- Email: [email protected]
A method for the specific proteolytic cleavage of protein chains
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
66, 156-163 (1956)
A Method for the Specific Proteolytic Cleavage of Protein Chains Christian B. Anfinsen, Michael Sela,’ and Harold Tritch2 From
the Laboratory of Cellular Physiology and Metabolism, National Institute, National Institutes of Health, Public Health Service, United States Department of Health, Education and Welfare, Bethesda, Maryland
Heart
Received June 5, 1956
The reproducible preparation of relatively large fragments of protein chains is of considerable importance as a preliminary step in the determination of the sequential arrangement of amino acid residues. Since partial hydrolyzates prepared with acid are generally far too complex (l-3), most of the proteins and large polypeptides (4-11) hitherto studied have been degraded with one or more proteolytic enzymes. The assignment of fragments, so produced, to their proper position along the peptide chain may then be accomplished by taking advantage of the overlapping compositions of peptides obtained from hydrolyzates of the protein using several different enzymes. Since the proper sequential alignment of peptides from particularly long chains is likely to become quite difficult, we have sought to devise methods for limiting, in a specific way, the number of peptide fragments produced in the reaction. In a previous paper (5) we have described the digestion of dinitrophenylated oxidized ribonuclease with trypsin. The substitution of a dinitrophenyl radical on the E-amino position of lysine prevents the cleavage, by trypsin, of bonds involving the carboxyl group of this amino acid. One obtains, then, a number of fragments, one greater than the number of arginine residues in the polypeptide chain. The N-terminally located peptide fragment contains, as its end group, the same amino acid as the 1 On leave of absence from the Weizmann Institute of Science, Rehovoth, Israel. e In partial fulfillment of the requirements for the M.S. degree at Georgetown University, Washington, D. C. 156
157
METHOD FOR CLEAVAGE OF PROTEIN CHAINS
original chain. The C-terminal peptide sequence is identified by its lack of arginine. Other fragments may be properly positioned in relation to one another by the isolation and characterization of arginine-containing “bridges,” from acid or enzyme hydrolyzates. The separation of large dinitrophenylated peptides is made difficult, however, by the strong adsorption of such molecules to filter paper and resin columns, and sharp separation has been achieved only by electrophoresis in urea-saturated buffers. This problem is avoided, to a large extent, by the use of carbobenzoxylation rather than dinitrophenylation. Following trypsin digestion of the carbobenzoxylated protein, the masking groups may be removed by treatment with anhydrous formic acid saturated with HBr (12). The general principle of the method is summarized in the following equation: CBZ I (N-terminal). (residue) CBZ
I
CBZ I .Lys. . .
..
CBZ I . .Arg. . . . .Lys. .
CBZ
I
N-term. . . . .LYs. +
Trypsin
CBZ
I
beneyl bromide
. . (C-terminal) (residue)
Arg + NHeR. GOa + unsubstituted MATERIALS
.Lys.
.
peptide
AND GENERAL
(C-term.)
fragments
HBr HCOOH
’
(as HBr salts)
METHODS
Commercial ribonuclease (Armour, Lot $4381-059) was oxidized with performic acid as previously described (5), and the reaction mixture was diluted with icecold water and lyophilized at once. Digestion of the carbenzoxylated protein was carried out with crystalline trypsin, treated in some experiments by preliminary solution in 8 M urea (13) before use to denature irreversibly any contaminating chymotrypsin which might be present. Ninhydrin determinations were performed by the method of Cocking and Yemm (14). The Levy modification (15) of the Sanger technique (16) was used for end-group analysis except in the case of the water-soluble dinitrophenyl derivatives. These, DNP-cysteic acid,3 and E-DNPlysine were extracted from the acidified aqueous solution with n-BOH, and separated by electrophoresis on Whatman %3 filter paper in phosphate buffer, 0.1 M, at pH 7.0. As in the case of the ether-soluble DNP-amino acids separated by the usual Levy solvent systems, the yellow spots were eluted from the paper with water and estimated in the Beckman spectrophotometer. * Abbreviations: DNP, dinitrophenyl; abbreviations for amino acids (e.g., alanine = Ala) are as recommended by E. Brand and J. Edsall [see Ref. (l)].
