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[69] Reaction with N-carboxy-α-amino acid anhydrides
580
[69]
MODIFICATION REACTION$
peptide cleavage with trypsin, it may be desirable to block c-NH~ groups of lysine so that the enzyme will attack only at arginyl peptide bonds. Covering of lysyl residues with acetyl groups frequently reduces the solubility of a protein substantially so that enzymatic attack is greatly slowed down. Succinylated proteins, however, in general are soluble at pH values /~7. (3) The succinic anhydride nucleus may provide a vehicle for the introduction of various functional groups into proteins under very mild conditions. As the entries in the table show, sulfur in various forms (protected mercaptan, disulfide, or thioether) or additional carboxyl groups or dimethylamino groups may be introduced. It is thus possible to examine the effects of these groups on the configuration and interactions of these macromolecules.
[69] Reaction with N-Carboxy-a-amino
By
Acid Anhydrides
MICHAEL SELA a n d RUTH ARNON
N-Carboxy-a-amino acid anhydrides readily undergo polymerization, with carbon dioxide evolution, to yield the corresponding poly-a-amino acids. 1 Amino groups are among the best initiators for this polymerization. Proteins, containing numerous free amino groups, may serve as multifunctional initiators, thus yielding polypeptidyl proteins. 2 The polymerization on proteins proceeds under mild conditions (aqueous media, low temperature, and neutral pH range), which do not as a rule cause denaturation of most proteins. It is thus possible by this approach to prepare chemically modified proteins which maintain the principal structural features of the native macromolecule. A considerable number of polypeptidyl enzymes have been prepared. These often retain enzymatic activity, although differing markedly from the native protein in physicochemical properties, such as solubility and electrophoretic mobility. 1 The peptidylation of enzymes may change their substrate specificity as well as pH and ionic strength dependence2 The investigation of a series of polypeptidyl derivatives of a given enzyme may shed light on the effect of charge, steric hindrance, and hydrophilic and hydrophobic groups on the catalytic activity of the enzyme. In studies of the conformation of an enzyme molecule and its rela1E. Katchalski, M. Sela, H. I. Silman and A. Berger, in "The Proteins" (H. Neurath, ed.), Vol. 2, p. 405. Academic Press, New York, 1964. R. R. Becker and M. A. Stahmann, J. Biol. Chem. 204, 745 (1953). D. Wellner, H. I. Silman, and M. Sela, J. Biol. Chem. 238, 1324 (1963).
[59]
N-CARBOXYAMIN ACID O ANHYDRIDES
581
tionship to enzymatic activity, reactions are often used that give rise to insoluble products. Peptides of DL-alanine, when attached to the molecule, have proven to act as a "solubilizing agent," and therefore recourse may be made in such studies to modification of the enzymes with N-carboxyDL-alanine anhydride. A case in point is the alanylation of trypsin, which permitted a study of reduction and reoxidation of this enzyme. In contrast to native trypsin, the poly-DL-alanyl trypsin yielded a soluble product upon complete reduction of its disulfide bonds? Ia connection with the preparation of water-insoluble enzymes, it is often advantageous to obtain a derivative of the enzyme enriched with a particular amino acid to facilitate binding to the insoluble carrier. The preparation of insoluble trypsin can serve as an example. The enzyme was coupled to the diazotized copolymer of p-amino-DL-phenylalanine and L-leucine. Since coupling is achieved through phenolic groups, the enzyme was reacted with N-carboxy-L-tyrosine anhydride, to yield polytyrosyl trypsin, prior to the coupling to the polymer? The study of polypeptidyl enzymes may also help to elucidate the role played by eamino groups in the unmodified enzymes. ~,7 Reaction of Enzymes with N-Carboxy-a-amino Acid Anhydrides In the reaction with the N-carboxy-a-amino acid anhydride, both the a-amino and the e-amino groups of the enzyme serve as initiators for the polymerization. The reaction proceeds according to the scheme given in Fig. 1. R
+ nl
I CO-CH--NH C
N~
t CO
"•NH-
(CO- CH-N: )rnH
"/~-- NH,-(CO'-iH -- NH),
FIO. 1. Reaction of an enzyme with an N-carboxy-a-amino acid anhydride.
