- Email: [email protected]
[66] Acetylation
[66]
ACETYL&TION
565
be expected that the reagents would react also with cysteine and probably with tryptophan under acidic conditions, and perhaps with other nucleophiles in base. Although not as yet investigated extensively, they represent reagents that may be useful in future modification studies.
[66] A c e t y l a t i o n
By J. F. RIORDAN and B. L. VALLEE Introduction Acetylation of amino groups is one of the most common means employed for the chemical modification of enzymes. Several reasons for this circumstance may be hypothesized. First, amino groups, being charged, tend to be located on the "surface" of the three-dimensional structure of proteins in contact with the ambient environment and hence readily accessible to chemical attack. Second, at least one acetylating agent is a~ailable that has the requisite properties of high specificity and rapid reactivity under mild conditions. Third, the extent of reaction can be assessed by relatively simple analytical means. Nonetheless the analytical characterization of acetylation presents certain difficulties, and consequently there are problems in interpretation of the relationship of acetylation to changes in biological activity. Methods
1. Acetylation with Acetic Anhydride. This reagent remains the one of choice for the substitution of acetyl groups on the amino groups of proteins. The standard procedure widely employed has been described previously (Vol. IV).1 Typically, a 2-10% protein solution or suspension in half-saturated sodium acetate is cooled in an ice bath and stirred with a magnetic mixer. An equal weight of acetic anhydride (10 meq per gram protein) is added in five equal porLions over the course of 1 hour at 0 ° and the stirring is continued for an additional hour. The product is isolated by gel filtration or dialysis. While this procedure has found general applicability the problem under study frequently requires alterations, and optimal conditions must be determined empirically in each instance. Sodium acetate presumably functions as a buffer of the acetylation reaction mixture and serves as a catalyst2 But the high concentration 1 H. Fraenkel-Conrat, Vol. IV, p. 247. 2 R. W. Green, K. P. Ang, and L. C. Lam, Biochem. J. 54, 181 (1953).
566
MODIFICATION REACTIONS
[66J
of acetate apparently plays an additional role by increasing the specificity of acetylation, a role not appreciated until recently. As pointed out (this volume [67]), acetic anhydride readily acetylates the phenolic hydroxyl group of tyrosyl residues in proteins, but O-acetyltyrosine undergoes hydrolysis rapidly in the presence of high concentrations of acetate, leaving only the amino groups acetylated. Acetylation under the conditions cited above can often lead to protein denaturation, s The high concentrations of anhydride employed may constitute one of the reasons. These concentrations, however, are required for several reasons. The reagent is rather unstable and undergoes spontaneous hydrolysis, thereby rapidly depleting the effective molar excess. In addition, the reaction is pH-dependent since only unprotonated amino groups are acetylated, and soon after the addition of anhydride there is a fall in pH from about 8 to 5.5. An excess of anhydride is therefore required if an appreciable rate of acetylation is to be achieved. When the reaction is carried out at constant pH using a pH-stat the procedure is much milder. In this instance a 30-60-fold molar excess of reagent is added to the protein solution (5-!0 mg/ml) in an appropriate buffer at pH 7.5. The reaction mixture is well stirred and the temperature maintained at 0-4 ° with an ice bath. The time course of acetylation is followed by recording the uptake of 1 N NaOH. The titration measures the release of protons due both to modification and to spontaneous hydrolysis of the anhydride and, of course, cannot therefore be used as a quantitative measure of the degree of acetylation. The reaction is generally completed within 20-30 minutes, and the product is isolated by gel filtration or dialysis. It is important to note that, owing to the absence of acetate, this method of acetylation generally results in the formation of both O-acetyltyrosine and N-acetyllysine. 2. Other Acetylating Agents. Several other acetylating agents have been employed for the modification of proteins. Acetyl chloride, while equally effective in reacting with amino groups as acetic anhydride, is somewhat more vigorous and consequently might be expected to result in greater denaturation. For many years ketene was employed commonly as an acetylating agent, but a number of disadvantages in comparison with acetic anhydride have discouraged its use. ~ It is an unstable, toxic gas requiring special generating equipment. The reaction is carried out by bubbling the gas through the protein solution. Hence, quantitation of the amount employed depends on maintaining a known rate of gas flow, and surface denaturation due to foaming becomes a major problem. 8j. L. Bethune, D. D. Ulmer, and B. L. Vallee, Biochemistry 3, 1764 (1964). 4F. W. Putnam, in "The Proteins" (H. Neurath and K. Bailey, eds.), Vol. 1B, p. 893. Academic Press, New York, 1953.