158
AmINSEN, SEW, AND TRITCH RESTJLTS 1. Carbobenzoxylation
In a typical experiment, 50 mg. of oxidized ribonuclease was dissolved in 10 ml. of 1 M NaHC03 at -3°C. Forty microliters of carbobenzoxychloride was added, and the reaction mixture was vigorously agitated. A second 40-~1. aliquot of reagent was added after 70 min. At various times during the course of the reaction, aliquots of 0.1 ml. were pipetted into a solution composed of 0.5 ml. of 0.2 M citrate buffer, pH 5.0, and 0.1 ml. of 1 iV HCl, and the content of free amino groups was estimated by reaction with ninhydrin. The data summarized in Fig. 1 show that the reaction is essentially complete after 50 min. The small residual content of free amino groups which persists during further reaction corresponds, approximately, to one such group per mole of protein and could represent a single, particularly hindered, lysine residue. However, as is discussed below, no end groups were detected in significant amounts in trypsin digests of the carbobenzoxylated protein other than those to be expected from the hydrolysis of peptide bonds formed by the carboxyl groups of the four arginine residues in ribonuclease. It seemsmore likely, therefore, that this residual ninhydrin color is a blank value not corrected for by the ordinary controls used in the Cocking and Yemm method.
60
120 180 MINUTES
240
FIG. 1. Disappearance Optical $455 filter.
of ninhydrin-reactive groups during carbobeneoxylation of oxidized ribonuclease. density was measured in the Klett-Summerson calorimeter
using
a
159
METHOD FOR CLEAVAQE OF PROTEIN CHAINS
The reaction mixture was acidified with HCl to pH 34, and the insoluble product was washed thoroughly by centrifugation with water, acetone, and ether,‘and dried in vacua. Yields were essentially quantitative. 2. Trypsin Digestion The carbobenzoxylated protein derivative was suspended in about 100 parts of water and, after adjustment of the pH to 8.0 with trimethylamine, was digested with crystalline trypsin, added at a level of 1% that of the protein substrate. The pH was maintained at 8.0 with 1% trimethylamine added from a buret, activated by a Coleman Auto-titrator. The relatively insoluble substrate was completely solubilized after 5-10 min. of digestion at 37°C. Table I lists the end-group analyses obtained on samples of the digest withdrawn from the reaction mixture after various lengths of incubation. Although not indicated in the table, traces of serine, leucine, valine, threonine, and alanine were always observed as end groups in trypsin digests of the carbobenzoxylated protein. When the digestion time was limited to 4 hr., the most prominent of these (serine) did not exceed 0.15 residues/mole. Similar results were observed in earlier experiments (5) on the digestion of dinitrophenylated ribonuclease and undoubtedly reflect the presence in the crystalline trypsin of small amounts of conTABLE I N-Terminal Amino Acid Analyses The general procedure of end-group estimation, together with the necessary correction factors for destruction during hydrolysis and losses during chromatography are as given elsewhere (5, 15). ‘4: Carbobenzoxy-oxidized ribonuclease following trypsin digestion Length of digestion
DNP-Asp
Moles of DNP-end group/mole of rilmnuclease DNP-Glu Bis-DNP-Lys cDNP-Lys DNP-CySOaH
hr.