When the N-carboxy-a-amino acid anhydrides contain blocking groups, these groups should be removed after polymerization, under mild conditions, to prevent the inactivation of the enzyme. Such conditions have been attained in the preparation of polylysyl enzymes (via the 4C. J. Epstein and C. B. Anfmsen, J. Biol. Chem. 237, 3464 (1962). ~A. Bar-Eli and E. Katchalski, Nature 188, 856 (1960). C. B. Anfinsen, M. Sela, and J. P. Cooke, J. Biol. Chem. 237, 1825 (1962). ' J . P. Cooke, C. B. Anfinsen, and M. Sela, J. Biol. Chem. 238, 2034 (1963).
582
MODIFICATION REACTIONS
[69]
poly-~,N-trifluoroacetyllysyl derivativesS). Although the peptide chains attached m a y differ somewhat in size, from a statistical analysis of the molecular weight distribution of multichain polyamino acids it m a y be predicted t h a t the homogeneity of the protein will not be greatly affected by the peptidylation reaction. 1 Details of the preparation of three different polypeptidyl enzyme derivatives are given below. Preparation o] Polytyrosyl Trypsin2 A solution of 1 g trypsin-50% MgS04 (i.e., 500 mg trypsin) in 36 ml 0.0025 N HC1 is introduced into a 250-ml flask, and 36 ml 0.1 M phosphate buffer p H 7.6 is added. The final p H of ~he mixture is 7.2. The mixture is chilled in an ice bath to 2 °. A solution of 0.8 g N-carboxy-T.-tyrosine anhydride 1° in 16 ml anhydrous dioxane is added dropwise with vigorous stirring to the cold protein solution. The milky reaction mixture formed is stirred magnetically for 16 hours at 4 °, and then dialyzed for 7 days against daily changes of 6 liters of 0.0025 N hydrochloric acid. The resultant clear solution (any precipitate formed is centrifuged off) 14 is lyophilized and the powder stored at 4 ° . The enrichment in tyrosine is determined by ultraviolet absorption measurements; the number of moles of tyrosine attached per mole of trypsin is calculated from the spectra of trypsin and polytyrosyl trypsin in acid and alkali. Amino-terminal residues are quantitated by dinitrophenylation; the number of moles of bis-dinitrophenyltyrosine obtained, after hydrolysis, per mole of polytyrosyl trypsin indicates the number of moles of peptide chains attached, thus permitting deduction of the average length of the polypeptide side chains. Preparation o] Poly-DL-aMnyl Ribonuclease. Several poly-DL-alanyl derivatives of ribonuc!ease, differing in extent of peptidylation, have been prepared and characterized. 8,8,7 The enrichment in alanine is a 8M. Sela, R. Arnon, and I. Jacobson, Biopolymers 1, 517 (1963). ' A. N. Glazer, A. Bar-Eli, and E. Katchalski, J. Biol. Chem. 237, 1832 (1962). l°N-Carboxy-L-tyros~ne anhydride,11 N-carboxy-DL-alanine anhydride,I~ and ,,N-trifluoroacetyl-a,Nocarboxy-L-lysine anhydrides are prepared by reacting L-tyrosine, DL-alanine, or e,N-trifluoroacetyl-L-lysine, respectively, with phosgene, according to Katchalski and Berge#1; e,N-trifluoroacetyl-L-lysine is prepared by coupling L-]ysine with ethyl thiotrifluoroacetate3 A. Berger, J. Kurtz, T. Sadeh, A. Yaron, R. Arnon and Y. Lapidoth, Bull. Res. Council Israel 7A, 98 (1958). M. Sela and S. Fuchs, in "Methods in Immunology and Immunochemistry," (M. W. Chase and C. A. Williams, eds.). Academic Press, New York, in press. la E. Katchalski and A. Berger, Vol. III, p. 546. t4A more extensive tyrosylation, achieved by using higher amounts of the N-carboxy anhydride, will lead to the formation of insoluble material.