[66]
ACETYLATION
567
Ketene is not recommended generally and should find a use largely for enzymes, such as pepsin, 5 which are particularly unstable at mildly alkaline pH. Other acetylating reagents and procedures have been employed for several proteins. N,S-Diacetylthioethanolamine acetylates proteins at pH 9--10, but these conditions limit its use to a relatively small group of proteins and are not favorable for the majority2 The technique employed is quite analogous to trifluoroacetylation (described in [34] ) and will therefore not be repeated here. N-Acetylimidazole has been used primarily for aeetylation of tyrosyl residues of proteins ([67]). However, in a number of proteins and polypeptides examined, acetylation of amino groups has been observed as well, although the degree of substitution was usually much less than with acetic anhydrideJ There is no evidence that p-nitrophenyl acetate acetylates amino groups of proteins in aqueous media, although nonpolar solvents might favor the reaction. Thus, p-nitrophenyl bromoacetate has been used to acylate ribonuclease in dimethylsulfoxide, and about 90% of the camino groups were modified,s The reagent is of interest since it introduces a cross-linking alkylating agent into the protein and presumably allows covalent bonding to antibody. This reaction exemplifies the use of organic solvents in allowing the exploration of new means of ,chemically modifying proteins, an approach that has been studied in detail in very few instances9 but offers great potential.1° Several other anhydrides have been employed as acylating agents of enzymes by utilizing either of the two methods described above. 11,1~ The reactivity of these anhydrides in general decreases with increasing chain length, perhaps due to decreased solubility. Dicarboxylic acid anhydrides offer interesting properties, not only because they have a much greater effect on protein charge but because they also afford a means of studying tyrosyl residues ([67]). A number of other means of acetylation of proteins have been reported, but in general do not appear ideally suited for studies of structure-function relationships in enzymes. Thus, procedures employing R. M. Herriott, Advan. Protein Chem. 3, 170 (1947).
ej. Baddiley, R. A. Keckwick, and E. M. Thain, Nature 170, 968 (1952). ft. F. Riordan, W. E. C. Wacker, and B. L. Vallee, Biochemistry 4, 1758 (1965). s j. Guldalian, W. B. Lawson,and R. K. Brown, J. Biol. Chem. 240, PC2758 (1965). 9S. M. Vratsanos, Arch. Bioehem. Biophys. 90, 132 (1960). ~°S. J. Singer, Advan. Protein Chem. 17, 1 (1962). 11L. Terminiello, J. Sri Ram, M. Bier, and F. F. Nord, Arch. Biochem. Biophys. 57, 252 (1955). ~J. F. Riordan and B. L. Vallee, Biochemistry 2, 1460 (1963).
568
MODIFICATION REACTIONS
[~{}]
acetic anhydride-ethyl acetate-formic acid, is acetic acid-acetic anhydride, 2 or hot 16% (v/v) acetic anhydride in acetic acid TM for acetylation reactions prove too much of an insult even for the most resistant of enzymes.
