DISULFIDE INTERCHANGE AND THE THREE-DIMENSIONAL STRUCTURE OF PROTEINS By Davip Grvot,* Francesco DE Lorenxzo,t Ropert F. GoLpBERGER, AND CHrisTian B. ANFINSEN LABORATORY OF CHEMICAL BIGLOGY, NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES, NATIONAL INSTITUTES OF HEALTH Communicated January 28, 1965 The ervstallographic studies of Kendrew and Perutz and their colleagues have demonstrated that the mvoglobins and hemoglobins of several species possess unique tertiary structures.' ? Such structural homogeneity probably characterizes most or all proteins, as indicated by the large body of physical and chemical informa- tion now available on many purified preparations. It has been suggested that the three-dimensional conformations of proteins are completely defined by the informa- tion present in the linear sequences of amino acids that make up the corresponding polypeptide chains. This suggestion has been supported by a number of studies on the reversible denaturation of proteins (summarized in ref. +). It has been shown that the renatured molecules, both those devoid of covalent cross linkages** and those possessing disulfide bonds which have been reductively cleaved prior to renaturation.*~!? exhibit physical and biological properties indistinguishable from those of the native molecules. These studies indicate that the tertiary struc- ture and specific disulfide bonds of a native protein molecule represent the most stable conformation of its polypeptide chain under physiologic conditions. If the information required for the pairing of half-cvstine residues in a polvpep- tide chain is inherent in the amino acid sequence, one may ask whether interrup- Vo. 53, 1965 BIOCHEMISTRY; GIVOL ET AL, 677 tion of the chain by cleavage of one or more peptide bonds might seriously modify this information. This could be the case, for example, for a protein that is syn- thesized as one polypeptide chain (e.g., chymotrypsinogen) and functional as a multichained protein (e.g., chymotrypsin). We have recently shown that a microsomal enzyme which accelerates the re- activation of the reduced form of bovine pancreatic ribonuclease (RNase) and of egg white lysozyme! is involved in the catalysis of sulfhydry|-disulfide interchange. Thus, the inactive product obtained by oxidation of reduced RNase in urea, con- taining random sets of half-cystine pairs, is rapidly converted to active RNase by the enzyme. If the “correct” disulfide bonds of a protein are a corollary of native tertiary structure, an enzyme that would catalyze their rearrangement could be used to test the stability of the sequence-directed pairing of half-eystine residues. We present, in this communication, the results of studies on the effect of the di- sulfide interchange enzyme on several RNase derivatives, chymotrypsin, and insulin. The results are consistent with the idea that the specific disulfide bonds of these pro- teins were formed according to the information present in single-chained pre- cursors which were subsequently converted, by peptide bond cleavage, to the meta- stable multichained proteins. Materials and Methods —Bovine pancreatic ribonuclease (Sigma Chemical Co.) was reduced as described previously’ and wax allowed to oxidize either spontaneously in 8 Jf urea at pH 8.2 for 100 hr," ur by incubation for 10 min with 107% W dehvdroascorbie acid (DHA) (Nutritional Biochemical Corp.) in 0.05 M bicarbonate buffer, pH 7.4.** After removal of the urea or the DHA under acidic conditions, '* both preparations were found to contain bo free sulfhydryl groups and to be enzymically inactive. The ‘C-protein” derivative of RNase was prepared by cleavage of methionyl bonds with cyanogen bromide and subsequent removal of the NH»-terminal tridecapeptide by gel filtration through a column of Sephadex G-25 (Pharmacia). The extents and rates of reactivation of re- duced RNase, and of the oxidized RNase preparations described above, were determined under various conditions by assay of RNase activity at pH 5.0. Chymotrypsinogen A and a-chymotrypsin (3 X erystallized) were purchased from the Wor- thington Biochemical Corp. Chymotrypsinogen was activated by incubation with trypsin ‘see Results). The esteratic activity of a-chymotrypsin was determined by the hydrolysis of benzoyl-tyrosyl-ethy! ester (Determatube BTEE, Worthington Biochemical Corp.