[69]
N-CARBOXYAMINO ACID ANHYDRIDES
583
function of the ratio of the N-carboxy-DL-alanine anhydride to ribonuclease used for the reaction. For a typical preparation, a solution of 1 g bovine pancreatic ribonuclease in 150 ml 0.05M phosphate buffer (pH 6.8) is introduced into a 500-ml Erlenmeyer flask, and cooled in an ice bath to 2% A solution of 3 g N-carboxy-DL-alanine anhydride 1° in 100 ml anhydrous dioxane is added dropwise while the mixture is stirred vigorously. The reaction proceeds at 4 ° with stirring for 24 hours. The entire mixture is then dialyzed in heat-treated cellophane tubing against several changes of distilled water to remove salts, alanine, small oligopeptides of alanine, and dioxane. The resultant solution is lyophilized (1.4 g). Any native ribonuclease or polypeptides of alanine present in the reaction product can be removed by chromatography on a column of phosphorylated cellulose, with a combined gradient of a salt and pH. 6 No native enzyme was found in the reaction product. The enrichment in alanine is determined by amino acid analysis, and the number of peptide chains attached is quantitated by dinitrophenylation or by deamination. The average chain length of the peptides attached may thus be calculated. Under the conditions cited above, 8 of the 11 amino groups of the protein are alanylated and the length of the side chains, which was found to be relatively uniform, is approximately 5. Two additional amino groups may be partially attacked by N-carboxyalanine anhydride upon more extensive alanylation, yielding a material that retains enzymatic activity. The last lysine residue, which was identified as residue 41 in the polypeptide chain of the enzyme, is vulnerable to the N-carboxy anhydride only when phosphate buffer is replaced by bicarbonate. Its alanylation brings about total loss of enzymatic activity. 7 Preparation o] Poly-L-lysyl Ribonuclease. ~5 The trifluoroacetyl function was found to be a useful reversible blocking agent for amino groups, being stable to the conditions of N-carboxy anhydride formation and polymerization, and easily removed under mild conditions. ~6 Consequently it was used in the synthesis of poly-L-lysyl ribonuclease. In a typical preparation, a solution of 0.5 g bovine pancreatic ribonuclease in 70 ml 0.05 M phosphate buffer (pH 7) is introduced into a 250-ml Erlenmeyer flask and cooled with ice to 2 °. A solution of 0.5 g a,N-carboxy~,N-trifluoroacetyl-L-lysine anhydride ~° in 20 ml anhydrous dioxane is added with stirring. The reaction is allowed to proceed for 24 hours in the cold room (4 ° ) and the entire mixture is dialyzed in heat-treated cellophane tubing for 3 days against several changes of 6 liters of distilled is A. Frensdorff, R. Arnon, and M. Sela, unpublished data. le F. Weygand and E. Csendes, Angew. Chem. 64, 136 (1952).
584
MODIFICATION REACTIONS
[70]
water at 4 °. During the reaction or the dialysis a precipitate often appears that contains a fraction of the protein which has been more extensively peptidylated. The precipitate is removed in the centrifuge and the supernatant fluid is lyophilized. The yield is approximately 0.45 g. The extent of the reaction m a y be estimated by fluorine determination, s To remove trifluoroacetyl groups, 100 mg poly-e,N-trifluoroacetyllysyl ribonuclease is introduced into a 25-ml flask and suspended in 7 ml 1 M aqueous piperidine. The suspension is stirred at 4 ° for 30 hours. 17 The clear solution obtained is neutralized with cold 0 . 5 N acetic acid, dialyzed in heat-treated cellophane tubing at 4 ° for 3 days against daily changes of 1 liter of distilled water, and lyophilized. Determinations of the enrichment in lysine, and the average length of the peptide side chains, are carried out by amino acid analysis of native and polylysyl ribonuelease before and after deamination. The number of moles of lysine residues obtained after hydrolysis of the deaminated polypeptidylated material indicates the actual number of moles of peptide chains attached. z~The material dissolves in the piperidine solution after 1 hour, but at this stage still contains about half the original trifluoroacetyl groups. Only after 30 hours in piperidine will the fluorine content drop to less than 1%.
[70] Guanidination
of Proteins
By JOE R. KIMMEL Principle The reaction of O-methylisourea with an amino group results in conversion of this group to a guanidino-group (guanidination, amidination) : R--NH~ q- CH30---C(NH)NH~ --. R--NH--C(NH)NH2 q- CHaOH When applied to lysine, 1,2 reaction can be made to occur only at the e-amino group by appropriate ma'sking of the ~-amino group. The product is the corresponding a-N-substituted homoarginine. The lysine residues of proteins react in the same manner since only the c-amino function is potentially available. Thus, guanidination of a protein converts some or all of the lysine residues to homoarginine residues and, on the basis of experience with several proteins, appears to occur to a minimal extent at the NH2-terminus of polypeptide chains, 1j. p. Greenstein, J. Org. Chem. 2, 480 (1938). 2G. M. Stevens and J. A. Bush, J. Biol. Chem. 183, 139 (1950).