Analytical Characterization The degree of acetylation of a protein can be determined either by measuring the number of acetyl groups introduced or by measuring the decrease of amino groups known to be present. By employing both methods, the specificity of the reaction can be assessed. The use of 1'C-labeled reagents is an obvious means of measuring acetyl incorporation, although the incorporation itself does not identify the reactive group acetylated. Hence, additional chemical identification is always necessary. Reaction with ninhydrin serves as the standard chemical method for determining the decrease in amino groups as a consequence of acetylation. 15 Absorbance at 570 m~ is an index of the free amino groups which can react with ninhydrin, and the degree of acetylation can therefore be calculated from the percent decrease of this absorbance. Usually the color yield of the protein is related to that for a standard amino acid such as leucine, for example, and is then expressed as leucine equivalents per g or micromole of protein. At best, the data are semiquantitative but in most instances serve as a convenient gauge of the' extent of reaction. More accurate data are obtained by gasometric analysis of amino groups with the method of Van Slyke26 Formol titrations have been used in some instances. Free amino groups have also been determined by reaction of the protein with fluorodinitrobenzene followed by 18-hour hydrolysis of the DNP-protein i n v a c u o at 105 ° with 6 N HC1. ~7 Chromatography of the aqueous phase after ether extraction allows quantitative determination of DNP-lysine, corresponding to those residues not acetylated. Amino acid analysis ~s of the hydrolyzate identifies the number of lysine residues acetylated and hence protected against reaction with FDNB. A number of procedures have been reported that are based on the titrimetric determination of the acetic acid released on hydrolysis of 1~S. M. Bose and K. T. Joseph, Arch. Biochem. Biophys. 74, 46 (1958). ~A. E. Brown and L. G. Beauregard, in "Sulfur in Proteins" (R. Benesch, et al., eds.), p. 59. Academic Press, New York, 1959. ~5S. Moore and W. H. Stein, J. Biol. Chem. 176, 367 (1948). ,e D. D. Van Slyke, J. Biol. Chem. 83, 425 (1929). ~7H. Fraenkel-Conrat, J. I. Harris, and A. L. Levy, Methods Biochem. Anal. 2, 359 (1955). 'SD. H. Spackman, W. H. Stein, and S. Moore, Anal. Chem. 30, 1190 (1958).
[66]
ACETYLAT1ON
569
the acetylated protein with p-toluenesulfonic acid. TM A sample containing 1-5 mg of acetyl groups is heated for 2-4 hours under reflux with 20 ml 25% aqueous solution of p-toluenesulfonic acid. The hydrolyzate is quantitatively transferred to a 100-ml micro-Kjeldahl flask and the acetic acid is steam distilled. The distillate is titrated after removal of C02 by bubbling with nitrogen. A variation of this procedure has been reported that is suitable for micro amounts of acetylated protein. 2° Other variations involve the use of sodium methoxide TM 22 or sulfuric acid 2 as the agent for hydrolysis. If analysis demonstrates that more acetyl groups have been incorporated into the protein than can be accounted for by amino group modification, the possibility of 0-acetylation must be examined. OAcetyltyrosine can be deacetylated by hydroxylamine at neutral pH, and can be quantitated either by spectral methods or by analysis of the resultant acethydroxamate (see [67]). Esters of serine or threonine can be cleaved with hydroxylamine above pH 10.5 and also quantitated from hydroxamate formation. 23 Thiol esters have a characteristic absorption maximum at 230-235 m~ 24 and are also cleaved by hydroxylamine. Discussion
I t has proven difficult in most instances to bring about complete acctylation of all the c-amino groups of proteins. For many proteins, 60-90% of the amino groups are acetylated with acetic anhydride. Those groups not susceptible to modification might be presumed to be buried in some manner in the interior of the three-dimensional structure of the protein. Acetylation of random copolymers of lysine was found to give complete amino group substitution and this circumstance could be cited in support of such a view. 7 I t is possible, however, t h a t incomplete acetylation also reflects randomness of the modification of the free amino groups. I t has been noted that complete acetylation of the S-peptide of ribonuclease required a 300-fold molar excess of anhydride. 2° When acetylation was performed with a 50-fold molar excess, mono-, di-, and triacetyl peptides were formed which could be separated by electrophoresis. Similarly, random acetylation of amino groups in proteins might be expected to yield a family of products wherein the number of ~ E. A. Kabat and M. M. Mayer, "Experimental Immunochemistry," 2nd. ed., p. 493. Thomas, Springfield, Illinois, 1961. P. Vithayathil and F. M. Richards, J. Biol. Chem. 235, 1029 (1960). ~ R. L. Whistler and A. Jeanes, Ind. Eng. Chem. 15, 317 (1943). "~F. B. Cramer, T. S. Gardner, and C. B. Purves, Ind. Eng. Chem. 15, 319 (1943). '3A. K. Balls and It. N. Wood, J. Biol. Chem. 219, 245 (1956). L. H. Noda, S. A. Kuby, and H. A. Lardy, J. Am. Chem. Soc. 75, 913 (1953).