B: Following
1 2 4
1.0 1.0 1.0
1.3 1.5 1.8
0 0 0
0 0 0
0.3 0.7 0.8
Theory
1.0
2.0
0
0
1.0
removal of carbobenzoxy Theory
groups from trypsin-digested
material
1.0
1.8
0.9
10.0
0.7
1.0
2.0
1.0
10.0
1.0
160
ANFINSEN,
SELA,
AND TRITCH
taminating proteolytic activity. Treatment of the trypsin by solution in 8 M urea prior to use (13) resulted in some decreasein the amounts of these nonspecific end groups, of about the order achieved with preliminary acid treatment according to Northrop and Kunitz (17). For the present study, however, the problem of nonspecific cleavageshas not been further pursued. As indicated in Table I, four new end groups are liberated during trypsin digestion in accordance with the presence in ribonuclease of four arginine residues. The Arg.Asp bond is most rapidly broken and the Arg.CySOs bond most slowly. The two Arg.Glu bonds are split at an intermediate rate. The kinetics of hydrolysis of these various bonds are similar to those observed in the case of the dinitrophenylated chain (5). The absence of bis-DNP-lysine confirms the completeness of the carbobenzoxylation reaction in the case of the N-terminal residue, and no E-DNP-lysine could be detected in hydrolyzates of samples of the carbobenzoxylated protein after dinitrophenylation. Evidence for the carbobenzoxylation of the hydroxyl groups of tyrosine was given by the fact that the quantitative Millon reaction for tyrosine was negative until the carbobenzoxy groups had been removed. Further, the spectrum of the carbobenzoxylated protein was devoid of the characteristic tyrosine absorption in the region of 280 rnp. 3. Removal of Carbobtmzoxy Groups
Although it was found in preliminary experiments that the carbobenzoxy groups could be removed by reduction with hydrogen gas in the presenceof palladium catalyst in anhydrous formic acid, removal of these groups has generally been carried out by the more convenient nonhydrolytic cleavage with anhydrous formic acid saturated with HBr. Upon solution of the trypsin digested, carbobenzoxylated material in this reagent, there occurs a vigorous evolution of Cot, and the typical lachrymatory properties of benzyl bromide may be detected. After 15-30 min. at room temperature, the reaction mixture is diluted with a large volume of anhydrous ether. The precipitated mixture of peptides is washed several times with ether and dried in vacua. End-group analysis by the Levy technique, and by electrophoresis of the water-soluble DNP derivatives as described in the section above on Materials and General Methods, yielded results such as shown in Table I. The yield of DNP-cysteic acid was somewhat low in the experiment shown. A similar low value is generally observed, however, in most ex-
METHOD
FOR
CLEAVAQE
OF
PROTEIN
CHAINS
161
periments and reflects the particular resistance of the Arg.CyEQH bond to trypsin digestion. We have, in general, avoided digestion conditions sufficiently severe to yield quantitative cleavage of this bond since the nonspecific cleavages due to contaminating proteolytic, enzymes may then reach disturbing proportions. The main consequence of this incomplete splitting, in the case of ribonuclease, is the presence in the digestion mixture of six, rather than five, peptide species, the sixth being composed of the aspartic acid terminal peptide still attached to the cysteic acid terminal peptide through an Arg.CyS03H bond. The separation of the peptide fragments produced by the method described in this paper has been carried out by the Michl (18) highvoltage paper-electrophoresis technique. Clean separation of four of the five main components is achieved by electrophoresis at 2000 v. for 2 hr., using the volatile pyridine-acetic acid buffer at pH 6.5 described by Ryle, Sanger, Smith, and Kitai (19). The