- Email: [email protected]
[55] Esterification
596
MODIFICATION REKCTIONS
[55]
untreated ribonuclease. 26 Such changes in the physical properties of the regenerated protein need not invalidate the use of amidination in sequence and cross-linking determinations. For example, removal of the amidino group has facilitated identification of the lysine residues crosslinked by reaction of ribonuclease with adipimidate. 4 In certain situations, however, it appears that reaction with maleic anhydride ~2 may have advantages over reaction with imidates.
Procedures ]or the Amidination o] Proteins 1. k method for exhaustive modification by reaction with methyl (or ethyl) acetimidate 3 has been given in Volume 11.6 2. The procedure for reaction of cytochrome b~ reductase with acetimidate 44 is useful for other small-scale preparations: Enzyme (0.03 ~mole, 22 lysine/FAD) is dissolved in 0.3 ml of pyrophosphate buffer, pH 8.5. Ethyl acetimidate (300 ~moles in 0.30 ml of 0.45 M borate buffer, pH 9.2) is added, and the mixture is left at 0 ° for 20 hours. The derivative can be separated from contaminating small molecules by passage through a Sephadex G-25 column. 3. Cross-linking using dimethyl suberimidate47: The pH is maintained at 8.5 (with 0.2M triethanolamine.HC1) to favor amidination over reagent hydrolysis. Dimethyl suberimidate, adjusted to pH 8.5 in the above buffer, is mixed with a solution of the protein to be crosslinked to give a final protein concentration of 0.5-5 mg/ml and a dimethyl suberimidate concentration of 1-12 mg/ml, and the reaction is allowed to proceed for 3 hours. ~2p. j. G. Butler, J. I. Harris, B. S. Hartley, and R. Leberman, Biochem. J. 112, 679 (1969).
[55] Esterification By PHILIP E. WILCOX~ In the short time that has elapsed since the publication of Volume 11, methods for the esterification of proteins have been extended substantially. Interest in modification of carboxyl groups has increased with each new discovery of the functional or structural role of a glutamic or aspartic acid side chain in an enzyme. The growing list of enzymes includes lysozyme, carboxypeptidase A, pepsin, trypsin and other serine proteases, ribonuclease T1, and triosephosphate isomerase. In Volume 11 [74], it was pointed out that only two methods of * Deceased.
[55]
ESTERIFICATION
597
esterification were known then to be group specific, namely, reaction with methanol-HC1 or ethanol-HC1 and reaction with derivatives of diazoacetic acid near pH 5. It now appears that reaction with triethyloxonium fluoroborate (Meerwein reagent) at pH values between 4 and 5 may be added to the repertoire of group-specific reagents. Furthermore, the use of aliphatic diazo compounds has been extended to include diazo ketones containing the grouping --CO--CH----N2. A remarkable advance in methodology has come from the discovery that copper ions promote the reaction between the carboxyl group of a specific aspartic acid residue in pepsin and a variety of aliphatic diazo compounds. 1,~ The mechanism is not presently clear, but it is likely that the method will be applicable to other enzymes. Indeed, the reaction has been shown to occur with a pepsinlike enzyme from the mold
Penicillium janthinellum2 Experience has furthermore shown that specific esterification of particular enzymes can be obtained with reagents that are not ordinarily specific for carboxyl groups alone. In most of these cases the reagent has been an active halogen compound, such as iodoacetate or a bromomethyl ketone. Specificity may result either from an enhanced reactivity of the carboxyl group due to the local environment in the native protein, or from specific binding of a particular reagent to a site in the enzyme, or perhaps from a combination of both effects. This article will be focused mainly on new methods that have been developed for reagents that are relatively specific for carboxyl groups. The other category of reagent, the reactive halogen compounds, will be given a more cursory treatment because the particular structure of the binding site in the enzyme must be of primary importance in each case. Esterification may be regarded as a special case of alkylation. It is therefore likely that particular compounds, which would ordinarily be classified among typical alkylating reagents, will be found to esterify uniquely situated carboxyl groups in enzymes and other proteins as the result of specific interactions. The reader is referred to other articles on alkylation (this volume [34a] and [39]).
Aliphatic Diazo Compounds The reactions of aliphatic diazo compounds are manifold and complex. A useful reference is the treatise by H. Zollinger. 4 In spite of their 1T. G. Rajagopalan, W. H. Stein, and S. Moore, J. Biol. Chem. 241, 4295 (1966). G. R. Delpierre and J. S. Fruton, Proc. Nat. Acad. Sci. U.S. 56, 1817 (1966). ' J . ~odek and T. Hofmann, J. Biol. Chem. 243, 450 (1968). 4H. Zollinger, "Azo and Diazo Chemistry: Aliphatic and Aromatic Compounds," Wiley (Interscience), New York, 1961.
598
MODIFICATION REACTIONS
[5S]
reactivity, aliphatic diazo compounds show a high degree of specificity for carboxyl groups in an aqueous m e d i u m when t h e y are partially stabilized b y the presence of a c a r b o n y l group a t t a c h e d to the diazotized carbon atom, as in the diazoacetyl or the d i a z o m e t h y l ketone moieties. T h e only exception is the ability of these reagents to alkylate free sulfhydryl groups (see ¥ol. 11 [74]). D i a z o compounds not so stabilized, for example, diazomethane, tend to be nonspecific. T h e e n z y m a t i c properties of pepsin suggested to a n u m b e r of investigators t h a t the active site of this e n z y m e contained at least one carboxyl group as an i m p o r t a n t component. Therefore, a v a r i e t y of diazo compounds were synthesized as site-specific reagents. Reliance was TABLE I ALKYL DIAZO COMPOUNDS FOUND TO INHIBIT PEPSIN 14C_
Compound I II III IV V VI VII VIII IX X XI XII XIII XIV XV
Diphenyldiazomethane N-Diazoacetyl-DL-norleucine N-Tosyl-L-phenylalanyldiazomethane N-Tosyl-D-phenylalanyldiazomethane N-Diazoacetyl-L-phenylalanine methyl ester N-Diazoacetyl-D-phenylalanine methyl ester N-Diazoacetylglyeine methyl (ethyl) ester N-Benzyloxycarbonyl-L-phenylalanyldiazomethane a-Diazo-p-bromoacetephenone 1-Diazo-4-phenylbutanone-2 1-Diazo-3-dinitrophenylaminopropanone-2 Ethyl 2-diazo-3-(p-hydroxyphenyl) propionate Phenylbenzoyldiazomethane N-Diazoacetyl-N'-2,4-dinitrophenylethylenediamine
Methyl (or ethyl) diazoacetate
labeled
Reference
Yes -Yes -Yes -Yes Yes -Yes ----
a b, c d d d, e, j d f g h e e e
--
i
Yes
c
e-e
a G. R. Delpierre and J. S. Fruton, Proc. Nat. Acad. Sci. U.S. 54, 1161 (1965). b T. G. Raiagopalan, W. H. Stein, and S. Moore, J. Biol. Chem. 241, 4295 (1966). c R. L. Lundblad and W. H. Stein, J. Biol. Chem. 244, 154 (1969). G. R. Delpierre and J. S. Fruton, Proc. Nat. Acad. Sci. U.S. 56, 1817 (1966). e L. ¥. Kozlov, L. M. Ginodman, and V. N. Orekhovich, Biokhimiya 32, 1011 (1967). f E. B. Ong and G. E. Perlmann, Nature (London) 215, 1492 (1967). B. F. Erlanger, S. M. Vratsanos, N. Wassermann, and A. G. Cooper, Biochem. Biophys. Res. Commun. 28, 203 (1967). h G. A. Hamilton, J. Spona, and L. D. Crowell, Biochem. Biophys. Res. Commun. 26, 193 (1967); K. T. Fry, O.-K. Kim, J. Spona, and G. A. Hamilton, Biochemistry 9, 4624 (1970). V. M. Stepanov, L. S. Lobareva, and N. I. Mal'tsev, Biochim. Biophys. Acta 151, 719 (1968). JR. S. Bayliss and J. R. Knowles, Chem. Commun: (1968) p. 196; R. S. Bayliss, J. R. Knowles, and G. B. Wybrandt, Biochem. J. 11$, 377 (1969).
[55]
ESTERIFICATION
599
placed on the fact that the specificity of pepsin is directed toward aromatic amino acid residues in peptide substrates. Although specific binding of a hydrophobic moiety may be one factor contributing to the reaction of many of these compounds with a specific aspartic acid residue in the enzyme, recent evidence suggests that the formation of a copper complex with the reagent is a much greater determinant of specificity. The procedures to be described have been selected from the work of a number of laboratories in order to provide a background that should be useful in planning a study of the modification of carboxyl groups in other enzymes. The basic procedure for the synthesis of derivatives of diazoacetic acid may be found in Volume 11 [74]. The application of this procedure to the synthesis of diazoacetyl-,~-phenylalanine methyl ester is given below, and references to similar compounds may be found in Table I. The synthesis of one diazo ketone, tosyl-L-phenylalanyldiazomethane, is given below as an exemplary case. A variety of analogous reagents may be synthesized by the same procedure.
Synthesis of Reagents N-Diazoacetyl-L-phenylalanine M e t h y l Ester 5
Glycyl-L-phenylalanine methyl ester hydrobromide is prepared by removing the blocking group from N-benzyloxycarbonyl-glycyl-,~-phenylalanine methyl ester with HBr in glacial acetic acid. The blocked peptide ester may be synthesized from N-benzyloxycarbonylglycine and phenylalanine methyl ester by one of the mixed anhydride methods. 6 The starting materials are readily obtained commercially. Dissolve 8.2 g (24 mmoles) glycyl-L-phenylalanine methyl ester hydrobromide in 40 ml of 2 M sodium acetate and add 2.0 ml of glacial acetic acid. Cool the mixture in an ice bath and add 3.0 g (43 mmoles) of NAN02 in small portions over 30 minutes. The mixture is allowed to stand for 3 hours in the cold, and the product is extracted with three 50-ml portions of ice-cold chloroform. The extracts are combined and dried over MgS04. Light petroleum ether (bp 40-60 °) is added until the solution becomes slightly turbid, and the mixture is allowed to stand overnight. The resulting yellow needles are collected by filtration and dried. The yield is about 70% (4.5 g), mp 126-128 °. The recrystallized material has an [a]~ = +186 ° (CHCl~). BR. S. Bayliss, J. R. Knowles, and G. B. Wybrandt, Biochem. J. 113, 377 (1969). 6N. F. Albertson, Org. React. 12, 157 (1962).