[69]
MODIFICATION REACTION$
peptide cleavage with trypsin, it may be desirable to block c-NH~ groups of lysine so that the enzyme will attack only at arginyl peptide bonds. Covering of lysyl residues with acetyl groups frequently reduces the solubility of a protein substantially so that enzymatic attack is greatly slowed down. Succinylated proteins, however, in general are soluble at pH values /~7. (3) The succinic anhydride nucleus may provide a vehicle for the introduction of various functional groups into proteins under very mild conditions. As the entries in the table show, sulfur in various forms (protected mercaptan, disulfide, or thioether) or additional carboxyl groups or dimethylamino groups may be introduced. It is thus possible to examine the effects of these groups on the configuration and interactions of these macromolecules.
[69] Reaction with N-Carboxy-a-amino
By
Acid Anhydrides
MICHAEL SELA a n d RUTH ARNON
N-Carboxy-a-amino acid anhydrides readily undergo polymerization, with carbon dioxide evolution, to yield the corresponding poly-a-amino acids. 1 Amino groups are among the best initiators for this polymerization. Proteins, containing numerous free amino groups, may serve as multifunctional initiators, thus yielding polypeptidyl proteins. 2 The polymerization on proteins proceeds under mild conditions (aqueous media, low temperature, and neutral pH range), which do not as a rule cause denaturation of most proteins. It is thus possible by this approach to prepare chemically modified proteins which maintain the principal structural features of the native macromolecule. A considerable number of polypeptidyl enzymes have been prepared. These often retain enzymatic activity, although differing markedly from the native protein in physicochemical properties, such as solubility and electrophoretic mobility. 1 The peptidylation of enzymes may change their substrate specificity as well as pH and ionic strength dependence2 The investigation of a series of polypeptidyl derivatives of a given enzyme may shed light on the effect of charge, steric hindrance, and hydrophilic and hydrophobic groups on the catalytic activity of the enzyme. In studies of the conformation of an enzyme molecule and its rela1E. Katchalski, M. Sela, H. I. Silman and A. Berger, in "The Proteins" (H. Neurath, ed.), Vol. 2, p. 405. Academic Press, New York, 1964. R. R. Becker and M. A. Stahmann, J. Biol. Chem. 204, 745 (1953). D. Wellner, H. I. Silman, and M. Sela, J. Biol. Chem. 238, 1324 (1963).
[59]
N-CARBOXYAMIN ACID O ANHYDRIDES
581
tionship to enzymatic activity, reactions are often used that give rise to insoluble products. Peptides of DL-alanine, when attached to the molecule, have proven to act as a "solubilizing agent," and therefore recourse may be made in such studies to modification of the enzymes with N-carboxyDL-alanine anhydride. A case in point is the alanylation of trypsin, which permitted a study of reduction and reoxidation of this enzyme. In contrast to native trypsin, the poly-DL-alanyl trypsin yielded a soluble product upon complete reduction of its disulfide bonds? Ia connection with the preparation of water-insoluble enzymes, it is often advantageous to obtain a derivative of the enzyme enriched with a particular amino acid to facilitate binding to the insoluble carrier. The preparation of insoluble trypsin can serve as an example. The enzyme was coupled to the diazotized copolymer of p-amino-DL-phenylalanine and L-leucine. Since coupling is achieved through phenolic groups, the enzyme was reacted with N-carboxy-L-tyrosine anhydride, to yield polytyrosyl trypsin, prior to the coupling to the polymer? The study of polypeptidyl enzymes may also help to elucidate the role played by eamino groups in the unmodified enzymes. ~,7 Reaction of Enzymes with N-Carboxy-a-amino Acid Anhydrides In the reaction with the N-carboxy-a-amino acid anhydride, both the a-amino and the e-amino groups of the enzyme serve as initiators for the polymerization. The reaction proceeds according to the scheme given in Fig. 1. R
+ nl
I CO-CH--NH C
N~
t CO
"•NH-
(CO- CH-N: )rnH
"/~-- NH,-(CO'-iH -- NH),
FIO. 1. Reaction of an enzyme with an N-carboxy-a-amino acid anhydride.