f&h band, which moves only slightly from the origin, is contaminated with the unsplit aspartic acid terminal fragment referred to above. Further studies are now in progress to improve the conditions of electrophoresis to permit complete separation in one run, and to investigate ion-exchange column procedures which might be particularly useful in the separation of peptides of the size likely to be obtained by the application of this general hydrolytic method to large protein molecules. DISCUSSION The specificity of trypsin, in comparison with other proteolytic enzymes, has been well established by a number of studies on various protein and polypeptide chains (11). These studies indicate that, when the time of digestion is not unnecessarily extended, transpeptidation does not seem to take place to a detectable degree. It should be emphasized, however, that this conclusion is based on purely negative evidence and that the translocations due to trypsin observed by Waley and Watson (20) and by Levin, Berger, and Katchalski (21) must be kept in mind during sequence studies which rely on proteolytic cleavage. The present method should be of particular value in the case of polypeptide chains of greater length than occurs in ribonuclease (124 residues) since in such instances the reconstruction of sequences from a consideration of overlapping compositions of peptide fragments obtained by a variety of cleavage methods might become extremely difficult. A problem which is certain to arise in many proteins is the destructive
162
ANFINSEN, SELA, AND TRITCH
effort of performic acid oxidation on tryptophan residues. Such oxidation might conceivably,-of itself, cause ruptures in the peptide chain. The full usefulness of the present method will only be achieved, therefore, when methods are developed which will exclusively rupture the disulfide bond without attacking other bonds in proteins. SUMMARY
Oxidized ribonuclease was reacted with carbobenzoxychloride, the carbobenzoxylated protein obtained was digested with trypsin, and the carbobenzoxy groups were removed from the tryptic digest by means of anhydrous hydrogen bromide. The analysis of the tryptic digest before and after decarbobenzoxylation showed that four new end groups were liberated during trypsin digestion, in accordance with the presence in ribonuclease of four arginine residues. The peptide fragments formed were separated by high-voltage paper electrophoresis. The experiments described suggest that this method may be of general use as a preliminary step in the determination of the sequential arrangement of amino acid residues in peptidic chains. REFERENCES 1. SANDER, F., Advances in Protein Chem. 7, 1 (1952). 2. DESNUELLE, P., in “The Proteins” (H. Neurath and K.
Bailey, eds.), Vol.
IA. AcademicPress,New York, 1953. 3. THOMPSON, A. R., Biochem. J. 90, 507 (1955). 4. SANGER, F., Symposia Sot. Exptl. Biol. 9. 10 (1955). 5. REDFIELD, R. R., AND ANFINSEN, C. B., J. Biol. Chem. 22l, 385 (1956). 6. HIRS, C. H. W., MOORE, S., AND STEIN, W., J. Biol. Chem. 219,623 (1956). 7. HOWARD, K. S., SHEPHERD, R. G., EIGNER, E. A., DAVIES, 0. S., AND BELL, P. H., J, Am. Chem. Sot. 77, 3419 (1955). 8. WHITE, W. F., AND LANDMANN, W. A., J. Am. Chem. Sot. 77, 1711 (1955). 9. LI, C. H., GESCIWIND, I., COLE, D., RAACKE, I., HARXUS, J., AND DIXON, J., Nature 176, 687 (1955). 10. BROMER, W. W., SINN, L. G., STAUB, A., AND BEHRENS, O., J. Am. Chem. Sot. 78, 3858 (1956). 11. ANFINSEN, C. B., AND REDFIELD, R. R., Advances in Protein Chem. 11, 1 12. 13. 14. 15. 16.
BERGER, A., AND BEN-ISHAI, D., J. Org. Chem. 17,1564 (1952). HARRIS, J. I., Nature 177, 471 (1956). COCKING, E. C., AND YEMY, E. W., Biochem. J. 69, xii (1954).
LEVY, A. L., Nature 174, 128 (1954). BANGER, F., Biochem. J. 39, 507 (1945).