600
MODIFICATION REACTIONS
[S5]
This procedure is readily adapted to the preparation of N-diazo[1-14C]-acetyl-L-phenylalanine methyl ester by starting with 1-14Clabeled glycine in the preparation of benzyloxycarbonylglycine.
Tosyl-L-phenylalanyldiazomethane (L-1-Diazo-~-phenyl-3tosylamidobutanone-2 ) The necessary intermediate, tosyl-L-phenylalanyl chloride, may be readily prepared from a commercial sample of tosylrL-phenylalanine. Suspend 3:2 g (10 mmoles) of the tosyl amino acid in 50 ml of anhydrous ethyl ether at 0 °, and add 2.3 g (11 mmoles) of PC15. Shake the mixture at 0 ° for 10 minutes and at room temperature for 10 minutes. Allow the product to crystallize during 1 hour at 0 °. Collect the crystalline material on a filter, wash it quickly with a small amount of cold ether and then with ice water, and dry the product in a desiccator under high vacuum. Yield, 80-90% (approximately 3.0 g), mp 128-129 ° (decomp.). The diazo ketone is prepared by adding 2.0 g (6.6 mmoles) of tosyl-L-phenylalanyl chloride to a solution of diazomethane (20 mmoles) in 50 ml of ethyl ether at 0 °. Keep the mixture in the dark at _room temperature for 16 hours, evaporate the solvent under vacuum, and recrystaltize the residue from a mixture of 1-butanol and cyclohexane. Yield, 0.8 g (35%), mp 109-111 ° (decomp.). A second recrystallization gives a product melting at 112-114 ° (decomp.), and [a]~ = - - 1 2 2 ° (approximately 0.4, ethanol). Procedures
Reaction o] Alkyl Diazo Compounds with Pepsin (Porcine) The conditions chosen by several groups of investigators have been similar. The range of conditions may be summarized as follows: Protein concentration Inhibitor concentration Cu~+concentration pH Temperature Time of reaction
o. 03-0.3 mM (1-10 mg/ml) 0.8-4.5 mM 1.0-4.5 mM 5.0-5.5 (acetate buffer) 15-38° 10-90 minutes
Within this range of conditions, the inhibition of pepsin was found to be greater than 90%. In many cases for which estimates were made, the reaction was specific for esterification of one carboxyl group with a stoiehiometry close to 1.0. Exceptions are noted below. The following procedure is taken from one of the successful experimentsY Pepsin is prepared by activation of pepsinogen (see Vol. XIX [20]).
[SS]
ESTERIFIC~TION
601
The esterification reaction is carried out at 15 °. Prepare a solution of the protein at a concentration of 1.25 mg/ml in a 6.25 mM acetate buffer, pH 5.4. To 4.0 ml of this solution, add 0.5 ml of 0.01 M CuC12 followed by 0.5 ml of a freshly prepared solution of the inhibitor in ethanol, in this example, a 1.43 mM solution of tosyl-L-phenylalanyldiazomethane. The molar ratios of protein:reagent:Cu are 1:5:35. The reaction is essentially complete in 45 minutes. At that time, the activity of the enzyme should be less than 10~, and the extent of incorporation of the inhibitor should be 1.0-+-0.1 mole per mole of protein. Assay methods for pepsin may be found in Vol. 19 [20]. Methods for determination of the extent of esterification will be described below. Comments
Subsequent to investigations of the reaction of diazo compounds with pepsin in a number of laboratories, a study by Lundblad and Stein ~ shed additional light on this reaction. Most significantly, they showed that preincubation of the diazo compound with cupric ion will eliminate a: time lag in enzyme inhibition which is found when the order of addition is the one prescribed above, namely, addition of cupric ion followed by the inhibitor. The change in the order of addition results in a shift in the maximum rate of reaction from pH 5.7 to 5.0, and the reaction is complete within 10 minutes at 15 °. It is concluded that the formation of a copper complex with the diazo compound is a rate-limiting step. Very likely this is a carbene complex which reacts very rapidly and specifically with the enzyme. A plot of the rate of this reaction as a function of pH is a bell-shaped curve with the maximum at pH 5.0, suggesting that the reagent reacts preferentially with a nonionized carboxyl group that in this case has an abnormal pK, at least as high as 5.6. Such a behavior, that is, a much higher rate of reaction with a nonionized carboxyl group compared with the ionized form, is consistent with what is known about the reactivity of aliphatic diazo compounds (see Vol. 11 [74]). Denatured pepsin is not esterified by diazo compounds under the conditions of the procedure given above. There is evidence that in the native enzyme a second carboxyl group is situated close to the aspartic acid residue that becomes esterified. It is postulated that this second carboxyl is ionized at pH 5 and that the negative charge may be involved in binding the copper complex, thus enhancing the rate of the specific reaction. This effect appears to predominate over stereochemical interactions which depend upon the structure of the inhibitor. Many R. L. Lundblad and W. H. Stein, J. Biol. Chem. 244, 154 (1969).
602
MODIFICATION REACTIONS
[5S]
different diazo compounds have been found to react with pepsin in the presence of cupric salts, and to inhibit activity. These various inhibitors are listed in Table I. Some degree of stereospecificity has been observed, however, in the case of tosylphenylalanyldiazomethane, in that the L-isomer reacts more rapidly with the enzyme than does the D-isomer. It will be noted that almost all the compounds in Table I incorporate at least one hydrophobic moiety, frequently a phenyl group. In general, they have been designed as affinity labels for pepsin, taking into account the known specificity of the enzyme toward aromatic side chains in peptide substrates. Incorporation of more than 1 mole of reagent per mole of pepsin is found in the case of diazoacetic acid methyl ester (compound XV). Evidently this compound, described in Vol. l l [74] as a group specific reagent for carboxyl groups, possesses a reactivity that is great enough to esterify not only the specific aspartic acid residue, but also other carboxyl groups as well. Diphenyldiazomethane (compound I) also shows a lack of specificity for the active site carboxyl group. Incorporation of greater than 1 mole of reagent has also been found with some of the most specific reagents if the conditions are much more vigorous, for example, if N-diazoacetyl-L-phenylalanine methyl ester is initially 80 mM, the protein concentration is 10 mg/ml, and the time of reaction is 90 minutes2 Finally, the homogeneity of the enzyme is a factor in the specificity. Some commercial samples of pepsin incorporate as many as 4 moles of reagent per mole of enzyme. Reaction with Triethyloxonium Fluoroborate Triethyloxonium fiuoroborate (Meerwein reagent) is a strong alkylating agent. In nonaqueous media, the reagent will ethylate such compounds as ethers, sulfides, nitriles, ketones, esters, and amides on oxygen, nitrogen, or sulfur, to give onium fluoroborates. In an aqueous medium, hydrolysis of the reagent occurs rapidly, the half-life being about 10 minutes at room temperature and pH values around 6. For this reason, only a small fraction of the reagent can be utilized for alkylation or esterification in aqueous medium, and it is difficult to cause any such reaction to go to completion. In this respect, the behavior of the Meerwein reagent resembles that of aliphatic diazo compounds (cf. Vol. 11 [74]). Although the Meerwein reagent is very reactive, recent investigations h a v e shown that it will specifically esterify carboxyl groups in lysozyme8 s S. M. Parsons, L, Jao, F. W. Dahlquist, C. L. Borders, Jr., T. Groff, J. Racs, and M. A.. Raftery, Biochemistry 8, 700 (1969).
ESTERIFICATION
[55]
603
and in trypsin ~ when the reaction is carried out at pH 4.5. Specificity was indicated by the observation that the amino acid analysis of the acid hydrolyzate of each protein gave results that were not appreciably different from the analysis of the corresponding native protein. Furthermore, estimates of the incorporation of ethyl groups into lysozyme were in agreement with measurements of the conversion of carboxyl groups into ester groups. Specificity may not be restricted, however, to carboxyl groups for all proteins or under different sets of conditions. Yonemitsu et al. l° have studied the esterification of a number of model peptides dissolved in bicarbonate buffer. With a molar ratio of Meerwein reagent to peptide of 20:1, esterification of carboxyl groups was obtained in yields greater than 80%. The side chains of serine, threonine, tyrosine, lysine, and arginine were completely nonreactive under these conditions. HQwever, the side chains of methionine and histidine were ethylated to give sulfonium and quaternary imidazolium salts, respectively. It would also be expected that a free sulfhydryl group of a cysteine residue would be ethylated. Although these possibilities for alkylation exist, one may expect that reaction at methionine in native enzymes will be only infrequently observed because this amino acid residue is usually buried, and reaction at exposed histidine residues can probably be inhibited by working at pH values between 4 and 5. Esterification by triethyloxonium fluoroborate proceeds by a nucleophilic attack of the negatively charged carboxylate group on the positively charged oxonium ion.
C--O f R
~ O--CI~--CH s • i CH~--CHs
~- C--OCI~CHs i R
+
i CH~--CHs
Therefore, one might expect that the reagent at a given pH would preferentially esterify carboxyl groups that are more highly ionized in comparison with those that are less ionized. Also, one might expect that an ionized carboxyl group in a hydrophobic pocket might be esterified preferentially because of a strong electrostatic interaction. The experimental results with lysozyme and trypsin are consistent with these postulated properties of the reagent. It is noteworthy that the apparent preference of the Meerwein reagent for ionized carboxyl groups over nonionized groups is just the 9H. Nakayama, K. Tanizawa, and Y. Kanaoka, Biochem. Biophys. Res. Commun. 40, 537 (1970). lo0. Yonemitsu, T. Hamada, and Y. Kanaoka, Tetrahedron Left. 23, 1819 (1969).