570
MODIFICATION REACTIONS
[57]
acetyl groups as well as their distribution throughout the molecule could vary over a wide range. Changes of enzymatic activity can be observed consequent to acetylation, which may be due to a variety of causes. Thus, loss of activity may be correlated with the modification of a single functional group of the enzyme. However, since amino groups are so reactive, usually several of them become modified and it then becomes necessary to differentiate functionally active residues from others equally reactive chemically. In such instances, protection experiments using substrates or inhibitors to prevent acetylation of active center residues have proven most helpful in determining the number of critical groups22, 25 C. H. W. Hirs, M. Halmann, and J. H. Kycia, Arch. Biochem. Biophys. Ill, 209 (1965).
[67] O - A c e t y l t y r o s i n e B y J. F. RIORDAN and B. L. VAT,LEE Introduction
There is no evidence at present suggesting that native proteins contain O-acetyltyrosyl residues. However, O-acetyl groups are readily introduced into the tyrosyl residues of proteins, in some instances leading to functional consequences. 1 That tyrosyl residues can be acetylated either with acetic anhydride or with N-acetylimidazole has been shown only recently2-s Acetic anhydride reacts with amino and sulfhydryl groups as well. N-Acetylimidazole also acetylates these residues, but reacts with amino groups much less readily than does the anhydrideY In certain instances, as in carboxypeptidase A, where the protein contains no free sulfhydryl groups, N-acetylimidazole selectively acetylates tyrosyl residues2 Other mono- and dicarboxylic acid anhydrides also acylate tyrosyl residues. The latter present a particularly interesting means of modification since the products undergo spontaneous deacylation at rates characteristic for the anhydride employed.4 ~J. F. Riordan and B. L. Vallee, Biochemistry 2, 1460 (1963). ~J. F. Riordan, W. E. C. Wacker, and B. L. Vallee, Biochemistry 4, 1758 (1965). R. T. Simpson, J. F. Riordan, and B. L. Vallee, Biochemistry 2, 616 (1963). J. F. Riordan and B. L. Vallee, Biochemistry 3, 1768 (1964).
ACETYL&TION
565
be expected that the reagents would react also with cysteine and probably with tryptophan under acidic conditions, and perhaps with other nucleophiles in base. Although not as yet investigated extensively, they represent reagents that may be useful in future modification studies.
[66] A c e t y l a t i o n
By J. F. RIORDAN and B. L. VALLEE Introduction Acetylation of amino groups is one of the most common means employed for the chemical modification of enzymes. Several reasons for this circumstance may be hypothesized. First, amino groups, being charged, tend to be located on the "surface" of the three-dimensional structure of proteins in contact with the ambient environment and hence readily accessible to chemical attack. Second, at least one acetylating agent is a~ailable that has the requisite properties of high specificity and rapid reactivity under mild conditions. Third, the extent of reaction can be assessed by relatively simple analytical means. Nonetheless the analytical characterization of acetylation presents certain difficulties, and consequently there are problems in interpretation of the relationship of acetylation to changes in biological activity. Methods
1. Acetylation with Acetic Anhydride. This reagent remains the one of choice for the substitution of acetyl groups on the amino groups of proteins. The standard procedure widely employed has been described previously (Vol. IV).1 Typically, a 2-10% protein solution or suspension in half-saturated sodium acetate is cooled in an ice bath and stirred with a magnetic mixer. An equal weight of acetic anhydride (10 meq per gram protein) is added in five equal porLions over the course of 1 hour at 0 ° and the stirring is continued for an additional hour. The product is isolated by gel filtration or dialysis. While this procedure has found general applicability the problem under study frequently requires alterations, and optimal conditions must be determined empirically in each instance. Sodium acetate presumably functions as a buffer of the acetylation reaction mixture and serves as a catalyst2 But the high concentration 1 H. Fraenkel-Conrat, Vol. IV, p. 247. 2 R. W. Green, K. P. Ang, and L. C. Lam, Biochem. J. 54, 181 (1953).