METHOD
FOR
CLEAVAGE
OF PROTEIN
CHAINS
163
17. NORTHROP, J. H., AND KTJNITZ, M., in “Handbuch der biol. Arbeitsmeth.” (Abderhalden, E.) Abt. IV, Teil 2, 2213. Urban und Schwarzenberg, Berlin, 1936. 18. MICHL, H., Mona&h. Chem. 82, 489 (1951). 19. RYLE, A. P., SANGER, F., SMITH, L. F., AND KITAI, It., Biochem. J. 60, 541 (1955). 20. WALEY, S. G., AND WATSON, J., Biochem. J. 66, 328 (1953). 21. LEVIN, Y., BERGER, A., AND KATCHAI,SKI, E., Biochem. J. 63, 308 (1966).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
66, 156-163 (1956)
A Method for the Specific Proteolytic Cleavage of Protein Chains Christian B. Anfinsen, Michael Sela,’ and Harold Tritch2 From
the Laboratory of Cellular Physiology and Metabolism, National Institute, National Institutes of Health, Public Health Service, United States Department of Health, Education and Welfare, Bethesda, Maryland
Heart
Received June 5, 1956
The reproducible preparation of relatively large fragments of protein chains is of considerable importance as a preliminary step in the determination of the sequential arrangement of amino acid residues. Since partial hydrolyzates prepared with acid are generally far too complex (l-3), most of the proteins and large polypeptides (4-11) hitherto studied have been degraded with one or more proteolytic enzymes. The assignment of fragments, so produced, to their proper position along the peptide chain may then be accomplished by taking advantage of the overlapping compositions of peptides obtained from hydrolyzates of the protein using several different enzymes. Since the proper sequential alignment of peptides from particularly long chains is likely to become quite difficult, we have sought to devise methods for limiting, in a specific way, the number of peptide fragments produced in the reaction. In a previous paper (5) we have described the digestion of dinitrophenylated oxidized ribonuclease with trypsin. The substitution of a dinitrophenyl radical on the E-amino position of lysine prevents the cleavage, by trypsin, of bonds involving the carboxyl group of this amino acid. One obtains, then, a number of fragments, one greater than the number of arginine residues in the polypeptide chain. The N-terminally located peptide fragment contains, as its end group, the same amino acid as the 1 On leave of absence from the Weizmann Institute of Science, Rehovoth, Israel. e In partial fulfillment of the requirements for the M.S. degree at Georgetown University, Washington, D. C. 156
157
METHOD FOR CLEAVAGE OF PROTEIN CHAINS
original chain. The C-terminal peptide sequence is identified by its lack of arginine. Other fragments may be properly positioned in relation to one another by the isolation and characterization of arginine-containing “bridges,” from acid or enzyme hydrolyzates. The separation of large dinitrophenylated peptides is made difficult, however, by the strong adsorption of such molecules to filter paper and resin columns, and sharp separation has been achieved only by electrophoresis in urea-saturated buffers. This problem is avoided, to a large extent, by the use of carbobenzoxylation rather than dinitrophenylation. Following trypsin digestion of the carbobenzoxylated protein, the masking groups may be removed by treatment with anhydrous formic acid saturated with HBr (12). The general principle of the method is summarized in the following equation: CBZ I (N-terminal). (residue) CBZ
I
CBZ I .Lys. . .
..
CBZ I . .Arg. . . . .Lys. .
CBZ
I
N-term. . . . .LYs. +
Trypsin
CBZ
I
beneyl bromide
. . (C-terminal) (residue)
Arg + NHeR. GOa + unsubstituted MATERIALS
.Lys.
.
peptide
AND GENERAL
(C-term.)
fragments
HBr HCOOH
’
(as HBr salts)
METHODS
Commercial ribonuclease (Armour, Lot $4381-059) was oxidized with performic acid as previously described (5), and the reaction mixture was diluted with icecold water and lyophilized at once. Digestion of the carbenzoxylated protein was carried out with crystalline trypsin, treated in some experiments by preliminary solution in 8 M urea (13) before use to denature irreversibly any contaminating chymotrypsin which might be present. Ninhydrin determinations were performed by the method of Cocking and Yemm (14). The Levy modification (15) of the Sanger technique (16) was used for end-group analysis except in the case of the water-soluble dinitrophenyl derivatives. These, DNP-cysteic acid,3 and E-DNPlysine were extracted from the acidified aqueous solution with n-BOH, and separated by electrophoresis on Whatman %3 filter paper in phosphate buffer, 0.1 M, at pH 7.0. As in the case of the ether-soluble DNP-amino acids separated by the usual Levy solvent systems, the yellow spots were eluted from the paper with water and estimated in the Beckman spectrophotometer. * Abbreviations: DNP, dinitrophenyl; abbreviations for amino acids (e.g., alanine = Ala) are as recommended by E. Brand and J. Edsall [see Ref. (l)].