}.'* The in- erease in absorbancy at 256 mp was followed during the first 4 min of the reaction. Trypsin i2 X erystallized, Worthington Biochemical Corp.) was treated with diisopropy!-fluorophosphate to destroy residual chymotryptic activity." Beef insulin ‘low zinc: lot #4096-836997) and the oxidized B chain and A chain of insulin were a gift from Eli Lilly Laboratories. Beef insulin labeled with I! (Abbott Laboratories) was used in a solution of 19 myg,‘ml in 0.025 VM phosphate buffer, pH 8, containing 50 mg of bovine serum albumin (Armour Co.) per ml. Pepsin (2 X crystallized) was obtained from Worthington Biochemical Corp. The disulfide interchange enzyme was prepared from beef liver microsomes as described pre- viously, except that Sephadex G-2U0 filtration was introduced into the procedure following the CM-Sephadex step.'+ The enzyme was assayed routinely by its effect on the rate of reactiva- tion of reduced R Nase.'$ Sephadex G-25 and G-200, DEAE-Sephadex, and CM-Sephadex were purchased from Phar- macia. Urea {Baker Chem. Co.} was recrystallized from 95°; ethanol before use. 3-Mercapto- ethanol ‘Eastman Co.) was used without further purification. Immunoassay of I'*'-labeled pork insulin was performed essentially as described by Berson vt al,” except that ascending chromatography in 0.025 VW phosphate buifer, pH 3, was used in- stead of electrophoresis. The samples (0.5 ml containing 0.5 mug of [}3!-labeled insulin) were incubated with 0.05 ml of guinea-pig antibovine inswin antiserum (diluted 1:40 with 0.025 MV phosphate buffer, pH 3.0, contaiming 50 mg bovine serum albumin per ml) for 48 hr at 4° and 9.2 67s BIOCHEMISTRY: GIVOL ET AL. Proc. N. A. S&S. ml was chromatographed on Whatman 3 1M paper for 31/2 hr at 4°. Radioactivity was meas- ured with a 4Pi automatic windowless paper chromatogram scanner ( Atomic Accessories, Inc.}. Changes in the pairing of half-cvstine residues in insulin were examined qualitatively on peptide maps after peptic digestion. The protein was dissolved in 5°, formic acid at 4 concentration of 10 mg/ml and pepsin (0.1 mg in 10 @l 5 formic acid; war added. After 6 hr of ineubation at 37° a second aliquot of pepsin was added. The reaction was stopped, after a total incubation time of 16 hr, by freezing and Ivophilization. The lyophilized material was dissolved in water at a con- centration of 50 mg/ml. and U.03 ml] were applied to Whatman 3 MM paper. Descending chroma- tography was performed in the organic phase of butanol: acetic acid: water (4:1:5}, and electro- phoresis was carried out at 2500 v in pyridine acetate buffer, pH 3.6, for 2 hr. The peptide maps were prepared in duplicate. One was stained with ninbydrin and the other with evanide-nitro- prusside for the detection of peptides containing disulfide bonds. 22 Oxidation with performic acid was carried out according to the method of Hirs.23 A Beckman /Spinco model 120 amino acid analyzer was utilized for amino acid analyses, Sam- ples for analysis were hydrolyzed in constant boiling HCI in evacuated. sealed tubes for 22 hr at 110°, Protein concentrations were determined by the method of Lawry ef al.2} Free sulfbydrv] groups were determined with 5,5'-dit hiobis(2-nitrobenzoic acidi ( Aldrich Chemical Co.) Results.