604
MODIFICATION REACTIONS
[55]
reverse of the behavior of alkyl diazo compounds, which react preferentially with the nonionized carboxyl group. These contrasting modes of reaction have a counterpart in the hydrolytic reactions. The reaction of diazo compounds with water is catalyzed by hydrogen ions, and there is not net consumption or production, whereas the reaction of water with the oxonium ion produces hydrogen ions. It appears that these two different types of reagents may be potentially useful for distinguishing and characterizing enzyme carboxyl groups with abnormal properties, the diazo compounds for such groups with abnormally high pK values, and the Meerwein reagent for those with abnormally low pK values.
Synthesis of Reagents Triethyloxonium Fluoroborate
A detailed procedure for the synthesis of the oxonium salt is given by Meerwein. 11 The starting materials are available from commercial sources. In brief, epichlorohydrin (1.51 moles) is added dropwise to a solution of freshly distilled boron fluoride etherate (2.00 moles) in 500 ml of anhydrous ethyl ether. After the exothermic reaction has subsided, the mixture is refluxed for 1 hour and is allowed to stand overnight at room temperature. The solvent is removed from the crystalline product by filter stick, and the product is washed by the addition of fresh portions of dry ether. The yield of colorless crystals Imp 91-92 ° (deeomp.)] is about 90%. The salt is very hygroscopic. It may be dried with a stream of nitrogen in a dry box and stored in a tightly closed bottle, but the material in this form should be used within a few days after bottling. Alternatively, the oxonium salt may be stored for longer periods under ether at a low temperature. If there is reason to use the methyl analog for esterification, a procedure is available for converting the triethyloxonium salt. 12 14C-Labeled Triethyloxonium Fluoroborate 8
Reagent labeled with ~4C may be prepared by exchange of ethyl groups between the oxonium salt and ~4C-labeled diethyl ether obtained from a commercial source. About 0.8 g of the oxonium salt is dissolved in 7 ml of methylene chloride containing 1.0 mmole of ~4C-labeled ethyl ether (1.0 mCi/mmole). The solution is placed in a heavy-walled glass ~IH. Meerwein, Org. Syn. 46, 113 (1966). It. Meerwein, Org. Syn. 46, 120 (1966).
[55]
ESTERIFICATION
605
ampoule, all manipulations being carried out under a dry nitrogen atmosphere and at a temperature of about --80 ° (solid C02-methylene chloride). The ampoule is sealed by torch and then warmed for about 20 hours in refluxing methylene chloride. After the ampoule has been cooled and opened, 1 ml of the solution is removed and added to 100 mg of 3,5-dinitrobenzoic acid dissolved in 15 ml of methylene chloride. The mixture is refiuxed for 4 hours, the solvent is evaporated, and the residue is washed with 5% NaHC03 solution. The ethyl 3,5-dinitrobenzoate (mp 83-84 °) is recrystallized from ethanol-water to provide material for the determination of specific activity of the ethyl group (approximately 5 X 10~ dpm/mole). The main bulk of the 14C-labeled oxonium salt is precipitated from methylene chloride solution as an oil by the addition of 20 ml of hexane. The supernatant solvent is drawn off and the oil is taken up in a small amount of dry acetonitrile. This solution, containing about 0.5 g of 14C-triethyl-oxonium fluoroborate, is the form of the labeled reagent that is added to an aqueous solution of protein for the esterification of carboxyl groups. Procedures 8
The conditions used for the esterification of lysozyme and trypsin are as follows: Protein concentration pH
Temperature Initial reagent concentration
10-12.5 mg/ml 4.5 or 4.0 20-25° 0.1 or 0.2 M
The protein is dissolved in water, and the pH is adjusted to the desired value by the addition of dilute perchloric acid. The pH during the reaction is maintained by a pH-stat using a solution of 4 N NaOH in the syringe. A portion of triethyloxonium fluoroborate is dried under a stream of nitrogen in a dry box and is weighted. An amount of acetonitrile equal to about one-fourth of the weight of the salt is added, and the resulting solution is weighed. All the solution is taken up into a dry, calibrated syringe, and the volume of solution that contains the amount of oxonium salt (MW 189.8) needed to bring the concentration of the reagent in the protein solution to 0.2 M (or 0.1 M) is calculated. Inject this amount of reagent into the protein solution while it is vigorously stirred in the pH-stat. The reaction and the consumption of base proceed for about 20 minutes. The modified protein is recovered by dialysis against distilled water (or 10-SM HC1) in order to remove small molecular reaction products, followed by lyophilization. In order to obtain
606
MODIFICATION REACTIONS
[55]
more extensive esterifieation, the dialyzed solution may be repeatedly treated with the oxonium salt, following the same procedure that was used for the first treatment. It may be advisable after each dialysis to remove any denatured protein that has precipitated. Comments
As in the case of esterification with dia~.oacetyl compounds (in the absence of cupric ions), an estimate of the extent of esterification as a function of the consumption of the reagent, expressed as millimoles per milliliter of reaction mixture, gives a useful comparative measure of the efficiency of modification (see Vol. 11 [74], p. 616). For example, the consumption of oxonium salt in the amount of 1.2 mmoles/ml at pH 4.5 resulted in the esterification of 22% (2.6 residues) of the carboxyl groups in lysozyme, and the consumption of 0.2 mmoles/ml resulted in the esterification of 12% (1.7 residues) of the carboxyl groups in trypsin. By comparison, the consumption of 0.6 mmoles of one or another of the diazoacetyl reagents resulted in the esterification of about 20% of the carboxyl groups in each of the proteins, chymotrypsinogen, ribonuclease, 7-globulin, and serum albumin. It appears that the efficiency is about the same for the two types of reagents. However, this comparison does not take into account the likelihood noted above, that the specificities of the two types of reagent are directed toward two different classes of carboxyl groups (high pK and low pK), and furthermore, that one or two carboxyl groups in a particular protein may possess an enhanced reactivity toward one type of reagent or the other (cf. Vol. 11 [74] pp. 605-606). The investigation of the esterification of lysozyme by triethyloxonium fluoroborate showed, indeed, that there are two carboxyl groups that have an enhanced reactivity toward this reagent2 By limiting the extent of modification to a level between 0.5 and 1.5 ester groups per mole, it was possible to obtain high yields of monoesterified derivatives. One of these, obtained in high yield by esterification at pH 4.0, proved to be a labile ester that completely reverted to native lysozyme during incubation at pH 7.2 and room temperature for 48 hours. A second specific carboxyl group became esterified in high yield by treatment with the oxonium salt at pH 4.5. After the labile ester group had been removed at pH 7.2, the stable monoesterified derivatives could be isolated and purified by chromotography on a cation-exchange resin. The monoesterified derivative which was labile at pH 7.2 showed a reduced enzymatic activity which resulted from a lowered binding affinity for substrates compared with the affinity of native enzyme.
[551
ESTERIFICATION
607
The second, stable monoesterified derivati.ve appeared to be devoid of enzymatic activity. It was possible to demonstrate that the modified residue was fl-ethylaspartic acid by means of total enzymatic hydrolysis and amino acid analysis. An examination of the peptide fragments produced by chymotryptic cleavage of the derivative showed that this residue was Asp-52 in the native enzyme. X-ray crystallographic studies had previously suggested that Asp-52 and Glu-35 may be involved in the catalytic mechanism of the enzyme. The location of the principal site of esterification in trypsin has not been determined at the time of this review. However, the extent of reaction at pH 4.5 is reduced from 1.7 to 1.0 ester groups per mole by the presence of 50 mM fl-naphthamidine. It is proposed that this inhibitor blocks esterification of Asp-177 which is situated in the binding pocket of this serine protease2 Reaction with Active Halogen Compounds A growing number of enzymes are now known that are inhibited in each case by esterification with an active halogen compound. Although reactivity of halogen compounds is ordinarily directed toward amino acid side chains containing nitrogen or sulfur atoms as nucleophiles, in each example of inhibition presented in Table II the carboxyl group of an aspartic or glutamic acid residue acts as the nucleophile. The reactions listed in Table II all have a high degree of specificity for the structure of the inhibitor. In some instances, the structure has been designed as a substrate analog, and it seems clear that the reagent is bound specifically to the active site of the enzyme. In other instances, such as ribonuclease T1, a relationship to the substrate binding site is not presently obvious, although a similarity to the inhibition of pancreatic ribonuclease with iodoacetate is suggestive. The inhibitor of elastase has no obvious structural similarity to good substrates of the enzyme, and the site of reaction is far removed from the substrate binding site, yet the stoichiometric incorporation of one equivalent of inhibitor results in complete inactivation o f the enzyme. Furthermore, the rate of the inhibition reaction is reduced by the presence of substrates. It would appear that this is an example of a conformational interaction between the substrate binding site and another site in t h e enzyme that specifically binds the inhibitor. The reaction of pepsin with p-bromophenacyl bromide is another interesting example. The aspartic acid residue that is specifically esterifled has been shown to be distinct from the aspartic acid that reacts with a variety of diazo compounds (see Table I). The site of reaction of the latter compounds has been located in the sequence, Ile-Val-Asp-Thr-Gly-
608
MODIFICATION REACTIONS
[5S]
M o 0J
O O O
¢n II ~
II oo II t.:
II
II
II ~
U
,-z
,..-.2
¢D
2:
2~ oo ~
O
o~ L)
.~.