158
AmINSEN, SEW, AND TRITCH RESTJLTS 1. Carbobenzoxylation
In a typical experiment, 50 mg. of oxidized ribonuclease was dissolved in 10 ml. of 1 M NaHC03 at -3°C. Forty microliters of carbobenzoxychloride was added, and the reaction mixture was vigorously agitated. A second 40-~1. aliquot of reagent was added after 70 min. At various times during the course of the reaction, aliquots of 0.1 ml. were pipetted into a solution composed of 0.5 ml. of 0.2 M citrate buffer, pH 5.0, and 0.1 ml. of 1 iV HCl, and the content of free amino groups was estimated by reaction with ninhydrin. The data summarized in Fig. 1 show that the reaction is essentially complete after 50 min. The small residual content of free amino groups which persists during further reaction corresponds, approximately, to one such group per mole of protein and could represent a single, particularly hindered, lysine residue. However, as is discussed below, no end groups were detected in significant amounts in trypsin digests of the carbobenzoxylated protein other than those to be expected from the hydrolysis of peptide bonds formed by the carboxyl groups of the four arginine residues in ribonuclease. It seemsmore likely, therefore, that this residual ninhydrin color is a blank value not corrected for by the ordinary controls used in the Cocking and Yemm method.
60
120 180 MINUTES
240
FIG. 1. Disappearance Optical $455 filter.
of ninhydrin-reactive groups during carbobeneoxylation of oxidized ribonuclease. density was measured in the Klett-Summerson calorimeter
using
a
159
METHOD FOR CLEAVAQE OF PROTEIN CHAINS
The reaction mixture was acidified with HCl to pH 34, and the insoluble product was washed thoroughly by centrifugation with water, acetone, and ether,‘and dried in vacua. Yields were essentially quantitative. 2. Trypsin Digestion The carbobenzoxylated protein derivative was suspended in about 100 parts of water and, after adjustment of the pH to 8.0 with trimethylamine, was digested with crystalline trypsin, added at a level of 1% that of the protein substrate. The pH was maintained at 8.0 with 1% trimethylamine added from a buret, activated by a Coleman Auto-titrator. The relatively insoluble substrate was completely solubilized after 5-10 min. of digestion at 37°C. Table I lists the end-group analyses obtained on samples of the digest withdrawn from the reaction mixture after various lengths of incubation. Although not indicated in the table, traces of serine, leucine, valine, threonine, and alanine were always observed as end groups in trypsin digests of the carbobenzoxylated protein. When the digestion time was limited to 4 hr., the most prominent of these (serine) did not exceed 0.15 residues/mole. Similar results were observed in earlier experiments (5) on the digestion of dinitrophenylated ribonuclease and undoubtedly reflect the presence in the crystalline trypsin of small amounts of conTABLE I N-Terminal Amino Acid Analyses The general procedure of end-group estimation, together with the necessary correction factors for destruction during hydrolysis and losses during chromatography are as given elsewhere (5, 15). ‘4: Carbobenzoxy-oxidized ribonuclease following trypsin digestion Length of digestion
DNP-Asp
Moles of DNP-end group/mole of rilmnuclease DNP-Glu Bis-DNP-Lys cDNP-Lys DNP-CySOaH
hr.