When the N-carboxy-a-amino acid anhydrides contain blocking groups, these groups should be removed after polymerization, under mild conditions, to prevent the inactivation of the enzyme. Such conditions have been attained in the preparation of polylysyl enzymes (via the 4C. J. Epstein and C. B. Anfmsen, J. Biol. Chem. 237, 3464 (1962). ~A. Bar-Eli and E. Katchalski, Nature 188, 856 (1960). C. B. Anfinsen, M. Sela, and J. P. Cooke, J. Biol. Chem. 237, 1825 (1962). ' J . P. Cooke, C. B. Anfinsen, and M. Sela, J. Biol. Chem. 238, 2034 (1963).
582
MODIFICATION REACTIONS
[69]
poly-~,N-trifluoroacetyllysyl derivativesS). Although the peptide chains attached m a y differ somewhat in size, from a statistical analysis of the molecular weight distribution of multichain polyamino acids it m a y be predicted t h a t the homogeneity of the protein will not be greatly affected by the peptidylation reaction. 1 Details of the preparation of three different polypeptidyl enzyme derivatives are given below. Preparation o] Polytyrosyl Trypsin2 A solution of 1 g trypsin-50% MgS04 (i.e., 500 mg trypsin) in 36 ml 0.0025 N HC1 is introduced into a 250-ml flask, and 36 ml 0.1 M phosphate buffer p H 7.6 is added. The final p H of ~he mixture is 7.2. The mixture is chilled in an ice bath to 2 °. A solution of 0.8 g N-carboxy-T.-tyrosine anhydride 1° in 16 ml anhydrous dioxane is added dropwise with vigorous stirring to the cold protein solution. The milky reaction mixture formed is stirred magnetically for 16 hours at 4 °, and then dialyzed for 7 days against daily changes of 6 liters of 0.0025 N hydrochloric acid. The resultant clear solution (any precipitate formed is centrifuged off) 14 is lyophilized and the powder stored at 4 ° . The enrichment in tyrosine is determined by ultraviolet absorption measurements; the number of moles of tyrosine attached per mole of trypsin is calculated from the spectra of trypsin and polytyrosyl trypsin in acid and alkali. Amino-terminal residues are quantitated by dinitrophenylation; the number of moles of bis-dinitrophenyltyrosine obtained, after hydrolysis, per mole of polytyrosyl trypsin indicates the number of moles of peptide chains attached, thus permitting deduction of the average length of the polypeptide side chains. Preparation o] Poly-DL-aMnyl Ribonuclease. Several poly-DL-alanyl derivatives of ribonuc!ease, differing in extent of peptidylation, have been prepared and characterized. 8,8,7 The enrichment in alanine is a 8M. Sela, R. Arnon, and I. Jacobson, Biopolymers 1, 517 (1963). ' A. N. Glazer, A. Bar-Eli, and E. Katchalski, J. Biol. Chem. 237, 1832 (1962). l°N-Carboxy-L-tyros~ne anhydride,11 N-carboxy-DL-alanine anhydride,I~ and ,,N-trifluoroacetyl-a,Nocarboxy-L-lysine anhydrides are prepared by reacting L-tyrosine, DL-alanine, or e,N-trifluoroacetyl-L-lysine, respectively, with phosgene, according to Katchalski and Berge#1; e,N-trifluoroacetyl-L-lysine is prepared by coupling L-]ysine with ethyl thiotrifluoroacetate3 A. Berger, J. Kurtz, T. Sadeh, A. Yaron, R. Arnon and Y. Lapidoth, Bull. Res. Council Israel 7A, 98 (1958). M. Sela and S. Fuchs, in "Methods in Immunology and Immunochemistry," (M. W. Chase and C. A. Williams, eds.). Academic Press, New York, in press. la E. Katchalski and A. Berger, Vol. III, p. 546. t4A more extensive tyrosylation, achieved by using higher amounts of the N-carboxy anhydride, will lead to the formation of insoluble material.