~.~
[55 ]
ESTERIFICATION
609
Thr-Ser-Leu, 5-18 whereas a small peptide esterified at aspartic acid with p-bromophenacyl bromide was isolated and shown to have the composition, (Gly~,Aspl,Ser~,Glu~)2 4 Compounds known to be competitive inhibitors of pepsin protect the enzyme from esterification by either diazo compounds or halogen compounds, and therefore both aspartic acid residues are presumed to be close to the substrate binding site. However, it is possible to prepare a diesterified derivative by using, successively, p-bromophenacyl bromide and a-diazo-p-bromoacetophenone. 15 An examination of the optimal conditions for obtaining nearly complete inhibition and stoiehiometric esterification with active halogen compounds indicates that at the present time no useful generalizations can be made concerning such specific reactions. It appears that each different enzyme must be considered as a unique case in which the enzyme structure and the special properties of certain carboxyl groups have a predominant influence. Methods of Analysis Although esterification of carboxyl groups in enzymes has been observed with very different types of reagents, the derivatives all have the ester linkage in common. In this section, methods that have been found useful for characterization of the various derivatives will be reviewed. Estimation of the Extent of Incorporation of Reagent The most common method for estimation of incorporation of reagent has utilized a 14C-labeled form of the reagent and application of scintillation spectrometry. The diazo compounds which were used in the study of pepsin and which have been synthesized with ~C-label are so indicated in Table I. The synthesis of [14C]triethyloxonium fluoroborate is described above. Among the reagents listed in Table II, the inhibitors for ribonuclease T1, elastase, and carboxypeptidase A were labeled with ~4C. A method used in the study of triose phosphate isomerase is of special interest. In this case, the label was introduced after the inhibitor had been reacted with the enzyme by reducing the derivative with sodium borohydride containing tritium label. The carbonyl group of the inhibitor moiety was thereby reduced to a secondary alcohol group. The result was the simultaneous incorporation of a tritium atom and the stabilization of the ester linkage. Since a ketonic function is u K. T. Fry, O.-K. Kim, J. Spona, and G. A. Hamilton, Biochemistry 9, 4625 (1970). 14B. F. Erlanger, S. M. Vratsanos, N. Wassermann, and A. G. Cooper, Biochem. Biophys. Res. Commun. 23, 243 (1966). ~B. F. Erlanger, S. M. Vratsanos, N. Wassermann, and A. G. Cooper, Biochem. Biophys. Res. Commun. 9.8, 203 (1967).
610
MODIFICATION REACTIONS
[55]
present in many of the compounds that have been used for the esterification of proteins, this method may have wide applicability. Another method for measuring the extent of incorporation of reagent has depended upon the synthesis of a reagent that contains an unnatural amino acid residue, namely norleucine. ~ Conventional amino acid analysis will give an accurate determination of the number of reagent molecules added to the enzyme. In some cases, advantage has been taken of some other type of label in the reagent. For example, bromine has been determined by elemental analysis ~5 or a chromophoric group has been determined by spectrometry. 1~ Characteristics and Estimation of Ester Groups
Analysis ]or Ethanol or Glycolic Acid alter Hydrolysis Lysozyme esterified with triethyloxonium fluoroborate has been analyzed for ethoxyl content by mild basic hydrolysis in sealed ampoules followed by gas-liquid partition chromatography using 10% Carbowax 20M on a 6-foot, 60-80-mesh column at 90o. 8 Esterification by haloacetate, as well as by diazoacetic acid derivatives, may be estimated after base hydrolysis by the method described in Volume 11 [74], p. 615.1~
Conditions ]or Basic Hydrolysis Typical esters are characteristically hydrolyzed by dilute sodium hydroxide, and lability to dilute base is indicative of an ester bond. Saponification of esterified lysozyme in a pH-stat at pH 10 has even been used as a quantitative method2 However, the stability of ester groups in protein derivatives has been shown to vary widely. At one extreme, it has been observed that the labile ethoxy group that is introduced into lysozyme at pH 4.0 by triethyloxonium fluoroborate is removed by incubation at pH 7.2 and room temperature for 48 hours. 8 A study of the rate of hydrolysis at pH 8.0 of the ester bond in pepsin inhibited with 1diazo-4-phenyl-2-butanone gave a half-life of 5.5 hours at 30 °, comparable to half-life of hydrolysis of the model compound, 1-acetoxy-4-phenyl2-butanone, under the same conditions. 18 At the other extreme of behavior, it was observed that incubation at pH 10 did not regenerate the activity of lysozyme esterified at Asp-52, whereas incubation at pH 2 resulted in reactivation of this derivative to the extent of 60%. 8 I'V. M. Stepanov, L. S. Labareva, N. I. Mol'tsev, Biochim. Biophys. Acta 151, 719 (1968). 1~K. Takahaski, W. H. Stein, and S. Moore, J. Biol. Chem. 242, 4682 (1967).
[55]
ESTERIFICATION
611
Reaction with Hydroxylamine and Estimation o] Hydroxamate Ester groups in enzyme derivatives have been determined by conversion to hydroxamic acid groups using 1 M hydroxylamine at pH values between 7.0 and 9.0, followed by estimation of the hydroxamate by the well-known colorimetric method that makes use of the ferric complex (e.g., see Vol. 3, 323). A positive color test for hydroxamate using N-l-naphthylethylenediamine in the Segal test is is also indicative. However, care must be taken because it is not possible to devise a control that will make certain that reaction of hydroxylamine with particular ester groups in a protein derivative or peptide fragment is quantitative. The most sensitive method of estimating hydroxamic acid groups is the one devised by Yasphe et al. 19 and effectively applied to the determination of ester groups in esterified lysozyme2
Reagents Sodium acetate, 5% Sulfanilic acid reagent (dissolve 10 g of sulfanilic acid in 1 liter of 30% acetic acid) Iodine reagent (dissolve 1.3 g I2 in 100 ml of glacial acetic acid) Sodium thiosulfate, 2.5% a-Naphthylamine reagent (dissolve 3 g of a-naphthylamine in 1 liter of 30% acetic acid) Hydrochloric acid, 3 N
Procedure. To 1.0 ml of sample containing from 0.05 to 0.1 t~eq of hydroxama*e, add 5 ml of 5% sodium acetate, 1 ml of sulfanilic acid reagent, and 0.5 ml of iodine reagent. Shake the mixture and allow it to stand for 5 minutes. Add 0.5 ml of 2.5% thiosulfate in order to remove excess I2, add 0.5 ml of 3 N HC1, and finally, add 1 ml of naphthylamine reagent. Make up the volume to 10 ml and determine the optical density at 530 nm after 90 minutes. Construct a calibration curve using a standard solution of acetylhydroxamic acid. The optical density for 0.1 ~mole is about 0.35. Estimation o] Esterified Residues by Di]]erence, Using the Lossen Rearrangement The conversion of ester groups in protein derivatives to hydroxamic acid groups, as indicated above, allows the identification of the residues 18F. Bergmann and R. Segal, Biochem. J. 62, 542 (1956). 1~j. Yasphe, Y. S. Halpern, and N. Grossowicz, Anal. Chem. 32, 518 (1960).
612
MODIFICATION REACTIONS
[SSl
as aspartic or glutamic esters by means of the Lossen rearrangement.2°-23 The hydroxamic acid groups are reacted with 2,4-dinitrofluorobenzene, the rearrangement is brought about by treatment with 0.1 N NaOH, the modified protein is hydrolyzed with 6 N HC1, and a sample is analyzed on the automatic amino acid analyzer. Those residues of aspartic acid which had been esterified in the protein derivative appear in the analysis as 2,3-diaminopropionic acid, and those residues of esterified glutamic acid appear as 2,4-diaminobutyric acid. Details of the procedure may be found in Volume 11 [74], pp. 610-611.
Estimation o] Esterified Residues by Di]]erence, U~ing the CDI Method ]or Free Carboxyl Groups A method for determination of the total content of free carboxyl groups in a protein has been developed by Hoare and Koshland 24 (see this volume [56]). The carboxyl groups are activated in the presence of an excess of glycine methyl ester by the use of a water-soluble carbodiimide in a denaturing solvent, such as 8 M urea or 6 M guanidinium chloride. After recovery of the modified protein, the incorporation of glycine may be determined by amino acid analysis. When this method is applied to a sample of esterified protein, the decrease in glycine incorporation compared to the amount obtained with native protein will give an estimate of the total number of esterified residues, s,9
Total Enzymatic Hydrolysis and Estimation o] Aspartic or Glutamic Esters Methods are now known by which esterified aspartic or glutamic acid residues may be liberated from certain modified proteins and identified by conventional chromatographic analysis.17,~5 It appears that several proteases attack the bonds on either side of the esterified residues at least as readily as the bonds to aspartic and glutamic acid residues themselves. For purposes of comparison, samples of ethyl or methyl esters of the acidic amino acids may be obtained commercially. However, two useful laboratory methods are given below, along with the procedure for the synthesis of the ~/-carboxymethyl ester of glutamic acid. A sample of ribonuclease T1 that had been inhibited by esterification with [14C]iodoacetate was fragmented by treatment with subtilisin BPN' O. O. Blumenfeld and P. M. Gallop, Biochemistry I, 947 (1962). 21E. Gross, and J. L. Morell, Y. Biol. Chem. 241, 3638 (1966). ~2L. Visser, D. S. Sigman, and E. R. Blout, Biochemistry 1O, 735 (1971). ~SF. C. Hartman, J. Amer. Chem. Soc. 92, 2170 (1970). ~D. G. Hoare and D. E. Koshland, Jr., J. Biol. Chem. 242, 2447 (1967). = S. M. Parsons and M. A. Raftery, Biochemistry 8, 4199 (1969).