B: Following
1 2 4
1.0 1.0 1.0
1.3 1.5 1.8
0 0 0
0 0 0
0.3 0.7 0.8
Theory
1.0
2.0
0
0
1.0
removal of carbobenzoxy Theory
groups from trypsin-digested
material
1.0
1.8
0.9
10.0
0.7
1.0
2.0
1.0
10.0
1.0
160
ANFINSEN,
SELA,
AND TRITCH
taminating proteolytic activity. Treatment of the trypsin by solution in 8 M urea prior to use (13) resulted in some decreasein the amounts of these nonspecific end groups, of about the order achieved with preliminary acid treatment according to Northrop and Kunitz (17). For the present study, however, the problem of nonspecific cleavageshas not been further pursued. As indicated in Table I, four new end groups are liberated during trypsin digestion in accordance with the presence in ribonuclease of four arginine residues. The Arg.Asp bond is most rapidly broken and the Arg.CySOs bond most slowly. The two Arg.Glu bonds are split at an intermediate rate. The kinetics of hydrolysis of these various bonds are similar to those observed in the case of the dinitrophenylated chain (5). The absence of bis-DNP-lysine confirms the completeness of the carbobenzoxylation reaction in the case of the N-terminal residue, and no E-DNP-lysine could be detected in hydrolyzates of samples of the carbobenzoxylated protein after dinitrophenylation. Evidence for the carbobenzoxylation of the hydroxyl groups of tyrosine was given by the fact that the quantitative Millon reaction for tyrosine was negative until the carbobenzoxy groups had been removed. Further, the spectrum of the carbobenzoxylated protein was devoid of the characteristic tyrosine absorption in the region of 280 rnp. 3. Removal of Carbobtmzoxy Groups
Although it was found in preliminary experiments that the carbobenzoxy groups could be removed by reduction with hydrogen gas in the presenceof palladium catalyst in anhydrous formic acid, removal of these groups has generally been carried out by the more convenient nonhydrolytic cleavage with anhydrous formic acid saturated with HBr. Upon solution of the trypsin digested, carbobenzoxylated material in this reagent, there occurs a vigorous evolution of Cot, and the typical lachrymatory properties of benzyl bromide may be detected. After 15-30 min. at room temperature, the reaction mixture is diluted with a large volume of anhydrous ether. The precipitated mixture of peptides is washed several times with ether and dried in vacua. End-group analysis by the Levy technique, and by electrophoresis of the water-soluble DNP derivatives as described in the section above on Materials and General Methods, yielded results such as shown in Table I. The yield of DNP-cysteic acid was somewhat low in the experiment shown. A similar low value is generally observed, however, in most ex-
METHOD
FOR
CLEAVAQE
OF
PROTEIN
CHAINS
161
periments and reflects the particular resistance of the Arg.CyEQH bond to trypsin digestion. We have, in general, avoided digestion conditions sufficiently severe to yield quantitative cleavage of this bond since the nonspecific cleavages due to contaminating proteolytic, enzymes may then reach disturbing proportions. The main consequence of this incomplete splitting, in the case of ribonuclease, is the presence in the digestion mixture of six, rather than five, peptide species, the sixth being composed of the aspartic acid terminal peptide still attached to the cysteic acid terminal peptide through an Arg.CyS03H bond. The separation of the peptide fragments produced by the method described in this paper has been carried out by the Michl (18) highvoltage paper-electrophoresis technique. Clean separation of four of the five main components is achieved by electrophoresis at 2000 v. for 2 hr., using the volatile pyridine-acetic acid buffer at pH 6.5 described by Ryle, Sanger, Smith, and Kitai (19). The f&h band, which moves only slightly from the origin, is contaminated with the unsplit aspartic acid terminal fragment referred to above. Further studies are now in progress to improve the conditions of electrophoresis to permit