[69]
N-CARBOXYAMINO ACID ANHYDRIDES
583
function of the ratio of the N-carboxy-DL-alanine anhydride to ribonuclease used for the reaction. For a typical preparation, a solution of 1 g bovine pancreatic ribonuclease in 150 ml 0.05M phosphate buffer (pH 6.8) is introduced into a 500-ml Erlenmeyer flask, and cooled in an ice bath to 2% A solution of 3 g N-carboxy-DL-alanine anhydride 1° in 100 ml anhydrous dioxane is added dropwise while the mixture is stirred vigorously. The reaction proceeds at 4 ° with stirring for 24 hours. The entire mixture is then dialyzed in heat-treated cellophane tubing against several changes of distilled water to remove salts, alanine, small oligopeptides of alanine, and dioxane. The resultant solution is lyophilized (1.4 g). Any native ribonuclease or polypeptides of alanine present in the reaction product can be removed by chromatography on a column of phosphorylated cellulose, with a combined gradient of a salt and pH. 6 No native enzyme was found in the reaction product. The enrichment in alanine is determined by amino acid analysis, and the number of peptide chains attached is quantitated by dinitrophenylation or by deamination. The average chain length of the peptides attached may thus be calculated. Under the conditions cited above, 8 of the 11 amino groups of the protein are alanylated and the length of the side chains, which was found to be relatively uniform, is approximately 5. Two additional amino groups may be partially attacked by N-carboxyalanine anhydride upon more extensive alanylation, yielding a material that retains enzymatic activity. The last lysine residue, which was identified as residue 41 in the polypeptide chain of the enzyme, is vulnerable to the N-carboxy anhydride only when phosphate buffer is replaced by bicarbonate. Its alanylation brings about total loss of enzymatic activity. 7 Preparation o] Poly-L-lysyl Ribonuclease. ~5 The trifluoroacetyl function was found to be a useful reversible blocking agent for amino groups, being stable to the conditions of N-carboxy anhydride formation and polymerization, and easily removed under mild conditions. ~6 Consequently it was used in the synthesis of poly-L-lysyl ribonuclease. In a typical preparation, a solution of 0.5 g bovine pancreatic ribonuclease in 70 ml 0.05 M phosphate buffer (pH 7) is introduced into a 250-ml Erlenmeyer flask and cooled with ice to 2 °. A solution of 0.5 g a,N-carboxy~,N-trifluoroacetyl-L-lysine anhydride ~° in 20 ml anhydrous dioxane is added with stirring. The reaction is allowed to proceed for 24 hours in the cold room (4 ° ) and the entire mixture is dialyzed in heat-treated cellophane tubing for 3 days against several changes of 6 liters of distilled is A. Frensdorff, R. Arnon, and M. Sela, unpublished data. le F. Weygand and E. Csendes, Angew. Chem. 64, 136 (1952).
584
MODIFICATION REACTIONS
[70]
water at 4 °. During the reaction or the dialysis a precipitate often appears that contains a fraction of the protein which has been more extensively peptidylated. The precipitate is removed in the centrifuge and the supernatant fluid is lyophilized. The yield is approximately 0.45 g. The extent of the reaction m a y be estimated by fluorine determination, s To remove trifluoroacetyl groups, 100 mg poly-e,N-trifluoroacetyllysyl ribonuclease is introduced into a 25-ml flask and suspended in 7 ml 1 M aqueous piperidine. The suspension is stirred at 4 ° for 30 hours. 17 The clear solution obtained is neutralized with cold 0 . 5 N acetic acid, dialyzed in heat-treated cellophane tubing at 4 ° for 3 days against daily changes of 1 liter of distilled water, and lyophilized. Determinations of the enrichment in lysine, and the average length of the peptide side chains, are carried out by amino acid analysis of native and polylysyl ribonuelease before and after deamination. The number of moles of lysine residues obtained after hydrolysis of the deaminated polypeptidylated material indicates the actual number of moles of peptide chains attached. z~The material dissolves in the piperidine solution after 1 hour, but at this stage still contains about half the original trifluoroacetyl groups. Only after 30 hours in piperidine will the fluorine content drop to less than 1%.
[70] Guanidination
of Proteins
By JOE R. KIMMEL Principle The reaction of O-methylisourea with an amino group results in conversion of this group to a guanidino-group (guanidination, amidination) : R--NH~ q- CH30---C(NH)NH~ --. R--NH--C(NH)NH2 q- CHaOH When applied to lysine, 1,2 reaction can be made to occur only at the e-amino group by appropriate ma'sking of the ~-amino group. The product is the corresponding a-N-substituted homoarginine. The lysine residues of proteins react in the same manner since only the c-amino function is potentially available. Thus, guanidination of a protein converts some or all of the lysine residues to homoarginine residues and, on the basis of experience with several proteins, appears to occur to a minimal extent at the NH2-terminus of polypeptide chains, 1j. p. Greenstein, J. Org. Chem. 2, 480 (1938). 2G. M. Stevens and J. A. Bush, J. Biol. Chem. 183, 139 (1950).