566
MODIFICATION REACTIONS
[66J
of acetate apparently plays an additional role by increasing the specificity of acetylation, a role not appreciated until recently. As pointed out (this volume [67]), acetic anhydride readily acetylates the phenolic hydroxyl group of tyrosyl residues in proteins, but O-acetyltyrosine undergoes hydrolysis rapidly in the presence of high concentrations of acetate, leaving only the amino groups acetylated. Acetylation under the conditions cited above can often lead to protein denaturation, s The high concentrations of anhydride employed may constitute one of the reasons. These concentrations, however, are required for several reasons. The reagent is rather unstable and undergoes spontaneous hydrolysis, thereby rapidly depleting the effective molar excess. In addition, the reaction is pH-dependent since only unprotonated amino groups are acetylated, and soon after the addition of anhydride there is a fall in pH from about 8 to 5.5. An excess of anhydride is therefore required if an appreciable rate of acetylation is to be achieved. When the reaction is carried out at constant pH using a pH-stat the procedure is much milder. In this instance a 30-60-fold molar excess of reagent is added to the protein solution (5-!0 mg/ml) in an appropriate buffer at pH 7.5. The reaction mixture is well stirred and the temperature maintained at 0-4 ° with an ice bath. The time course of acetylation is followed by recording the uptake of 1 N NaOH. The titration measures the release of protons due both to modification and to spontaneous hydrolysis of the anhydride and, of course, cannot therefore be used as a quantitative measure of the degree of acetylation. The reaction is generally completed within 20-30 minutes, and the product is isolated by gel filtration or dialysis. It is important to note that, owing to the absence of acetate, this method of acetylation generally results in the formation of both O-acetyltyrosine and N-acetyllysine. 2. Other Acetylating Agents. Several other acetylating agents have been employed for the modification of proteins. Acetyl chloride, while equally effective in reacting with amino groups as acetic anhydride, is somewhat more vigorous and consequently might be expected to result in greater denaturation. For many years ketene was employed commonly as an acetylating agent, but a number of disadvantages in comparison with acetic anhydride have discouraged its use. ~ It is an unstable, toxic gas requiring special generating equipment. The reaction is carried out by bubbling the gas through the protein solution. Hence, quantitation of the amount employed depends on maintaining a known rate of gas flow, and surface denaturation due to foaming becomes a major problem. 8j. L. Bethune, D. D. Ulmer, and B. L. Vallee, Biochemistry 3, 1764 (1964). 4F. W. Putnam, in "The Proteins" (H. Neurath and K. Bailey, eds.), Vol. 1B, p. 893. Academic Press, New York, 1953.
[66]
ACETYLATION
567
Ketene is not recommended generally and should find a use largely for enzymes, such as pepsin, 5 which are particularly unstable at mildly alkaline pH. Other acetylating reagents and procedures have been employed for several proteins. N,S-Diacetylthioethanolamine acetylates proteins at pH 9--10, but these conditions limit its use to a relatively small group of proteins and are not favorable for the majority2 The technique employed is quite analogous to trifluoroacetylation (described in [34] ) and will therefore not be repeated here. N-Acetylimidazole has been used primarily for aeetylation of tyrosyl residues of proteins ([67]). However, in a number of proteins and polypeptides examined, acetylation of amino groups has been observed as well, although the degree of substitution was usually much less than with acetic anhydrideJ There is no evidence that p-nitrophenyl acetate acetylates amino groups of proteins in aqueous media, although nonpolar solvents might favor the reaction. Thus, p-nitrophenyl bromoacetate has been used to acylate ribonuclease in dimethylsulfoxide, and about 90% of the camino groups were modified,s The reagent is of interest since it introduces a cross-linking alkylating agent into the protein and presumably allows covalent bonding to antibody. This reaction exemplifies the use of organic solvents in allowing the exploration of new means of ,chemically modifying proteins, an approach that has been studied in detail in very few instances9 but offers great potential.1° Several other anhydrides have been employed as acylating agents of enzymes by utilizing either of the two methods described above. 11,1~ The reactivity of these anhydrides in general decreases with increasing chain length, perhaps due to decreased solubility. Dicarboxylic acid anhydrides offer interesting properties, not only because they have a much greater effect on protein charge but because they also afford a means of studying tyrosyl residues ([67]). A number of other means of acetylation of proteins have been reported, but in general do not appear ideally suited for studies of structure-function relationships in enzymes. Thus, procedures employing R. M. Herriott, Advan. Protein Chem. 3, 170 (1947).
