Reprinted from the Procegpinas oF THE NATIONAL ACADEMY OF SCLENCES Vol. 64, Na. 2, pp. 428-435. October, 1969. SOLID PHASE SYNTHESIS OF A 42-RESIDUE FRAGMENT OF STAPHYLOCOCCAL NUCLEASE: PROPERTIES OF A SEMISYNTHETIC ENZYME By Davin A. ONTJES AND CurisTiIAN B. ANFINSEN NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES, NATIONAL INSTITUTES OF HEALTH, BETHESDA, MARYLAND Communicated May 14, 1969 Abstract.—The polypeptide corresponding to the amino acid sequence from residue 6 through 47 in staphylococcal nuclease has been synthesized by the solid-phase method. The synthetic product closely resembles the correspond- ing native polypeptide in both physical and chemical properties. The synthetic peptide may be recombined with the complimentary native peptide comprising residues 49 through 149 to form an active, semisynthetic enzyme. The “func- tional purification” of the crude, synthetic polypeptide by affinity chromatog- raphy was found to yield a synthetic fraction of greatly enhanced specific activity. This purification was accomplished on a column of Sepharose to which the complimentary native peptide had been covalently bound. The major extracellular nuclease of Staphylococcus aureus is relatively re- sistant to the action of proteolytic enzymes, when digestion is carried out in the presence of Ca++ and the nucleotide 3’,5’-deoxythymidine diphosphate (pdTp). Under these conditions nuclease may be selectively cleaved by trypsin into an amino-terminal pentapeptide and two inactive, structureless polypeptide frag- ments termed P, (residues 6 through 48) and Ps; (residues 49 through 149). Under appropriate conditions, Pz and P; ean recombine through noncovalent interactions to form an active, structurally ordered enzyme derivative, termed nuclease-T.!: 2. This complex is analogous to the extensively studied ribonucle- ase-S system.?: 4 While disulfide bonds play an important role in the structure of ribonuclease-S, such bonds are absent in nuclease-T. The nuclease-T complex, which possesses about 8% of the activity of native nuclease, provides an excellent model enzyme for the study of the relationships between primary amino acid sequence, three-dimensional conformation, and catalytic activity. Preliminary X-ray diffraction studies of native nuclease indicate that the P. and Ps; polypeptide chains occupy opposite hemispheres in the crystalline protein. Both the P. and Ps chains contain residues which ap- pear well situated for interaction with the substrate.’ The three-dimensional structures of nuclease and nuclease-T have been compared by a number of in- direct methods, including heat and urea denaturation, circular dichroism, fluorescence spectroscopy, and tritium exchange.’ ?)® Hf appears that the structure of nuclease-T is quite similar to that of the native enzyme, but that. if ts less stable. The organic synthesis of the P, fragment of the nuclease-T complex has been based on the solid phase method developed by Merrifield.’ This technique has been used by Gutte and Merrifield in the synthesis of an active pancreatic ribo- 428 Vou. 64, 1969 CHEMISTRY: ONTJES AND ANFINSEN 429 nuclease.’ The synthesis of nuclease fragment P: provides another example of the application of the solid-phase method to the reproduction of a large poly- peptide sequence. The “functional purification” of the crude polypeptide prod- uct by affinity chromatography has been found to be a useful adjunct to this type of synthesis. Materials and Methods.—N-t-Butyloxycarbonyl (BOC)-L-amino acid derivatives were purchased from either Fox Chemical Co. or Cyclo Chemical Co. and were determined to be of suitable purity by thin-layer chromatography. Side-chain blocking groups included the 6 or y benzyl esters (OBzl) of aspartic and glutamic acids, the benzyl ethers (OBzl) of threonine and tyrosine, the N*-trifluoroacetyl (eTFA) group of lysine, the nitro-guani- dino (NO:) group of arginine, and the imidazole-N-carbobenzoxy (im-Cbz) group of histidine. a-BOC-im-Cbz-histidine was synthesized by dissolving BOC histidine (7.5 mmoles) in 25 ml water containing 25 mmoles of NaHCO ;. The solution was chilled to 5°C