basis for experiments on pro- tein synthesis in this tissue can be devised. Materials and Analytical Methods The homogeneity of each lot of commercial erystalline ribonuclease (Armour and Company or Worthington Biochemical Laboratory) used in these studies was checked ultracentrifugally, and, in most cases, by electrophoresis. These preparations showed a single N terminal amino acid (lysine) by the dinitrofluorobenzene (DNFB) method. Their specific enzyme activity corresponded to the accepted values in the literature (6). Analytical values obtained for phenylalanine (7) (3 residues per mole) and for cysteine estimated as cysteic acid (8 residues per mole) agreed within 5 to 10 per cent with the values obtained on ion exchange columns by Hirs, Moore, and Stein. The preparations of pepsin, chymotrypsin, and trypsin used for degrada- ‘ Hirs, W., Moore, 8., and Stein, W. H., personal communication. 201 202 STRUCTURE OF RIBONUCLEASE tion were crystalline commercial products. Worthington carboxypep- tidase was recrystallized eight times and treated (8) with diisopropyl fluorophosphate (DFP) before use. In view of the laborious and time-consuming nature of the commonly used methods for ribonuclease assay, a rapid and fairly accurate procedure was developed for use in these studies. Ribonuclease (0 to 14 y) in 1.50 ec. of 0.1 mM acetate buffer, pH 5.0, is added to 1.0 cc. of yeast nucleic acid (Schwarz) dialyzed 48 hours against water, final concentration 0.8 per cent. After incubation for 25 minutes at 25° the reaction is stopped with 0.5 ec. of 0.75 per cent uranium acetate in 25 per cent perchloric acid. Following removal of precipitated protein and substrate by centrifugation, 0.10 cc. of the supernatant fluid is diluted to 3.1 ce. with water and read 500 ] J ) t T T © FEB.1953 400+ =o MAR. * 4 AAPR. " ae ° eMAY " e 0 300; =m SEPT." 7 oO N W 200; s + AOOF + \ | ! 1 | 0 10 .20 30 40 .50 .60 .70 KUNITZ UNITS Fic. 1. Calibration curve for the determination of ribonuclease activity at 260 my in the Beckman spectrophotometer. A standard curve with known levels of pure ribonuclease is run with each set of determinations, although this is probably unnecessary in view of the excellent reproduci- bility of the procedure (see Fig. 1). Correction is made for the reagent blank determined by incubation without enzyme. This blank increases slowly during storage of the substrate in the cold, but appears to have no effect on reproducibility. The method gives linear results up to 0.60 Kunitz unit (45 units per mg. of ribonuclease). The data of Fig. 1 indicate the stability of the reagents employed over a 6 month period. Results Gross Structure—The ribonuclease molecule is characterized by a high degree of geometrical symmetry. Its low f/fy ratio ((9) and Table I) and unit cell dimensions (30 « 18 & 48 A (10)) indicate the probable presence of an extensive system of cross-linkage. Earlier chemical studies by Brand ANFINSEN, REDFIELD, CHOATE, PAGE, AND CARROLL 203 and his colleagues (11) and other experiments from this laboratory (12) made possible calculations suggesting the presence of three N terminal groups, and consequently three peptide chains per mole (mol. wt. 13,500). The N terminal and C terminal end-group analyses reported below, how- ever, point almost certainly to a single chain structure, and this con- clusion is confirmed by ultracentrifugal and diffusion experiments. Native ribonuclease sediments, under the conditions described in Table I, with an S20,w Of 1.9 to 2.2 (see also (9, 12)). Following treatment with performic TABLe I Physical and Chemical Data on Native and Oxidized Ribonuclease Chemical data Physical data Ribonuclease = Protein or | | Calculated —SH —SO; ivativ 20. XK sw permci™) Speomoe™ [auivaive u%) 5% | catettea i rn | ome || gm. er mote Native........ 0 0 150 | 9.9 1.88 15,800 1.31 Oxidized...... | 0 | 7.8 + 0.1 90 | 7.0 | 1.35 15, 800 1.87 Native ribonuclease and oxidized enzyme prepared as described in the text were dialyzed overnight against phosphate buffer, 1/2 = 0.1, pH 7.2. sao. was deter- mined in the Spinco ultracentrifuge and Deo, in the Aminco-Stern electrophoresis apparatus with boundary sharpening by the method of Kahn and Polson (33). The analytical data of Hirs, Moore, and Stein indicate a molecular weight of about 13,500 gm. per mole. Upon performic acid oxidation, this value should increase 500 to 1000 gm. per mole owing to introduction of oxygen into cysteine, methionine, and tyrosine residues. The data above have been chosen from an experiment in which all figures reported were derived from studies on a single batch of enzyme. Calcula- tions of moles are based on finite concentrations of protein, These data, when com- pared with those of Rothen (9), indicate the desirability of more extensive studies on the physical properties of ribonuclease, particularly in regard to diffusion measure- ments, and the estimation of frictional ratios; such experiments are being carried out by one of us (W. R. C.). acid, this constant falls to 