ej. Baddiley, R. A. Keckwick, and E. M. Thain, Nature 170, 968 (1952). ft. F. Riordan, W. E. C. Wacker, and B. L. Vallee, Biochemistry 4, 1758 (1965). s j. Guldalian, W. B. Lawson,and R. K. Brown, J. Biol. Chem. 240, PC2758 (1965). 9S. M. Vratsanos, Arch. Bioehem. Biophys. 90, 132 (1960). ~°S. J. Singer, Advan. Protein Chem. 17, 1 (1962). 11L. Terminiello, J. Sri Ram, M. Bier, and F. F. Nord, Arch. Biochem. Biophys. 57, 252 (1955). ~J. F. Riordan and B. L. Vallee, Biochemistry 2, 1460 (1963).
568
MODIFICATION REACTIONS
[~{}]
acetic anhydride-ethyl acetate-formic acid, is acetic acid-acetic anhydride, 2 or hot 16% (v/v) acetic anhydride in acetic acid TM for acetylation reactions prove too much of an insult even for the most resistant of enzymes.
Analytical Characterization The degree of acetylation of a protein can be determined either by measuring the number of acetyl groups introduced or by measuring the decrease of amino groups known to be present. By employing both methods, the specificity of the reaction can be assessed. The use of 1'C-labeled reagents is an obvious means of measuring acetyl incorporation, although the incorporation itself does not identify the reactive group acetylated. Hence, additional chemical identification is always necessary. Reaction with ninhydrin serves as the standard chemical method for determining the decrease in amino groups as a consequence of acetylation. 15 Absorbance at 570 m~ is an index of the free amino groups which can react with ninhydrin, and the degree of acetylation can therefore be calculated from the percent decrease of this absorbance. Usually the color yield of the protein is related to that for a standard amino acid such as leucine, for example, and is then expressed as leucine equivalents per g or micromole of protein. At best, the data are semiquantitative but in most instances serve as a convenient gauge of the' extent of reaction. More accurate data are obtained by gasometric analysis of amino groups with the method of Van Slyke26 Formol titrations have been used in some instances. Free amino groups have also been determined by reaction of the protein with fluorodinitrobenzene followed by 18-hour hydrolysis of the DNP-protein i n v a c u o at 105 ° with 6 N HC1. ~7 Chromatography of the aqueous phase after ether extraction allows quantitative determination of DNP-lysine, corresponding to those residues not acetylated. Amino acid analysis ~s of the hydrolyzate identifies the number of lysine residues acetylated and hence protected against reaction with FDNB. A number of procedures have been reported that are based on the titrimetric determination of the acetic acid released on hydrolysis of 1~S. M. Bose and K. T. Joseph, Arch. Biochem. Biophys. 74, 46 (1958). ~A. E. Brown and L. G. Beauregard, in "Sulfur in Proteins" (R. Benesch, et al., eds.), p. 59. Academic Press, New York, 1959. ~5S. Moore and W. H. Stein, J. Biol. Chem. 176, 367 (1948). ,e D. D. Van Slyke, J. Biol. Chem. 83, 425 (1929). ~7H. Fraenkel-Conrat, J. I. Harris, and A. L. Levy, Methods Biochem. Anal. 2, 359 (1955). 'SD. H. Spackman, W. H. Stein, and S. Moore, Anal. Chem. 30, 1190 (1958).