— Ribonuclease derivatives: The enzymically inactive products ob- tained by the oxidation in 8 Af urea or with DHA have been shown to be rapidly activated by the disulfide interchange enzyme in the presence of 8-mercaptoethanol (optimal concentration, 10-3 M) (Fig. 1, curves J and 2), as contrasted with the long periods (16-24 hr) required without the enzyme." The requirement for 6- mercaptoethanol in the reactivation mixture could be abolished by previous partial reduction of these molecules (Fig. 1, curve 3).4 Since we have shown that the enzyme does not accelerate the oxidation of sulfhydrv] groups,’4 and since it acti- vates “incorrectly” cross-linked RNase as well as fully reduced R Nase, it may be con- cluded that the process catalyzed is a sulfhydry]-disulfide interchange. The effect of the enzyme on the “C-protein’' derivative of RNase described by Gross and Witkop” is illustrated in Figure 2. The C-protein is composed of three polypeptide chains held together by one intrachain and three interchain disulfide bonds, all present in the original RNase molecule, The aggregation and precipita- tion produced by the enzyme in the presence of 10-3 Af 8-mereaptoethanol was so rapid that the 8-mercaptoethano! concentration had to be diminished to 10~* AY. At the latter level the concentration of 8-mercaptoethanol was less than 5 per cent that of half-cystine residues in the C-protein emploved (4 mg protein-ml). Thus, the precipitation cannot be due to simple reduction. No precipitation occurred over a 24-hr period in the presence of either enzyme alone or 8-mercaptocthanol alone (10~? or 10-* Af) under these conditions (0.1 Mf Tris, pH 7.2). (Precipitation did occur after 1 hr at pH 7.8 in the presence of 1073 1f 8-mercaptoethanol alone.) The enzyme-catalvzed aggregation and precipitation of C-protein is presumably caused by disulfide interchange, which leads to random pairing of half-cvstine residues forming a cross-linked network of chains. In previous experiments on the reactivation of reduced RNase it was difficult to demonstrate euzyme activity at low ratios of enzyme to reduced RNase" since con- centrations of the substrate (reduced RNase) greater than 0.02 mg ml led to ex- tensive intermolecular disulfide bonding. In the experiments with C-protein and with insulin, described below, catalysis by the disulfide interchange enzyme is easily demonstrated at weight ratios of enzyme to substrate of less than 1:100. Chymotrypsin: ~Chymotrypsin rapidly inactivates the disulfide interchange en- Vou. 53, 1965 BIOCHEMISTRY: GIVOL ET AL. 679 f / pO eo ee — - _ { oo / / ool 5 / - \ i \ : ; a - / z 2 2 ! 3 ‘ 5 a / 2 = ~ / 3 3 < , 4a 60> ~ - 3 : z 3 > | & = f a 40° - a i a MINUTES WeNUTES MINUTES 1 2 3 Fic. 1.—Reactivation of oxidized RNase derivatives. Curve I: DH A-oxidized RNase, 107? W 3-mercaptoethanol. Curve 2: Urea-oxidized RNase, 107? VW 3-mercaptoethanol. Curve 3: Urea-oxidized RNase containing 2-4 sulfhydry! groups per mole. Curve 4: DHA-oxidized or urea-oxidized RNase with either 1073 M 3-mercaptoethanol or the disulfide interchange enzyme. ‘All incubations were carried out at 37° in 0.1 UF Tris, pH 7.5, and contained 20 ag of RNase deriva- tive per mi and (except as indicated for curve 4) 20 ug of the disulfide interchange enzyme per ml. Fic. 2.—Treatment of “C-protein” with the disulfide interchange enzyme. The incubation mixture contained 4 mg of “C-protein”’ and 80 ug of the disulfide interchange enzyme in 1 mlof 0.1 Mi Tris, pH 7.2, 107! VWs 3-mercaptoethanol. Turbidity was followed with a Cary model 15 spectrophotometer. Fic. 3.—Inactivation of chymotrypsio by the disulfide interchange enzyme. The upper curve ( @—@) was obtained with an incubation mixture containing 10 gg of chymotrypsin. diluted from 2 urea solution (see text), in 1 ml of 0.1 W Tris. pH 7.5, with or without 3-mercaptoethanol at a con- centration of 1073 M. The lower curve (O—O) was obtuined with an incubation mixture prepared as above (with é-mercaptoethanol) plus 100 yg of the disulfide interchange enzyme. Aliquots of 0.1 ml were removed from the incubation mixtures for assay of chymotrypsin activity. PERCENT acTiVGTY pe AL Shame 5, Tee de a a o