[55]
ESTERIFICATION
613
(Nagarse) for 3 hours at pH 8 and 37 °. A radioactive peptide was isolated by paper chromatography. The peptide consisted of the sequence of residues 57-62, Try-Glu-Trp-Pro-Ile-Leu. A sample of the peptide was hydrolyzed with aminopeptidase M for 24 hours at pH 7 and 37 °. Analysis of the hydrolyzate on the automatic amino acid analyzer (0.9 X 60-cm column) showed the presence of tyrosine, isoleucine, leucine, small amounts of tryptophan and proline, and a new ninhydrin-positive peak which emerged at 4 3 _ 3 ml, just ahead of the position for S-carboxymethylcysteine. The new component was identical in its properties with a synthetic sample of the ~,-carboxymethyl ester of glutamic acid. The stable, monoethyl ester of lyozyme inhibited by Meerwein's reagent has been hydrolyzed with subtilisin (Carlsberg) for 4 hours at pH 7 and 25 °, followed by aminopeptidase M for 20 hours at pH 7.0 and 25 °. An aliquot of the hydrolyzate was subiected to amino acid analysis using 0.3M lithium citrate buffer, pH 2.80 (Spinco Technical Bulletin A-TB-044), at 25 ° in a Beckman-Spinco Model 120B analyzer. Compared with the analysis of native lysozyme, the derivative gave one less aspartic acid residue and a new peak appeared in the chromatogram between the positions of glutamic acid (plus glutamine) and proline. The position of this peak corresponded to that found for an authentic sample of fl-ethyl aspartate. This compound is completely resolved from peaks of the common amino acids by using the lithium buffer system. A chymotryptic peptide was isolated from a sample of the monoethyl ester of lysozyme. This peptide corresponded to fragment C-15 obtained by the action of chymotrypsin on native enzyme and was composed of residues 45-53, Arg-Asn-Thr-Asp-Gly-Ser-Thr-Asp-Tyr. Treatment of the esterified peptide with carboxypeptidase A resulted in the rapid liberation of the COOH-terminal tyrosine residue, followed by a somewhat slower liberation of fl-ethyl aspartate from the penultimate position. This result is in contrast to the slow liberation of the penultimate aspartic acid residue from the peptide obtained from native protein. The rate of formation of free amino acids was determined by amino acid analysis with the automatic analyzer. Synthesis o] the ~,-Carboxymethyl Ester o] Glutamic Acid. 17 Suspend 1.0 g of L-glutamic acid and 20 g of sodium glycolate in 60 ml of dioxane in a round-bottom flask. Chill the mixture in an ice bath and add 10 ml of thionyl chloride. Stopper the flask and allow it to stand for 5 hours at 25 °. An additional 5 ml of thionyl chloride are then added, followed 6 hours later by 25 ml of thionyl chloride and 5 g of sodium glycolate. After the mixture has stood overnight, the dioxane is removed in a rotary evaporator and about 100 ml of ethyl ether is added. The precipitate is collected and washed with ether. About one-sixth of the product
614
MODIFICATION REACTIONS
[55]
is purified on a column (4 }( 19 era) of Dowex 50-X2 (200-400 mesh) using 0.1 M pyridinium acetate buffer, pH 3.2, as eluent at a flow rate of 20 ml per hour. The elution of the product is followed by analyses for amino acid with ninhydrin and for glycolic acid with chromotropic acid reagent (see Vol. 11 [74] p. 615). The peak fractions which give positive tests for both amino acid and glycolic acid are pooled and evaporated to dryness in a rotary evaporator at 40 °. The residue is transferred to a test tube with 1 ml of water, and about 5 volumes of acetone is added. The precipitate is collected, and the material is crystallized from a small amount of water and acetone at 0 °. The product is collected by centrifugation, washed with a little acetone and ether, and stored in a vacuum desiccator over sulfuric acid. Synthesis of fl-Ethyl Aspartate Hydrochloride3 ~ Reflux a mixture of 5.0 g of DL-aspartic acid hydrochloride and 1.1 g of anhydrous HC1 in 15 ml of absolute ethanol for 15 minutes. Add dry ether to the warm solution until a slight turbidity develops, and allow the product to crystallize at room temperature. The crystals are collected on a filter, washed with dry ether, and stored in a vacuum desiccator. Yield, 2.8 g, mp 174-177 °. Synthesis of ~,-Ethyl L-Glutamate. 2~ This compound may be readily synthesized by esterification of poly-L-glutamic acid with triethyloxonium fluoroborate according to the procedure described above for the esterification of lysozyme. The polymer is then hydrolyzed with subtilisin and aminopeptidase M to yield a solution containing essentially only L-glntamic acid and v-ethyl L-glutamate in a ratio of 1:3. An aliquot of this solution may be used as a convenient reference for the amino acid analysis of enzymatic hydrolyzates of modified protein.
Stabigty of Ester Linkages during Fragmentation and Isolation o] Peptides Because the stability of the ester linkage in different enzyme derivatives has been shown to vary considerably depending upon the nature of the enzyme and the type of esterifying reagent, no general procedures for the fragmentation of these derivatives can be given. Nevertheless, a summary of successful methods may be useful' to other efforts directed toward the location of the site of esterification in an enzyme derivative. The ester linkage in pepsin inhibited with diazo compounds appears to be labile to incubation at pH values above 72 ,13 The linkage becomes even more labile upon denaturation of the protein. Therefore, the derivative has been denatured either with acetone at low pH or by treatment at pH values around 7 for only 2 hours at 25 °. The denatured protein can then be fragmented by digestion with native pepsin at pH values
[55 ]
ESTERIFICATION
615
near 3 with no cleavage of ester bonds. Esterified peptides have been isolated by gel filtration, paper electrophoresis, and paper chromatography, using acidic buffers, several containing pyridine and acetic acid. The lability of the ester bond was put to some use by adapting the cleavage to the method of "diagonal electrophoresis." The mobility of the esterified peptide was changed between two electrophoretic runs by treating the paper with an aqueous solution saturated with triethylamine2 The ester linkage in several other enzyme derivatives is more normal in stability. The linkage in lysozyme ethylated at Asp-52 is stable to the conditions used for oxidation of cystine by performic acid at --10% The oxidized derivative is stable to incubation for 9 hours at pH 7.2 and 25 °. Therefore, enzymatic digestion with trypsin, chymotrypsin, and carboxypeptidase k could be usefully applied? ~ The ester bond at Glu-6 in elastase reacted with a bromoketone is stable to cyanogen bromide cleavage in 70% formic acid, followed by chromatography on SE-Sephadex using a pH gradient from 4.5 to about 7.0.22 In the case of ribonuelease T1, the ester bond in y-carboxymethylGlu-58 was found to be cleaved to the extent of 30% during reduction and carboxymethylation of the protein at pH 8.5, using mercaptoethanol and iodoacetate in 8 M urea over a period of 20.5 hours. The ester bond was stable during digestion with substilisin for 3 hours at pH 8.0.17 Addendum
2G
In a study of the esterification of bovine insulin by treatment with anhydrous methanol-HC1 (0.1M), a specifie N-to-O aeyl shift was found to occur at the bond between Tyr-B26 and Thr-B27. The equilibrium mixture at 25 ° contained about 60% of the protein with an aeyl ester linkage to the oxygen of the threonine residue. This derivative could be isolated by chromatography in 7 M urea at pH 4.75. Deamination with nitrous acid and treatment with 0.1 M sodium carbonate resulted in a specific cleavage of the ester bond between the tyrosine and threonine residues. On the other hand, treatment of the derivative in aqueous solution at pH values above 2.2 resulted in an irreversible O-to-N acyl shift, giving a derivative of the native structure with all six carboxyl groups esterified.
D. Levy and F. g . Carpenter, Biochemistry 9, 3215 (1970).
MODIFICATION REKCTIONS
[55]
untreated ribonuclease. 26 Such changes in the physical properties of the regenerated protein need not invalidate the use of amidination in sequence and cross-linking determinations. For example, removal of the amidino group has facilitated identification of the lysine residues crosslinked by reaction of ribonuclease with adipimidate. 4 In certain situations, however, it appears that reaction with maleic anhydride ~2 may have advantages over reaction with imidates.
Procedures ]or the Amidination o] Proteins 1. k method for exhaustive modification by reaction with methyl (or ethyl) acetimidate 3 has been given in Volume 11.6 2. The procedure for reaction of cytochrome b~ reductase with acetimidate 44 is useful for other small-scale preparations: Enzyme (0.03 ~mole, 22 lysine/FAD) is dissolved in 0.3 ml of pyrophosphate buffer, pH 8.5. Ethyl acetimidate (300 ~moles in 0.30 ml of 0.45 M borate buffer, pH 9.2) is added, and the mixture is left at 0 ° for 20 hours. The derivative can be separated from contaminating small molecules by passage through a Sephadex G-25 column. 3. Cross-linking using dimethyl suberimidate47: The pH is maintained at 8.5 (with 0.2M triethanolamine.HC1) to favor amidination over reagent hydrolysis. Dimethyl suberimidate, adjusted to pH 8.5 in the above buffer, is mixed with a solution of the protein to be crosslinked to give a final protein concentration of 0.5-5 mg/ml and a dimethyl suberimidate concentration of 1-12 mg/ml, and the reaction is allowed to proceed for 3 hours. ~2p. j. G. Butler, J. I. Harris, B. S. Hartley, and R. Leberman, Biochem. J. 112, 679 (1969).
[55] Esterification By PHILIP E. WILCOX~ In the short time that has elapsed since the publication of Volume 11, methods for the esterification of proteins have been extended substantially. Interest in modification of carboxyl groups has increased with each new discovery of the functional or structural role of a glutamic or aspartic acid side chain in an enzyme. The growing list of enzymes includes lysozyme, carboxypeptidase A, pepsin, trypsin and other serine proteases, ribonuclease T1, and triosephosphate isomerase. In Volume 11 [74], it was pointed out that only two methods of * Deceased.
[55]
ESTERIFICATION
597
esterification were known then to be group specific, namely, reaction with methanol-HC1 or ethanol-HC1 and reaction with derivatives of diazoacetic acid near pH 5. It now appears that reaction with triethyloxonium fluoroborate (Meerwein reagent) at pH values between 4 and 5 may be added to the repertoire of group-specific reagents. Furthermore, the use of aliphatic diazo compounds has been extended to include diazo ketones containing the grouping --CO--CH----N2. A remarkable advance in methodology has come from the discovery that copper ions promote the reaction between the carboxyl group of a specific aspartic acid residue in pepsin and a variety of aliphatic diazo compounds. 1,~ The mechanism is not presently clear, but it is likely that the method will be applicable to other enzymes. Indeed, the reaction has been shown to occur with a pepsinlike enzyme from the mold
Penicillium janthinellum2 Experience has furthermore shown that specific esterification of particular enzymes can be obtained with reagents that are not ordinarily specific for carboxyl groups alone. In most of these cases the reagent has been an active halogen compound, such as iodoacetate or a bromomethyl ketone. Specificity may result either from an enhanced reactivity of the carboxyl group due to the local environment in the native protein, or from specific binding of a particular reagent to a site in the enzyme, or perhaps from a combination of both effects. This article will be focused mainly on new methods that have been developed for reagents that are relatively specific for carboxyl groups. The other category of reagent, the reactive halogen compounds, will be given a more cursory treatment because the particular structure of the binding site in the enzyme must be of primary importance in each case. Esterification may be regarded as a special case of alkylation. It is therefore likely that particular compounds, which would ordinarily be classified among typical alkylating reagents, will be found to esterify uniquely situated carboxyl groups in enzymes and other proteins as the result of specific interactions. The reader is referred to other articles on alkylation (this volume [34a] and [39]).