1.35. Such a change in sedimentation might be accounted for either by oxidative division of the molecule into two essen- tially equal fragments or by rupture of cross-linking disulfide bonds, re- sulting in the production of a derivative so coiled as to impart greater frictional characteristics. Although the first alternative is almost. ruled out by the fact that dialysis of oxidized ribonuclease results in no loss of nitrogen from the dialysis sac, this point was more thoroughly established by diffusion measurements. Table I summarizes the data from an experi- ment in which the molecular weights of native and performic acid-treated ribonuclease are compared. Ribonuclease contains 8 cysteine residues, * The presence of 8 cysteine residues permits the theoretical presence of sixteen 204 STRUCTURE OF RIBONUCLEASE all bound in disulfide linkage as evidenced by the absence of —SH groups when tested by the method of Boyer (14). Solution in 85 per cent formic acid for 30 minutes at room temperature causes minimal, if any, change in the protein, since, upon removal of the solvent in vacuo, the enzyme activity is essentially completely recovered. The presence of HO. during this 80 minute period (1 part of 30 per cent H,O2 to 9 parts of HCOOH), however, results in the complete oxidation of cysteine sulfur to the cysteic acid form. Thus, following hydrolysis in a sealed tube with 6 N acid, 8 moles of cysteic acid per mole of protein (Table IL) can be separated chro- matographically on Dowex 50 columns (H+ form, pH 7) (13) and deter- mined colorimetrically (15). The above results strongly suggest that this protein is cross-linked through four disulfide bridges. The physical studies described above and TaBLe IT Number of Cysteine Residues per Mole of Ribonuclease Oxidized ribonuclease Cysteic acid per uw Experiment No. Cysteic acid determined hydrolyzed ribonuclease BM uM \ uM 1 : 0.0722 0.563 7.8 2 | 0.504 3.90 7.7 3 | 0.119 0.940 7.9 In Experiment 1, ribonuclease calculated from the Kjeldahl nitrogen value, as™ suming 13,400 gm. per mole of ribonuclease and 16.5 per cent nitrogen. In Exper- iments 2 and 3, ribonuclease calculated from the dry weight of sample. All values orrected for 10 per cent loss of cysteic acid during oxidation of protein (34). the end-group analysis below lead one to the tentative postulation of a gross structure, such as is depicted in Fig. 2. Some support for this gen- eral picture is derived from the x-ray diffraction experiments of Carlisle and Scouloudi (10) which indicated five crystallographie chains.2 Com- plete amino acid analyses by Hirs, Moore, and Stein! lead to an estimate of 121 amino acid residues per mole of ribonuclease. Thus in this prelimi- nary suggestion, each of the five peptide folds depicted should contain, on the average, 24 amino acid residues with disulfide cross-links as in- dipeptide sequences of this amino acid in ribonuclease. Using the general method described by Flavin (13), we have, at present, direct, degradative evidence for seven different cysteic acid sequences from a considerably larger family of chromatograph- ically separable di- and tripeptides of this amino acid. 3 In a more recent paper (16), Carlisle, Scouloudi, and Spier state that further examination of the x-ray data suggests the presence of six crystallographic chains rather than five. We have, nevertheless, schematized the molecule as shown in Fig. 2, with five folds, since the present chemical data are compatible with such a struc- ture. ry ANFINSEN, REDFIELD, CHOATE, PAGE, AND CARROLL 205 dicated. It is clear that such a general structure, when further spatially compressed by arrangement of the peptide chain in the a-helix coils sug- gested by Pauling and Corey (17), would result in a highly compact, sym- metrical molecule. The presence of 4 proline residues! in a five fold struc- ture is also compatible with the postulated (18) réle of this amino acid as a center of direction reversal in peptide chains. N Terminal Residue of Ribonuclease—Dinitrophenyl! ribonuclease (DNP ribonuclease) was prepared according to the usual methods for DNP pro- tein (5). Acid hydrolysis was performed either in concentrated HCI or constant boiling HCl in sealed tubes at 105° for varying intervals of 2 to 18 hours. Identification of the DNP amino acids was made by paper chromatog- raphy, by the systems of Blackburn and Lowther (19), Biserte and Os- [NH] LY S-GLU-THR-AL Amp 5 yo Pio) | z S é [PRO] so E [PRO] CS {MET,TYR, ALA, LEU, PHE]-VAL Fic. 2. Generalized gross structure of ribonuclease teaux (20), Monier and Pénasse (21), and finally the two-dimensional technique of Levy.t For quantitative determination, the DNP spots were eluted with 1 per cent sodium bicarbonate and their absorption measured at 350 mu in the Beckman spectrophotometer (20, 22). As previously reported (23), bis-DNP lysine was the only DNP amino acid detected in the ether extracts of the hydrolysates. No a-DNP argi- nine or bis-DNP histidine could be detected in the aqueous phase. Determination of Moles Bis-DNP Lysine per Mole DNP Ribonuclease— Weighed samples (approximately 0.15 to 0.2 ym of DNP ribonuclease dried over P03) were submitted to acid hydrolysis at 105° in sealed tubes with 0.5 ce. of constant boiling HCl. After 2, 5, and 8 hours, duplicate samples were diluted with water, extracted with ether, and chromato- graphed by the two-dimensional technique of Levy.t Duplicate 0.20 um aliquots of a standard bis-DNP lysine solution were subjected to the same procedures, and the unknowns compared to the standards after identical hydrolysis times. Using Hirs, Moore, and Stein’s figures for the amino acid composition 4 Levy, A. L., personal communication. 