and two 4.5 mmole aliquots of carbobenzoxychloride (Cyclo) were added at 15-min intervals, accompanied by vigorous stirring. After 1 hr, excess CbzCl was removed by ether extraction, and the solution was acidified by addition of 30 mmoles of citric acid. The desired product was extracted with two 25-ml portions of ethyl acetate. After wash- ing with water, the ethyl acetate solution was dried over NarSO, and flash evaporated to an oil. The oil was dissolved in an appropriate volume of CH»Clefor addition to the resin in a coupling step. The product showed a major spot, R; 0.68, on thin-layer chromatog- raphy (n-butanol, 4: water, 2: pyridine, 1: HAc, 1) with a minor leading contaminant. Staphylococcal nuclease, Foggi strain, and its purified native peptide derivatives, P, and P3, were prepared as previously described.1: 9 Coupling procedure: The general scheme of synthesis is shown in Figure 1. It has been shown that treatment of native nuclease-P, with carboxypeptidase B will remove lysine residue 48 without loss of activity.2 Synthesis was therefore begun at proline 47. BOC proline (9 mmoles) was esterified to 10 gm of chloromethylated resin (Cyclo, 1.04 mmoles Cl/gm) in the presence of triethylamine (9 mmoles) by refluxing in ethanol for 52hr. Synthesis was begun with 5 gm of BOC-prolyl resin, which contained 0.31 mmoles proline/gm. In the repeated coupling cycles the BOC group was removed by treatment for Cleavage from Resin by Anhydrous HF PARTIALLY DEBLOCKED PEPTIDE Removal of eTFA Groups by 1M Piperidine in Urea DEBLOCKED PEPTIDE esolting and Preliminary Purification an Sephadex CRUDE PEPTIDE Fig. 1—Scheme of Solid Phase Synthesis of Nuclease Fragment Pr, Residues 6-47. Ex- perimental details are given in the text. The amino acid sequence is that of the Vs strain,?! with one revision from published data. Glutamic acid, rather than glutamine, has been found in position 43 in both the Vs and Foggi strains.?? 430 CHEMISTRY: ONTJES AND ANFINSEN Proc. N. A. 5S. '/, hr with 4 N HCl in dioxane. The resulting peptide hydrochloride was converted to the free base by treatment for 10 min with 10% triethylamine in CHCl;. Five millimoles of the appropriate BOC amino acid derivative (threefold excess) was added in a suitable solvent, 5 mmoles of dicyclohexylcarbodiimide (Aldrich) was added, and coupling was allowed to proceed for 2hr. Glutamine was added as the p-nitrophenyl ester in dimethyl- formamide with a reaction time of 12 hr. Detailed accounts of reagent preparation and rinsing procedures have been published elsewhere.” 1° ‘The progress of the synthesis was followed by removal of resin samples after coupling of residue 33 and residue 18. These samples were cleaved from the resin and subjected to amino acid analysis after a stan- dard 20-hr hydrolysis in vacuo in 6 N HCl at 110°C. Cleavage procedure: The blocked peptide was cleaved from the resin by treatment with anhydrous HF for i hr at 0°C, in the presence of anisole."!: 42, A typical procedure used 1 gm peptidy] resin, 0.5 ml of anisole, and 10 ml of HF. After removal of the excess HF in vacuo, the peptide-resin mixture was extracted 3 times with ethyl acetate to remove remaining anisole, and the e-TFA peptide was extracted into glacial HAc and lyophilized. One gram of completed peptidyl resin typically yielded 450 mg (0.075 mmoles) of --TFA peptide and 400 mg of residual polymer. Since 400 mg of starting polymer contained 0.124 mmoles of the carboxyterminal amino acid, the yield at this stage was approxi- mately 60%. Removal of «TFA groups: The e-TFA groups were removed by dissolving 100 mg of crude e-TFA peptide in 5 ml of 1 Af aqueous piperidine (pH 12.5) and 8 W urea for 7 hr at O°C. This reaction was terminated by application of the mixture to a 2.4 * 100 em column of Sephadex G-25, followed by elution with 0.05 4f HAc. The completeness of e-TFA removal was estimated by the deamination of peptide samples with nitrous acid. The number of intact lysines found by amino acid analysis after this procedure was found to correspond elosely to the «TFA content in the deblocked peptide as estimated by analysis for elemental