Aliphatic Diazo Compounds The reactions of aliphatic diazo compounds are manifold and complex. A useful reference is the treatise by H. Zollinger. 4 In spite of their 1T. G. Rajagopalan, W. H. Stein, and S. Moore, J. Biol. Chem. 241, 4295 (1966). G. R. Delpierre and J. S. Fruton, Proc. Nat. Acad. Sci. U.S. 56, 1817 (1966). ' J . ~odek and T. Hofmann, J. Biol. Chem. 243, 450 (1968). 4H. Zollinger, "Azo and Diazo Chemistry: Aliphatic and Aromatic Compounds," Wiley (Interscience), New York, 1961.
598
MODIFICATION REACTIONS
[5S]
reactivity, aliphatic diazo compounds show a high degree of specificity for carboxyl groups in an aqueous m e d i u m when t h e y are partially stabilized b y the presence of a c a r b o n y l group a t t a c h e d to the diazotized carbon atom, as in the diazoacetyl or the d i a z o m e t h y l ketone moieties. T h e only exception is the ability of these reagents to alkylate free sulfhydryl groups (see ¥ol. 11 [74]). D i a z o compounds not so stabilized, for example, diazomethane, tend to be nonspecific. T h e e n z y m a t i c properties of pepsin suggested to a n u m b e r of investigators t h a t the active site of this e n z y m e contained at least one carboxyl group as an i m p o r t a n t component. Therefore, a v a r i e t y of diazo compounds were synthesized as site-specific reagents. Reliance was TABLE I ALKYL DIAZO COMPOUNDS FOUND TO INHIBIT PEPSIN 14C_
Compound I II III IV V VI VII VIII IX X XI XII XIII XIV XV
Diphenyldiazomethane N-Diazoacetyl-DL-norleucine N-Tosyl-L-phenylalanyldiazomethane N-Tosyl-D-phenylalanyldiazomethane N-Diazoacetyl-L-phenylalanine methyl ester N-Diazoacetyl-D-phenylalanine methyl ester N-Diazoacetylglyeine methyl (ethyl) ester N-Benzyloxycarbonyl-L-phenylalanyldiazomethane a-Diazo-p-bromoacetephenone 1-Diazo-4-phenylbutanone-2 1-Diazo-3-dinitrophenylaminopropanone-2 Ethyl 2-diazo-3-(p-hydroxyphenyl) propionate Phenylbenzoyldiazomethane N-Diazoacetyl-N'-2,4-dinitrophenylethylenediamine
Methyl (or ethyl) diazoacetate
labeled
Reference
Yes -Yes -Yes -Yes Yes -Yes ----
a b, c d d d, e, j d f g h e e e
--
i
Yes
c
e-e
a G. R. Delpierre and J. S. Fruton, Proc. Nat. Acad. Sci. U.S. 54, 1161 (1965). b T. G. Raiagopalan, W. H. Stein, and S. Moore, J. Biol. Chem. 241, 4295 (1966). c R. L. Lundblad and W. H. Stein, J. Biol. Chem. 244, 154 (1969). G. R. Delpierre and J. S. Fruton, Proc. Nat. Acad. Sci. U.S. 56, 1817 (1966). e L. ¥. Kozlov, L. M. Ginodman, and V. N. Orekhovich, Biokhimiya 32, 1011 (1967). f E. B. Ong and G. E. Perlmann, Nature (London) 215, 1492 (1967). B. F. Erlanger, S. M. Vratsanos, N. Wassermann, and A. G. Cooper, Biochem. Biophys. Res. Commun. 28, 203 (1967). h G. A. Hamilton, J. Spona, and L. D. Crowell, Biochem. Biophys. Res. Commun. 26, 193 (1967); K. T. Fry, O.-K. Kim, J. Spona, and G. A. Hamilton, Biochemistry 9, 4624 (1970). V. M. Stepanov, L. S. Lobareva, and N. I. Mal'tsev, Biochim. Biophys. Acta 151, 719 (1968). JR. S. Bayliss and J. R. Knowles, Chem. Commun: (1968) p. 196; R. S. Bayliss, J. R. Knowles, and G. B. Wybrandt, Biochem. J. 11$, 377 (1969).
[55]
ESTERIFICATION
599
placed on the fact that the specificity of pepsin is directed toward aromatic amino acid residues in peptide substrates. Although specific binding of a hydrophobic moiety may be one factor contributing to the reaction of many of these compounds with a specific aspartic acid residue in the enzyme, recent evidence suggests that the formation of a copper complex with the reagent is a much greater determinant of specificity. The procedures to be described have been selected from the work of a number of laboratories in order to provide a background that should be useful in planning a study of the modification of carboxyl groups in other enzymes. The basic procedure for the synthesis of derivatives of diazoacetic acid may be found in Volume 11 [74]. The application of this procedure to the synthesis of diazoacetyl-,~-phenylalanine methyl ester is given below, and references to similar compounds may be found in Table I. The synthesis of one diazo ketone, tosyl-L-phenylalanyldiazomethane, is given below as an exemplary case. A variety of analogous reagents may be synthesized by the same procedure.
Synthesis of Reagents N-Diazoacetyl-L-phenylalanine M e t h y l Ester 5
Glycyl-L-phenylalanine methyl ester hydrobromide is prepared by removing the blocking group from N-benzyloxycarbonyl-glycyl-,~-phenylalanine methyl ester with HBr in glacial acetic acid. The blocked peptide ester may be synthesized from N-benzyloxycarbonylglycine and phenylalanine methyl ester by one of the mixed anhydride methods. 6 The starting materials are readily obtained commercially. Dissolve 8.2 g (24 mmoles) glycyl-L-phenylalanine methyl ester hydrobromide in 40 ml of 2 M sodium acetate and add 2.0 ml of glacial acetic acid. Cool the mixture in an ice bath and add 3.0 g (43 mmoles) of NAN02 in small portions over 30 minutes. The mixture is allowed to stand for 3 hours in the cold, and the product is extracted with three 50-ml portions of ice-cold chloroform. The extracts are combined and dried over MgS04. Light petroleum ether (bp 40-60 °) is added until the solution becomes slightly turbid, and the mixture is allowed to stand overnight. The resulting yellow needles are collected by filtration and dried. The yield is about 70% (4.5 g), mp 126-128 °. The recrystallized material has an [a]~ = +186 ° (CHCl~). BR. S. Bayliss, J. R. Knowles, and G. B. Wybrandt, Biochem. J. 113, 377 (1969). 6N. F. Albertson, Org. React. 12, 157 (1962).
600
MODIFICATION REACTIONS
[S5]
This procedure is readily adapted to the preparation of N-diazo[1-14C]-acetyl-L-phenylalanine methyl ester by starting with 1-14Clabeled glycine in the preparation of benzyloxycarbonylglycine.
Tosyl-L-phenylalanyldiazomethane (L-1-Diazo-~-phenyl-3tosylamidobutanone-2 ) The necessary intermediate, tosyl-L-phenylalanyl chloride, may be readily prepared from a commercial sample of tosylrL-phenylalanine. Suspend 3:2 g (10 mmoles) of the tosyl amino acid in 50 ml of anhydrous ethyl ether at 0 °, and add 2.3 g (11 mmoles) of PC15. Shake the mixture at 0 ° for 10 minutes and at room temperature for 10 minutes. Allow the product to crystallize during 1 hour at 0 °. Collect the crystalline material on a filter, wash it quickly with a small amount of cold ether and then with ice water, and dry the product in a desiccator under high vacuum. Yield, 80-90% (approximately 3.0 g), mp 128-129 ° (decomp.). The diazo ketone is prepared by adding 2.0 g (6.6 mmoles) of tosyl-L-phenylalanyl chloride to a solution of diazomethane (20 mmoles) in 50 ml of ethyl ether at 0 °. Keep the mixture in the dark at _room temperature for 16 hours, evaporate the solvent under vacuum, and recrystaltize the residue from a mixture of 1-butanol and cyclohexane. Yield, 0.8 g (35%), mp 109-111 ° (decomp.). A second recrystallization gives a product melting at 112-114 ° (decomp.), and [a]~ = - - 1 2 2 ° (approximately 0.4, ethanol). Procedures
Reaction o] Alkyl Diazo Compounds with Pepsin (Porcine) The conditions chosen by several groups of investigators have been similar. The range of conditions may be summarized as follows: Protein concentration Inhibitor concentration Cu~+concentration pH Temperature Time of reaction
o. 03-0.3 mM (1-10 mg/ml) 0.8-4.5 mM 1.0-4.5 mM 5.0-5.5 (acetate buffer) 15-38° 10-90 minutes
Within this range of conditions, the inhibition of pepsin was found to be greater than 90%. In many cases for which estimates were made, the reaction was specific for esterification of one carboxyl group with a stoiehiometry close to 1.0. Exceptions are noted below. The following procedure is taken from one of the successful experimentsY Pepsin is prepared by activation of pepsinogen (see Vol. XIX [20]).
[SS]
ESTERIFIC~TION
601
The esterification reaction is carried out at 15 °. Prepare a solution of the protein at a concentration of 1.25 mg/ml in a 6.25 mM acetate buffer, pH 5.4. To 4.0 ml of this solution, add 0.5 ml of 0.01 M CuC12 followed by 0.5 ml of a freshly prepared solution of the inhibitor in ethanol, in this example, a 1.43 mM solution of tosyl-L-phenylalanyldiazomethane. The molar ratios of protein:reagent:Cu are 1:5:35. The reaction is essentially complete in 45 minutes. At that time, the activity of the enzyme should be less than 10~, and the extent of incorporation of the inhibitor should be 1.0-+-0.1 mole per mole of protein. Assay methods for pepsin may be found in Vol. 19 [20]. Methods for determination of the extent of esterification will be described below. Comments
Subsequent to investigations of the reaction of diazo compounds with pepsin in a number of laboratories, a study by Lundblad and Stein ~ shed additional light on this reaction. Most significantly, they showed that preincubation of the diazo compound with cupric ion will eliminate a: time lag in enzyme inhibition which is found when the order of addition is the one prescribed above, namely, addition of cupric ion followed by the inhibitor. The change in the order of addition results in a shift in the maximum rate of reaction from pH 5.7 to 5.0, and the reaction is complete within 10 minutes at 15 °. It is concluded that the formation of a copper complex with the diazo compound is a rate-limiting step. Very likely this is a carbene complex which reacts very rapidly and specifically with the enzyme. A plot of the rate of this reaction as a function of pH is a bell-shaped curve with the maximum at pH 5.0, suggesting that the reagent reacts preferentially with a nonionized carboxyl group that in this case has an abnormal pK, at least as high as 5.6. Such a behavior, that is, a much higher rate of reaction with a nonionized carboxyl group compared with the ionized form, is consistent with what is known about the reactivity of aliphatic diazo compounds (see Vol. 11 [74]). Denatured pepsin is not esterified by diazo compounds under the conditions of the procedure given above. There is evidence that in the native enzyme a second carboxyl group is situated close to the aspartic acid residue that becomes esterified. It is postulated that this second carboxyl is ionized at pH 5 and that the negative charge may be involved in binding the copper complex, thus enhancing the rate of the specific reaction. This effect appears to predominate over stereochemical interactions which depend upon the structure of the inhibitor. Many R. L. Lundblad and W. H. Stein, J. Biol. Chem. 244, 154 (1969).