206 STRUCTURE OF RIBONUCLEASE of bovine pancreatic ribonuclease,! and adding 167 for each e-lysine (ten), N terminal] residue (one), O-tyrosine (six), and imidazoly! group of histidine (four), as used by Porter (5) and Sanger (22), to the molecular weight of ribonuclease based on Hirs, Moore, and Stein’s figures, one arrives at a molecular weight of 13,841 + 21(167) = 16,848 for DNP ribonuclease. An average of 0.90 mole of bis-DNP lysine per mole DNP ribonuclease was detected. The 2 hour sample was disregarded because solution of the sample was incomplete (Table IIT). No spots other than bis-DNP lysine and bis-DNP lysylglutamic acid (see below) were detected. As noted below, since the next residue was found to be threonine, it is a safe assumption that the N terminal group was completely hydrolyzed. In addition to the 5 to 10 per cent error in Tass III Quantitative Determination of N Terminal Amino Acid Residue | Sample | Amount detected after hydrolysis Time of hydrolysis “7 oO | Weight Lysine | MSE. | Total terminal a ! a a hrs. | mg. BM BM uM eM mole 5 2.35 0.140 | 0.100 0.086 0.136 0.97 5 3.35 0.199 | 0.150 0.037 0.187 | 0.94 8 3.22 0.191 | 0.141 0.016 0.157 0.82 8 | 3.04 | 0.181 | 0.141 | 0.017 | 0.158 | 0.87 wee on | AVELABE cece bbe b ent ttre enn ene es | 0.90 the method (22), it is conceivable that the figure of 0.9 rather than 1.0 end-group per mole might be due to a higher rate of destruction when the DNP derivative is present in the protein than that which prevails when the amino acid derivative (24) is free in solution. Alternatively, it may be that the presence of other products of hydrolysis catalyzes the destruc- tion of the derivative. Determination of N Terminal Sequence—40 mg. of DNP ribonuclease were subjected to partial acid hydrolysis in 11 n HCl! for 72 hours at 37°. After dilution, the hydrolysate was extracted with ether, ethyl acetate, and n-butanol (25). The extracts were concentrated in vacue and then subjected to electrophoresis on Munktell No. 20 paper with 0.033 m_ phos- phate buffer at pH 7.0 (26) as electrolyte. The main zone present in the ether extract, migrating at the rate of bis-DNP lysine, was extracted with bicarbonate and identified as such by paper chromatography. A fainter zone present in the ether and ethyl acetate extracts proved, upon complete hydrolysis and chromatography, to be bis-DNP lysylglutamic acid. ANFINSEN, REDFIELD, CHOATE, PAGE, AND CARROL. 207 No zones containing bis-DNP lysine were found on electrophoresis of the n-butanol extracts. A second hydrolysis for 20 hours, rather than 72 hours, showed again only the two components, bis-DNP lysine and bis-DNP lysylglutamic acid, the latter compound being present in greater amount. Pepsin Hydrolysis of DNP Ribonuclease—40 mg. of DNP-ribonuclease were suspended in 0.01 w HCl, the pH was adjusted to 1.8 with 0.1 mu HCl, and the suspension diluted to 4.0 cc. with 0.01 m HCl. Pepsin (Worthington, crystallized four times), 0.28 mg., was dissolved in a drop of water and added to the suspension, which was then incubated with shaking, one-fourth of the initial volume being withdrawn after 4 and after 22 hours. These aliquots were extracted with ethyl acetate until no further color was removed. The extracts were subjected to paper electrophoresis, the zones eluted and hydrolyzed, and the resulting ether-extractable DNP derivatives and the water-soluble residues (27) were chromatographed. Free bis-DNP lysine, dinitrophenol, and an electrophoretic band con- taining bis-DNP lysine plus glutamic acid, threonine, and alanine in ap- proximately equimolar concentrations were identified. In order to determine the sequence of the amino acids in the tetrapeptide component, the remaining material from the above incubation was ex- tracted with ethyl acetate. After removal of the ethyl acctate, the ex- tracted material was hydrolyzed for 18 hours at 37° with 11 nw HCl. The hydrolysate was diluted with water and extracted with ethyl acetate. This extract contained bis-DNP lysine (2+) and bis-DNP lysylglutamic acid (4+-) determined by the methods used in the previous section. The aque- ous portion, after removal of HCl in vacuo, was treated with fluorodinitro- benzene in 1 per cent trimethylamine acetate buffer, pH 9.5. The excess reagent was extracted from the alkaline solution, and the solution was dried under high vacuum.