fluorine. Preparation of Sepharose-P; column: Native nuclease fragment P; was bound eo- valently to Sepharose (agarose) by the cyanogen bromide activation method.!4 Sepharose 4-B (8 ml bed volume) was suspended by gentle stirring in 20 ml 0.1 Jf NaHCO, at 5°C and the pH was adjusted to 11 by addition of 2N NaOH. About 150 mg of CNBr (East- man) was added and stirring continued for 10 min, while pH was maintained at 11 by dropwise addition of 2. N NaOH. The suspension was transferred to a chilled sintered glass filter and rinsed quickly with 3 portions each of cold 0.1 4f NaHCO, water, and 0.1 Af phosphate buffer, pH 6.5. This activated Sepharose was stirred gently in 15 ml of phosphate buffer containing 22 mg of fragment P; for 3 hr at 5°C. At the end of this time the 280 my absorbancy of the supernatant P; solution had stabilized at 27% of its starting level, indicating a content of approximately 2 mg of bound P3/ml of bed volume. The reaction was terminated by exhaustive rinsing of the P;-Sepharose in a small column with phosphate buffer, water, 0.1 44 HAc, and water. The column could then be stored indefinitely at 5°C. The relatively low pH of the buffer during the coupling step was found to be important. Coupling of P3; to the Sepharose proceeded efficiently at pH 9, but the resulting P;-Sepharose was unable to bind fragment P,. Coupling at the higher pH probably encourages bond formation at multiple sites on the P; molecule, through unionized e-NH2 groups. A P; molecule bound to Sepharose at several points would not be expected to be capable of free complex formation with Ps. Results —Characterization of crude synthetic product: The elution pattern of the deblocked peptide from Sephadex G-25 (I*ig. 2) showed a minor peak, be- ginning at the void volume of the column, and a major retained peak. The ratio of the elution volume of the retained peak to the void volume was similar to that found for native Pe, averaging 1.46. Enzyme generating activity was confined to this fraction. Amino acid molar ratios in an acid hydrolyzed sample of the de- blocked and desalted peptide (fraction B) were Lys 6.9 (6), His 2.2 (2), Arg 1.1 Vou. 64, 1969 CHEMISTRY: ONTJES AND ANFINSEN 431 (1), Asp 3.2 (3), Thr 4.7 (5), Glu 2.9 a 8 (3), Pro 4.6 (4), Gly 2.4 (3), Ala 2.0 (2), Val 1.9 (2), Met 1.8 (2), Ile 2.0 (2), Leu 6.9 (6), Tyr 0.8 (1), Phe 1.1 (1). Theoretical values are given in parenthesis. After nitrous acid de- amination, only 0.2 moles of lysine were found, indicating efficient re- oo 150 moval of the eT FA blocking groups. An enzymatic hydrolysis of the same fraction was performed according to Hill and Schmidt® using a combina- tion of pronase, leucine aminopepti- dase, and prolidase. Ratios of amino acid were Lys 6.1 (6), His 1.9 (2), Arg 1.2 (1), Asp 3.1 (3), Thr 5.0 (5), Gin 1.1 (1), Glu 2.1 (2), Pro 3.6 (4), Gly 2.7 (2), Ala 2.6 (2), Val 1.9 (2), Met 2.0 (2), Ile 2.1 (2), Leu 6.4 (6), Tyr 0.96 (1), Phe 0.96 (1). The partially purified peptide (fraction B) was digested with trypsin in parallel with a sample of native P,. The fingerprint patterns of the two samples, as shown in Figure 3, were quite similar. Two components present in the syn- thetic digest but not in the native digest are outlined by dashed lines. Further purification of crude peptide: Synthetic fraction B was purified fur- ther, either by ion exchange or affinity chromatography. Ion-exchange chroma- tography was carried out on phosphorylated cellulose with a concentration gra- dient of ammonium acetate. The elution pattern, seen in Figure 4, shows hetero- 3 ' ABSORBANCY, 280 mu 2-Oe . i a ed 200 250 300 ELUTION VOLUME Fie. 2—Desalting and Preliminary Purification of Crude Synthetic Peptide. 100 mg of deblocked 6-47 peptide was applied to a 2.4 x 100-cm column of Sephadex G-25 (fine) and eluted with 0.05 M HAc. The column void volume is 150 ml. ABSORBANCY, 280 mu OYWU "ALIALLONGNOD ¥34dNg FRACTION NUMBER Fie. 4—Fractionation of Native P. and Synthetic 6-47 Peptide on Phosphorylated Cellulose. 