602
MODIFICATION REACTIONS
[5S]
different diazo compounds have been found to react with pepsin in the presence of cupric salts, and to inhibit activity. These various inhibitors are listed in Table I. Some degree of stereospecificity has been observed, however, in the case of tosylphenylalanyldiazomethane, in that the L-isomer reacts more rapidly with the enzyme than does the D-isomer. It will be noted that almost all the compounds in Table I incorporate at least one hydrophobic moiety, frequently a phenyl group. In general, they have been designed as affinity labels for pepsin, taking into account the known specificity of the enzyme toward aromatic side chains in peptide substrates. Incorporation of more than 1 mole of reagent per mole of pepsin is found in the case of diazoacetic acid methyl ester (compound XV). Evidently this compound, described in Vol. l l [74] as a group specific reagent for carboxyl groups, possesses a reactivity that is great enough to esterify not only the specific aspartic acid residue, but also other carboxyl groups as well. Diphenyldiazomethane (compound I) also shows a lack of specificity for the active site carboxyl group. Incorporation of greater than 1 mole of reagent has also been found with some of the most specific reagents if the conditions are much more vigorous, for example, if N-diazoacetyl-L-phenylalanine methyl ester is initially 80 mM, the protein concentration is 10 mg/ml, and the time of reaction is 90 minutes2 Finally, the homogeneity of the enzyme is a factor in the specificity. Some commercial samples of pepsin incorporate as many as 4 moles of reagent per mole of enzyme. Reaction with Triethyloxonium Fluoroborate Triethyloxonium fiuoroborate (Meerwein reagent) is a strong alkylating agent. In nonaqueous media, the reagent will ethylate such compounds as ethers, sulfides, nitriles, ketones, esters, and amides on oxygen, nitrogen, or sulfur, to give onium fluoroborates. In an aqueous medium, hydrolysis of the reagent occurs rapidly, the half-life being about 10 minutes at room temperature and pH values around 6. For this reason, only a small fraction of the reagent can be utilized for alkylation or esterification in aqueous medium, and it is difficult to cause any such reaction to go to completion. In this respect, the behavior of the Meerwein reagent resembles that of aliphatic diazo compounds (cf. Vol. 11 [74]). Although the Meerwein reagent is very reactive, recent investigations h a v e shown that it will specifically esterify carboxyl groups in lysozyme8 s S. M. Parsons, L, Jao, F. W. Dahlquist, C. L. Borders, Jr., T. Groff, J. Racs, and M. A.. Raftery, Biochemistry 8, 700 (1969).
ESTERIFICATION
[55]
603
and in trypsin ~ when the reaction is carried out at pH 4.5. Specificity was indicated by the observation that the amino acid analysis of the acid hydrolyzate of each protein gave results that were not appreciably different from the analysis of the corresponding native protein. Furthermore, estimates of the incorporation of ethyl groups into lysozyme were in agreement with measurements of the conversion of carboxyl groups into ester groups. Specificity may not be restricted, however, to carboxyl groups for all proteins or under different sets of conditions. Yonemitsu et al. l° have studied the esterification of a number of model peptides dissolved in bicarbonate buffer. With a molar ratio of Meerwein reagent to peptide of 20:1, esterification of carboxyl groups was obtained in yields greater than 80%. The side chains of serine, threonine, tyrosine, lysine, and arginine were completely nonreactive under these conditions. HQwever, the side chains of methionine and histidine were ethylated to give sulfonium and quaternary imidazolium salts, respectively. It would also be expected that a free sulfhydryl group of a cysteine residue would be ethylated. Although these possibilities for alkylation exist, one may expect that reaction at methionine in native enzymes will be only infrequently observed because this amino acid residue is usually buried, and reaction at exposed histidine residues can probably be inhibited by working at pH values between 4 and 5. Esterification by triethyloxonium fluoroborate proceeds by a nucleophilic attack of the negatively charged carboxylate group on the positively charged oxonium ion.
C--O f R
~ O--CI~--CH s • i CH~--CHs
~- C--OCI~CHs i R
+
i CH~--CHs
Therefore, one might expect that the reagent at a given pH would preferentially esterify carboxyl groups that are more highly ionized in comparison with those that are less ionized. Also, one might expect that an ionized carboxyl group in a hydrophobic pocket might be esterified preferentially because of a strong electrostatic interaction. The experimental results with lysozyme and trypsin are consistent with these postulated properties of the reagent. It is noteworthy that the apparent preference of the Meerwein reagent for ionized carboxyl groups over nonionized groups is just the 9H. Nakayama, K. Tanizawa, and Y. Kanaoka, Biochem. Biophys. Res. Commun. 40, 537 (1970). lo0. Yonemitsu, T. Hamada, and Y. Kanaoka, Tetrahedron Left. 23, 1819 (1969).
604
MODIFICATION REACTIONS
[55]
reverse of the behavior of alkyl diazo compounds, which react preferentially with the nonionized carboxyl group. These contrasting modes of reaction have a counterpart in the hydrolytic reactions. The reaction of diazo compounds with water is catalyzed by hydrogen ions, and there is not net consumption or production, whereas the reaction of water with the oxonium ion produces hydrogen ions. It appears that these two different types of reagents may be potentially useful for distinguishing and characterizing enzyme carboxyl groups with abnormal properties, the diazo compounds for such groups with abnormally high pK values, and the Meerwein reagent for those with abnormally low pK values.
Synthesis of Reagents Triethyloxonium Fluoroborate
A detailed procedure for the synthesis of the oxonium salt is given by Meerwein. 11 The starting materials are available from commercial sources. In brief, epichlorohydrin (1.51 moles) is added dropwise to a solution of freshly distilled boron fluoride etherate (2.00 moles) in 500 ml of anhydrous ethyl ether. After the exothermic reaction has subsided, the mixture is refluxed for 1 hour and is allowed to stand overnight at room temperature. The solvent is removed from the crystalline product by filter stick, and the product is washed by the addition of fresh portions of dry ether. The yield of colorless crystals Imp 91-92 ° (deeomp.)] is about 90%. The salt is very hygroscopic. It may be dried with a stream of nitrogen in a dry box and stored in a tightly closed bottle, but the material in this form should be used within a few days after bottling. Alternatively, the oxonium salt may be stored for longer periods under ether at a low temperature. If there is reason to use the methyl analog for esterification, a procedure is available for converting the triethyloxonium salt. 12 14C-Labeled Triethyloxonium Fluoroborate 8
Reagent labeled with ~4C may be prepared by exchange of ethyl groups between the oxonium salt and ~4C-labeled diethyl ether obtained from a commercial source. About 0.8 g of the oxonium salt is dissolved in 7 ml of methylene chloride containing 1.0 mmole of ~4C-labeled ethyl ether (1.0 mCi/mmole). The solution is placed in a heavy-walled glass ~IH. Meerwein, Org. Syn. 46, 113 (1966). It. Meerwein, Org. Syn. 46, 120 (1966).