[66]
ACETYLAT1ON
569
the acetylated protein with p-toluenesulfonic acid. TM A sample containing 1-5 mg of acetyl groups is heated for 2-4 hours under reflux with 20 ml 25% aqueous solution of p-toluenesulfonic acid. The hydrolyzate is quantitatively transferred to a 100-ml micro-Kjeldahl flask and the acetic acid is steam distilled. The distillate is titrated after removal of C02 by bubbling with nitrogen. A variation of this procedure has been reported that is suitable for micro amounts of acetylated protein. 2° Other variations involve the use of sodium methoxide TM 22 or sulfuric acid 2 as the agent for hydrolysis. If analysis demonstrates that more acetyl groups have been incorporated into the protein than can be accounted for by amino group modification, the possibility of 0-acetylation must be examined. OAcetyltyrosine can be deacetylated by hydroxylamine at neutral pH, and can be quantitated either by spectral methods or by analysis of the resultant acethydroxamate (see [67]). Esters of serine or threonine can be cleaved with hydroxylamine above pH 10.5 and also quantitated from hydroxamate formation. 23 Thiol esters have a characteristic absorption maximum at 230-235 m~ 24 and are also cleaved by hydroxylamine. Discussion
I t has proven difficult in most instances to bring about complete acctylation of all the c-amino groups of proteins. For many proteins, 60-90% of the amino groups are acetylated with acetic anhydride. Those groups not susceptible to modification might be presumed to be buried in some manner in the interior of the three-dimensional structure of the protein. Acetylation of random copolymers of lysine was found to give complete amino group substitution and this circumstance could be cited in support of such a view. 7 I t is possible, however, t h a t incomplete acetylation also reflects randomness of the modification of the free amino groups. I t has been noted that complete acetylation of the S-peptide of ribonuclease required a 300-fold molar excess of anhydride. 2° When acetylation was performed with a 50-fold molar excess, mono-, di-, and triacetyl peptides were formed which could be separated by electrophoresis. Similarly, random acetylation of amino groups in proteins might be expected to yield a family of products wherein the number of ~ E. A. Kabat and M. M. Mayer, "Experimental Immunochemistry," 2nd. ed., p. 493. Thomas, Springfield, Illinois, 1961. P. Vithayathil and F. M. Richards, J. Biol. Chem. 235, 1029 (1960). ~ R. L. Whistler and A. Jeanes, Ind. Eng. Chem. 15, 317 (1943). "~F. B. Cramer, T. S. Gardner, and C. B. Purves, Ind. Eng. Chem. 15, 319 (1943). '3A. K. Balls and It. N. Wood, J. Biol. Chem. 219, 245 (1956). L. H. Noda, S. A. Kuby, and H. A. Lardy, J. Am. Chem. Soc. 75, 913 (1953).
570
MODIFICATION REACTIONS
[57]
acetyl groups as well as their distribution throughout the molecule could vary over a wide range. Changes of enzymatic activity can be observed consequent to acetylation, which may be due to a variety of causes. Thus, loss of activity may be correlated with the modification of a single functional group of the enzyme. However, since amino groups are so reactive, usually several of them become modified and it then becomes necessary to differentiate functionally active residues from others equally reactive chemically. In such instances, protection experiments using substrates or inhibitors to prevent acetylation of active center residues have proven most helpful in determining the number of critical groups22, 25 C. H. W. Hirs, M. Halmann, and J. H. Kycia, Arch. Biochem. Biophys. Ill, 209 (1965).
[67] O - A c e t y l t y r o s i n e B y J. F. RIORDAN and B. L. VAT,LEE Introduction
There is no evidence at present suggesting that native proteins contain O-acetyltyrosyl residues. However, O-acetyl groups are readily introduced into the tyrosyl residues of proteins, in some instances leading to functional consequences. 1 That tyrosyl residues can be acetylated either with acetic anhydride or with N-acetylimidazole has been shown only recently2-s Acetic anhydride reacts with amino and sulfhydryl groups as well. N-Acetylimidazole also acetylates these residues, but reacts with amino groups much less readily than does the anhydrideY In certain instances, as in carboxypeptidase A, where the protein contains no free sulfhydryl groups, N-acetylimidazole selectively acetylates tyrosyl residues2 Other mono- and dicarboxylic acid anhydrides also acylate tyrosyl residues. The latter present a particularly interesting means of modification since the products undergo spontaneous deacylation at rates characteristic for the anhydride employed.4 ~J. F. Riordan and B. L. Vallee, Biochemistry 2, 1460 (1963). ~J. F. Riordan, W. E. C. Wacker, and B. L. Vallee, Biochemistry 4, 1758 (1965). R. T. Simpson, J. F. Riordan, and B. L. Vallee, Biochemistry 2, 616 (1963). J. F. Riordan and B. L. Vallee, Biochemistry 3, 1768 (1964).