50 mg of 6-47 peptide (fraction B Fig. 3—Peptide Maps of Tryptic Digests of Native P: and Synthetic 6-47 Peptide. Chroma- tography was run in n-butanol, acetic acid, and water (4:1:5); electrophoresis was run in pyridinium acetate at pH 3.6, 2500 v for 70 minutes.2> Position of phenol red marker after chromatography is indicated by spot- labeled PR. from Sephadex G-25) and 10 mg of native Pz. were applied to separate 1.5 & 10 cm columns of phosphorylated cellulose (Whatman) and eluted with a gradient of 100 ml of 0.38 M ammonium acetate, pH 6.0, and 100 ml of 1.0 M ammonium acetate, pH 8.0. —O, native P.; —@, synthetic 6-47; —A, buffer con- ductivity. ; 432 CHEMISTRY: ONTJES AND ANFINSEN Proc. N. ALS. geneous behavior. The broad peak corresponding to the typical elution range of native P; (85-39 mmho conductivity) was found to possess the highest specific activity. Amino acid analysis of this fraction was not different from that of the crude starting material. Affinity chromatography was carried out on a 1 X 5 em column of Sepha- rose-P;. In preliminary experiments, binding of small quantities of native Pz was seen when an excess of P, was applied to the column in pH 8 buffer with addi- tion of CaCl, and pdTp for stabilization of the P:-P; complex. Bound P2 could be eluted from the column with dilute HAc at pH 3, where fragments P; and P; are known not to associate. A 5-mg sample of crude synthetic peptide was ap- plied to a similar column and eluted in the same way. As seen in Figure 5, the specific activity of the small bound fraction was approximately 10 times greater than that of the material which failed to bind. This fraction comprised about 7 per cent of the total material applied, and did not differ significantly from the starting material in its amino acid composition. Disk gel electro- phoresis of the starting material, the nonbound and the bound peaks showed diminished heterogeneity in the bound peak and a mobility similar to that of Elution ° Start pH3 40 ABSORBANCY, 750 my WH SHUN ALIALLOV °o a FRACTION NUMBER Fig. 5—Fractionation of Synthetic 6-47 Pep- tide on Sepharose-P3. 5 mg of synthetic pep- tide was applied to the column in 0.05 M borate buffer, pH 8, containing 0.01 M CaCh Fic. 6—Disk Gel Electrophoresis of Native Ps and 0.001 M pdTp, and eluted with the same and Synthetic 6-47 Peptides Before and After buffer. After collection of 19 ml of eluate, Fractionation’ on Sepharose-P;. Polyacryl- the buffer was changed to 0.001 Mf HAc, pH 3. amide gel electrophoresis (71/2% cross-linking) Lowry* protein determinations were run on was run at pH 3.3, 3 ma/tube, for 3 hr at 5°C. 0.1 ml aliquots of each fraction (—@—). Direction of migration is toward the anode Activities were measured by incubating 0.05 (bottom of tubes). Tube 1 shows native P2; ml of each fraction with 0.05 mT of a5 mg/ml tube 2 native P2 not binding to Sepharose-P3; solution of Ps in 0.1 Af Tris, pIL8, and assay- tube 3 native P, binding to Sepharose-P3; ing against DNA (-O-). The graph in the tube 4 crude 6-47 synthetic peptide; tube 5 upper left corner shows the results of the re- synthetic peptide not binding to Sepharose- application of the unbound fraction to the Ps; tube 6 synthetic peptide binding to same column. Sepharose-P3. Vou. 64, 1069 CHEMISTRY: ONTJES AND ANFINSEN 433 native P, (Fig. 6). When the nonbound synthetic peak was reapplied to the same column, no further binding could be detected. This rejected fraction had a low activity when added to P; in free solution but was apparently unable to bind to P; on the solid-phase support. Enzymatic properties of synthetic P,: The relative specific activities of the crude peptide and of fractions purified by phosphocellulose or Sepharose-P; are shown in Figure 7. The DNase activity generated by native P, is nearly maxi- mal when it is added to P; in a one-to-one molar ratio. The Sepharose-P; purified synthetic material, which was the most active of all synthetic derivatives, pro- duced about 30 per cent of full activation when added to P; in a one-to-one ratio. When added in a 20- to 40-fold molar excess, material purified by either ion ex- change or affinity chromatography could generate 90 per cent of full activity. (Working with larger excesses was difficult because of the limited solubility of both native and synthetic P.) Activities measured against RNA (Fig. 8) were 20 Native P, Do 30 Notive 5 Synthetic (rom Sepharose R,) 5 9 o ynthetic 8 (rom Phosphocellulose) DNase ACTIVITY, Units/mt Synthetic 8, (Crudel Synthetic B, (from Sepharose-Py) Si nthetic 8% {from Phosphocelluiose) yy m : ! 