complete separation in one run, and to investigate ion-exchange column procedures which might be particularly useful in the separation of peptides of the size likely to be obtained by the application of this general hydrolytic method to large protein molecules. DISCUSSION The specificity of trypsin, in comparison with other proteolytic enzymes, has been well established by a number of studies on various protein and polypeptide chains (11). These studies indicate that, when the time of digestion is not unnecessarily extended, transpeptidation does not seem to take place to a detectable degree. It should be emphasized, however, that this conclusion is based on purely negative evidence and that the translocations due to trypsin observed by Waley and Watson (20) and by Levin, Berger, and Katchalski (21) must be kept in mind during sequence studies which rely on proteolytic cleavage. The present method should be of particular value in the case of polypeptide chains of greater length than occurs in ribonuclease (124 residues) since in such instances the reconstruction of sequences from a consideration of overlapping compositions of peptide fragments obtained by a variety of cleavage methods might become extremely difficult. A problem which is certain to arise in many proteins is the destructive
162
ANFINSEN, SELA, AND TRITCH
effort of performic acid oxidation on tryptophan residues. Such oxidation might conceivably,-of itself, cause ruptures in the peptide chain. The full usefulness of the present method will only be achieved, therefore, when methods are developed which will exclusively rupture the disulfide bond without attacking other bonds in proteins. SUMMARY
Oxidized ribonuclease was reacted with carbobenzoxychloride, the carbobenzoxylated protein obtained was digested with trypsin, and the carbobenzoxy groups were removed from the tryptic digest by means of anhydrous hydrogen bromide. The analysis of the tryptic digest before and after decarbobenzoxylation showed that four new end groups were liberated during trypsin digestion, in accordance with the presence in ribonuclease of four arginine residues. The peptide fragments formed were separated by high-voltage paper electrophoresis. The experiments described suggest that this method may be of general use as a preliminary step in the determination of the sequential arrangement of amino acid residues in peptidic chains. REFERENCES 1. SANDER, F., Advances in Protein Chem. 7, 1 (1952). 2. DESNUELLE, P., in “The Proteins” (H. Neurath and K.
Bailey, eds.), Vol.
IA. AcademicPress,New York, 1953. 3. THOMPSON, A. R., Biochem. J. 90, 507 (1955). 4. SANGER, F., Symposia Sot. Exptl. Biol. 9. 10 (1955). 5. REDFIELD, R. R., AND ANFINSEN, C. B., J. Biol. Chem. 22l, 385 (1956). 6. HIRS, C. H. W., MOORE, S., AND STEIN, W., J. Biol. Chem. 219,623 (1956). 7. HOWARD, K. S., SHEPHERD, R. G., EIGNER, E. A., DAVIES, 0. S., AND BELL, P. H., J, Am. Chem. Sot. 77, 3419 (1955). 8. WHITE, W. F., AND LANDMANN, W. A., J. Am. Chem. Sot. 77, 1711 (1955). 9. LI, C. H., GESCIWIND, I., COLE, D., RAACKE, I., HARXUS, J., AND DIXON, J., Nature 176, 687 (1955). 10. BROMER, W. W., SINN, L. G., STAUB, A., AND BEHRENS, O., J. Am. Chem. Sot. 78, 3858 (1956). 11. ANFINSEN, C. B., AND REDFIELD, R. R., Advances in Protein Chem. 11, 1 12. 13. 14. 15. 16.
BERGER, A., AND BEN-ISHAI, D., J. Org. Chem. 17,1564 (1952). HARRIS, J. I., Nature 177, 471 (1956). COCKING, E. C., AND YEMY, E. W., Biochem. J. 69, xii (1954).
LEVY, A. L., Nature 174, 128 (1954). BANGER, F., Biochem. J. 39, 507 (1945).
METHOD
FOR
CLEAVAGE
OF PROTEIN
CHAINS
163
17. NORTHROP, J. H., AND KTJNITZ, M., in “Handbuch der biol. Arbeitsmeth.” (Abderhalden, E.) Abt. IV, Teil 2, 2213. Urban und Schwarzenberg, Berlin, 1936. 18. MICHL, H., Mona&h. Chem. 82, 489 (1951). 19. RYLE, A. P., SANGER, F., SMITH, L. F., AND KITAI, It., Biochem. J. 60, 541 (1955). 20. WALEY, S. G., AND WATSON, J., Biochem. J. 66, 328 (1953). 21. LEVIN, Y., BERGER, A., AND KATCHAI,SKI, E., Biochem. J. 63, 308 (1966).