1 Lb L | n Fie. 7—DNase Activity Generated by Synthetic 0 02 04 06 08 iO 1z 147 16 1B BO . . . MOLAR RATIO; P, PR, P, Preparations. Synthetic Pz preparations a were incubated for 1 hr in varying molar excess RNase ACTIVITY, Units/ml 3 0 n a 1 i | 1 4 oO o2 04 O68 O08 10 he 14 16 8 20 MOLAR RATIO, f to P, with 0.2 mg/ml native P; in 0.05 M Tris, pH 8, Fic. 8—RNase Activity Generated by and assayed against DNA by the method of Synthetic P; Preparations. The same solu- Cuatrecasas ef al.25 tions shown in Fig. 7 were used. lower, relative to native P,. Figure 9 shows the specific activity of control samples of native P: after treatment with HF, or with HF followed by piperidine. Successive treatment with both reagents reduced activity by approximately 50 per cent. The conformational stabilities of native and semisynthetic nu- clease-T complex were evaluated by trypsin digestion in the presence and absence of Cat+ and pdTp (Fig. 10). Both native and semisynthetic complexes showed trypsin resistance only in the presence of metal ion and ligand. Discussion.—Rationale and limitations in synthetic tactics: Ideal blocking groups for all amino acid side chains in the P; sequence have not been found. The im-Cbz group was accepted for histidine even though BOC-im-Cbz histidine is relatively unstable and difficult to purify. In our work BOC histidine (un- protected imidazole) has given unacceptably low coupling efficiency. The TFA group for lysine has the disadvantage of requiring rather basic (pH 12.5) conditions for its removal, but has the advantage of complete acid stability. The more frequently used e-Cbz group is removed to a significant extent upon prolonged exposure to the acid conditions of BOC removal. With repeated 434 CHEMISTRY: ONTJES AND ANFINSEN Proc. N. A. 8. 20 ~ Untreated E— < a é > HF Alone z ‘ NATIVE SEMISYNTHETIC 5 10 aon NUCLEASE -T NUCLEASE -T bE HF + Piperidine % a Ez With Co, pdTp < x ro . z < £ 3 , With Co, pdTp a ~ f ad bE $ 10}- : ° ——L 6 \ 0 02 O4 O68 08 io 4 MOLAR RATIO, P, to P, 3 z a Without Co, pdTp Without Ca, pdTp _ . oG—- at - Fig. 9—DNase Activity of Native ° i , ae ° 60 r20 . MI INCU iT P, Before and After Treatment with NUTES INCUBATION WITH TRYPSIN Deblocking Reagents. Deblocking conditions were the same as those Fig. 10—Trypsin Inactivation of Native and Semisyn- described in the text. After treat- thetic Nuclease-T in the Presence and Absence of Catt ment with reagents, the native P, and pdTp. A twofold molar excess of native Ps, or samples were desalted on Sephadex Sepharose-P; purified synthetic Pz, was incubated with G-25, lyophilized, and assayed after 0.2 mg/ml P; in 0.05 M Tris, pH 8, and 0.05 mg/ml incubation with native Ps, as pre- D¥FP-treated trypsin. Concentration of CaCh was viously described for the synthetic 0.01 M and concentration of pdTp 0.0001 M where material. indicated. acid exposures in a growing polypeptide chain, there is increasing danger of epeptidyl side chain formation.” Neither of the deblocking reagents used in P, synthesis, HF or piperidine, proved to be innocuous for enzyme activity. The nature of the chemical changes induced in native P, is not known. Enzymic hydrolysis of deblocked synthetic material has shown no evidence of racemization or of a-8 peptide bond shifts. Fragment P, has a tendency to form an inactive aggregate in aqueous solution, particularly at basic pH. Such aggregation may well account for a part of the activity loss observed during deblocking. It is interesting that native nuclease fares somewhat better than fragment P, upon exposure to both HF and piperi- dine, with 70 to 80 per cent of its original activity being recoverable. Heterogeneity and purification: In large synthetic polypeptides heterogeneity will exist, whatever the means of synthesis. With lengthy solid-phase products only relatively gross errors are detectable by the analytical techniques reported here. A 2 per cent amino acid deletion at each coupling step would pass un- noticed. In such cases it may be desirable to exploit certain functional prop- erties of the synthetic product to improve its purity and specific activity. This principle was used by Hofmann in a purification of ribonuclease-S-peptide." Kato and Anfinsen have recently used a Sepharose-S-peptide column to purify a crude mixture of synthetic S-peptide derivative (residues 1 through 15)."? With synthetic preparations of complete enzymes, affinity chromatography upon a column bearing a suitable ligand should prove useful. A Sepharose column bear- ing pdTp has proved very effective in purifying native nuclease.” In the current state of the art, solid-phase synthesis offers a promising tech- nique for studying relationships between structure and function in polypeptides Vot. 64, 1969 CHEMISTRY: ONTJES AND ANFINSEN 435 and proteins of biological interest. It should be emphasized that the problem of heterogeneity, together with low or variable yield of the correct sequence in the synthetic product, dictates caution in the interpretation of results. Neverthe- less, valuable information may be obtained when synthetic peptide analogs differ grossly in their biologic activities. Preliminary studies with synthetic Pz analogs indicate that residues 6 through 9 (including the histidine residue at position 8) may be deleted without significant loss of binding to P; or enzymatic activity. Deletion of residues 6 through 17 leaves an inactive derivative which still binds to a P;-Sepharose column. Fur- ther deletion leads to loss of binding as well. Both methionines in the 6 to 47 sequence may be replaced with norleucine to yield an active analog. Replace- ment of glutamic acid in position 43 by glutamine leads to complete loss of cata- lytie activity, while binding to P; is retained. A full account of the results of these studies on analogs will be published elsewhere. ' Taniuchi, H., C. B. Anfinsen, and A. Sodja, these ProceEpines, 58, 1235 (1967). ? Taniuchi, H., and C, B. Anfinsen, J. Biol. Chem., 243, 4778 (1968). * Richards, F. M., and P. J. Vithayathil, J. Biol. Chem., 234, 1459 (1959). *Finn, F. M., and K. Hofmann, J. Am. Chem. Soc., 87, 645 (1965). 5 Arnone, A., C. J. Bier, F. A. Cotton, E. E. Hazen, D. C. Richardson, and J. 8. Richardson, these Procexpinas, 64, 420 (1969). * Schechter, A. N., L. Mordvek, and C. B. Anfinsen, these PRocEEDINGS, 61, 1478 (1968). * Merrifield, R. B., Science, 150, 178 (1965). * Gutte, B., and R. B. Merrifield, J. Am. Chem. Soc., 91, 501 (1969). * Mordvek, L., C. B. Anfinsen, J. L. Cone, and H. Taniuchi, J. Biol. Chem., 244, 497 (1969). ” Anfinsen, C. B., D. Ontjes, M. Ohno, L. Corley, and A. Eastlake, these PRocEEDINGs, 58, 1806 (1967). 1 Sakakibara, S., Y. Shimonishi, Y. Kishida, M. Okada, and H. Sugihara, Bull. Chem. Soc. Japan, 40, 2164 (1967). : ? Lenard, J., and A. B. Robinson, J. Am. Chem. Soc., 89, 181 (1967). 8 Anfinsen, C. B., M. Sela, and J. P. Cooke, J. Biol. Chem., 237, 1826 (1962). * Porath, J., R. Axen, and S. Emback, Nature, 215, 1491 (1967). © Hill, R. L., and W. R. Schmidt, J. Biol. Chem., 237, 389 (1962). The prolidase used in these studies was the gift of Professor R. L. Hill, Duke University Medical School. * Ontjes, D. A., and C. B. Anfinsen, Proceedings of the 1st American Peptide Symposium, in press. 7 Yaron, A., and 8. Schlossman, Biochem. J., 7, 2673 (1968). * Hofmann, K., M, J. Smithers, and F. M. Finn, J. Am. Chem. Soc., 88, 4107 (1966). ° Kato, L, and C. B. Anfinsen, J. Biol. Chem., in press. * Cuatrecasas, P., M. Wilchek, and C. B. Anfinsen, these ProceepiNas, 61, 636 (1968). 1 'Taniuchi, H., C. L. Cusumano, C. B. Anfinsen, and J. L. Cone, J. Biol. Chem., 243, 4775 (1968). 2? Cone, J. L., H. Taniuchi, and C. B. Anfinsen, unpublished observations. *® Katz, A., W. J. Dreyer, and C. B. Anfinsen, J. Biol. Chem., 234, 2897 (1959). * Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem., 193, 265 (1951). *® Cuatrecasas, P., 8. Fuchs, and C. B. Anfinsen, J. Biol. Chem., 242, 1541 (1967).