[55]
ESTERIFICATION
605
ampoule, all manipulations being carried out under a dry nitrogen atmosphere and at a temperature of about --80 ° (solid C02-methylene chloride). The ampoule is sealed by torch and then warmed for about 20 hours in refluxing methylene chloride. After the ampoule has been cooled and opened, 1 ml of the solution is removed and added to 100 mg of 3,5-dinitrobenzoic acid dissolved in 15 ml of methylene chloride. The mixture is refiuxed for 4 hours, the solvent is evaporated, and the residue is washed with 5% NaHC03 solution. The ethyl 3,5-dinitrobenzoate (mp 83-84 °) is recrystallized from ethanol-water to provide material for the determination of specific activity of the ethyl group (approximately 5 X 10~ dpm/mole). The main bulk of the 14C-labeled oxonium salt is precipitated from methylene chloride solution as an oil by the addition of 20 ml of hexane. The supernatant solvent is drawn off and the oil is taken up in a small amount of dry acetonitrile. This solution, containing about 0.5 g of 14C-triethyl-oxonium fluoroborate, is the form of the labeled reagent that is added to an aqueous solution of protein for the esterification of carboxyl groups. Procedures 8
The conditions used for the esterification of lysozyme and trypsin are as follows: Protein concentration pH
Temperature Initial reagent concentration
10-12.5 mg/ml 4.5 or 4.0 20-25° 0.1 or 0.2 M
The protein is dissolved in water, and the pH is adjusted to the desired value by the addition of dilute perchloric acid. The pH during the reaction is maintained by a pH-stat using a solution of 4 N NaOH in the syringe. A portion of triethyloxonium fluoroborate is dried under a stream of nitrogen in a dry box and is weighted. An amount of acetonitrile equal to about one-fourth of the weight of the salt is added, and the resulting solution is weighed. All the solution is taken up into a dry, calibrated syringe, and the volume of solution that contains the amount of oxonium salt (MW 189.8) needed to bring the concentration of the reagent in the protein solution to 0.2 M (or 0.1 M) is calculated. Inject this amount of reagent into the protein solution while it is vigorously stirred in the pH-stat. The reaction and the consumption of base proceed for about 20 minutes. The modified protein is recovered by dialysis against distilled water (or 10-SM HC1) in order to remove small molecular reaction products, followed by lyophilization. In order to obtain
606
MODIFICATION REACTIONS
[55]
more extensive esterifieation, the dialyzed solution may be repeatedly treated with the oxonium salt, following the same procedure that was used for the first treatment. It may be advisable after each dialysis to remove any denatured protein that has precipitated. Comments
As in the case of esterification with dia~.oacetyl compounds (in the absence of cupric ions), an estimate of the extent of esterification as a function of the consumption of the reagent, expressed as millimoles per milliliter of reaction mixture, gives a useful comparative measure of the efficiency of modification (see Vol. 11 [74], p. 616). For example, the consumption of oxonium salt in the amount of 1.2 mmoles/ml at pH 4.5 resulted in the esterification of 22% (2.6 residues) of the carboxyl groups in lysozyme, and the consumption of 0.2 mmoles/ml resulted in the esterification of 12% (1.7 residues) of the carboxyl groups in trypsin. By comparison, the consumption of 0.6 mmoles of one or another of the diazoacetyl reagents resulted in the esterification of about 20% of the carboxyl groups in each of the proteins, chymotrypsinogen, ribonuclease, 7-globulin, and serum albumin. It appears that the efficiency is about the same for the two types of reagents. However, this comparison does not take into account the likelihood noted above, that the specificities of the two types of reagent are directed toward two different classes of carboxyl groups (high pK and low pK), and furthermore, that one or two carboxyl groups in a particular protein may possess an enhanced reactivity toward one type of reagent or the other (cf. Vol. 11 [74] pp. 605-606). The investigation of the esterification of lysozyme by triethyloxonium fluoroborate showed, indeed, that there are two carboxyl groups that have an enhanced reactivity toward this reagent2 By limiting the extent of modification to a level between 0.5 and 1.5 ester groups per mole, it was possible to obtain high yields of monoesterified derivatives. One of these, obtained in high yield by esterification at pH 4.0, proved to be a labile ester that completely reverted to native lysozyme during incubation at pH 7.2 and room temperature for 48 hours. A second specific carboxyl group became esterified in high yield by treatment with the oxonium salt at pH 4.5. After the labile ester group had been removed at pH 7.2, the stable monoesterified derivatives could be isolated and purified by chromotography on a cation-exchange resin. The monoesterified derivative which was labile at pH 7.2 showed a reduced enzymatic activity which resulted from a lowered binding affinity for substrates compared with the affinity of native enzyme.
[551
ESTERIFICATION
607
The second, stable monoesterified derivati.ve appeared to be devoid of enzymatic activity. It was possible to demonstrate that the modified residue was fl-ethylaspartic acid by means of total enzymatic hydrolysis and amino acid analysis. An examination of the peptide fragments produced by chymotryptic cleavage of the derivative showed that this residue was Asp-52 in the native enzyme. X-ray crystallographic studies had previously suggested that Asp-52 and Glu-35 may be involved in the catalytic mechanism of the enzyme. The location of the principal site of esterification in trypsin has not been determined at the time of this review. However, the extent of reaction at pH 4.5 is reduced from 1.7 to 1.0 ester groups per mole by the presence of 50 mM fl-naphthamidine. It is proposed that this inhibitor blocks esterification of Asp-177 which is situated in the binding pocket of this serine protease2 Reaction with Active Halogen Compounds A growing number of enzymes are now known that are inhibited in each case by esterification with an active halogen compound. Although reactivity of halogen compounds is ordinarily directed toward amino acid side chains containing nitrogen or sulfur atoms as nucleophiles, in each example of inhibition presented in Table II the carboxyl group of an aspartic or glutamic acid residue acts as the nucleophile. The reactions listed in Table II all have a high degree of specificity for the structure of the inhibitor. In some instances, the structure has been designed as a substrate analog, and it seems clear that the reagent is bound specifically to the active site of the enzyme. In other instances, such as ribonuclease T1, a relationship to the substrate binding site is not presently obvious, although a similarity to the inhibition of pancreatic ribonuclease with iodoacetate is suggestive. The inhibitor of elastase has no obvious structural similarity to good substrates of the enzyme, and the site of reaction is far removed from the substrate binding site, yet the stoichiometric incorporation of one equivalent of inhibitor results in complete inactivation o f the enzyme. Furthermore, the rate of the inhibition reaction is reduced by the presence of substrates. It would appear that this is an example of a conformational interaction between the substrate binding site and another site in t h e enzyme that specifically binds the inhibitor. The reaction of pepsin with p-bromophenacyl bromide is another interesting example. The aspartic acid residue that is specifically esterifled has been shown to be distinct from the aspartic acid that reacts with a variety of diazo compounds (see Table I). The site of reaction of the latter compounds has been located in the sequence, Ile-Val-Asp-Thr-Gly-
608
MODIFICATION REACTIONS
[5S]
M o 0J
O O O
¢n II ~
II oo II t.:
II
II
II ~
U
,-z
,..-.2
¢D
2:
2~ oo ~
O
o~ L)
.~.
~.~
[55 ]
ESTERIFICATION
609
Thr-Ser-Leu, 5-18 whereas a small peptide esterified at aspartic acid with p-bromophenacyl bromide was isolated and shown to have the composition, (Gly~,Aspl,Ser~,Glu~)2 4 Compounds known to be competitive inhibitors of pepsin protect the enzyme from esterification by either diazo compounds or halogen compounds, and therefore both aspartic acid residues are presumed to be close to the substrate binding site. However, it is possible to prepare a diesterified derivative by using, successively, p-bromophenacyl bromide and a-diazo-p-bromoacetophenone. 15 An examination of the optimal conditions for obtaining nearly complete inhibition and stoiehiometric esterification with active halogen compounds indicates that at the present time no useful generalizations can be made concerning such specific reactions. It appears that each different enzyme must be considered as a unique case in which the enzyme structure and the special properties of certain carboxyl groups have a predominant influence. Methods of Analysis Although esterification of carboxyl groups in enzymes has been observed with very different types of reagents, the derivatives all have the ester linkage in common. In this section, methods that have been found useful for characterization of the various derivatives will be reviewed. Estimation of the Extent of Incorporation of Reagent The most common method for estimation of incorporation of reagent has utilized a 14C-labeled form of the reagent and application of scintillation spectrometry. The diazo compounds which were used in the study of pepsin and which have been synthesized with ~C-label are so indicated in Table I. The synthesis of [14C]triethyloxonium fluoroborate is described above. Among the reagents listed in Table II, the inhibitors for ribonuclease T1, elastase, and carboxypeptidase A were labeled with ~4C. A method used in the study of triose phosphate isomerase is of special interest. In this case, the label was introduced after the inhibitor had been reacted with the enzyme by reducing the derivative with sodium borohydride containing tritium label. The carbonyl group of the inhibitor moiety was thereby reduced to a secondary alcohol group. The result was the simultaneous incorporation of a tritium atom and the stabilization of the ester linkage. Since a ketonic function is u K. T. Fry, O.-K. Kim, J. Spona, and G. A. Hamilton, Biochemistry 9, 4625 (1970). 14B. F. Erlanger, S. M. Vratsanos, N. Wassermann, and A. G. Cooper, Biochem. Biophys. Res. Commun. 23, 243 (1966). ~B. F. Erlanger, S. M. Vratsanos, N. Wassermann, and A. G. Cooper, Biochem. Biophys. Res. Commun. 9.8, 203 (1967).
610
MODIFICATION REACTIONS
[55]
present in many of the compounds that have been used for the esterification of proteins, this method may have wide applicability. Another method for measuring the extent of incorporation of reagent has depended upon the synthesis of a reagent that contains an unnatural amino acid residue, namely norleucine. ~ Conventional amino acid analysis will give an accurate determination of the number of reagent molecules added to the enzyme. In some cases, advantage has been taken of some other type of label in the reagent. For example, bromine has been determined by elemental analysis ~5 or a chromophoric group has been determined by spectrometry. 1~ Characteristics and Estimation of Ester Groups
Analysis ]or Ethanol or Glycolic Acid alter Hydrolysis Lysozyme esterified with triethyloxonium fluoroborate has been analyzed for ethoxyl content by mild basic hydrolysis in sealed ampoules followed by gas-liquid partition chromatography using 10% Carbowax 20M on a 6-foot, 60-80-mesh column at 90o. 8 Esterification by haloacetate, as well as by diazoacetic acid derivatives, may be estimated after base hydrolysis by the method described in Volume 11 [74], p. 615.1~
Conditions ]or Basic Hydrolysis Typical esters are characteristically hydrolyzed by dilute sodium hydroxide, and lability to dilute base is indicative of an ester bond. Saponification of esterified lysozyme in a pH-stat at pH 10 has even been used as a quantitative method2 However, the stability of ester groups in protein derivatives has been shown to vary widely. At one extreme, it has been observed that the labile ethoxy group that is introduced into lysozyme at pH 4.0 by triethyloxonium fluoroborate is removed by incubation at pH 7.2 and room temperature for 48 hours. 8 A study of the rate of hydrolysis at pH 8.0 of the ester bond in pepsin inhibited with 1diazo-4-phenyl-2-butanone gave a half-life of 5.5 hours at 30 °, comparable to half-life of hydrolysis of the model compound, 1-acetoxy-4-phenyl2-butanone, under the same conditions. 18 At the other extreme of behavior, it was observed that incubation at pH 10 did not regenerate the activity of lysozyme esterified at Asp-52, whereas incubation at pH 2 resulted in reactivation of this derivative to the extent of 60%. 8 I'V. M. Stepanov, L. S. Labareva, N. I. Mol'tsev, Biochim. Biophys. Acta 151, 719 (1968). 1~K. Takahaski, W. H. Stein, and S. Moore, J. Biol. Chem. 242, 4682 (1967).
[55]
ESTERIFICATION
611
Reaction with Hydroxylamine and Estimation o] Hydroxamate Ester groups in enzyme derivatives have been determined by conversion to hydroxamic acid groups using 1 M hydroxylamine at pH values between 7.0 and 9.0, followed by estimation of the hydroxamate by the well-known colorimetric method that makes use of the ferric complex (e.g., see Vol. 3, 323). A positive color test for hydroxamate using N-l-naphthylethylenediamine in the Segal test is is also indicative. However, care must be taken because it is not possible to devise a control that will make certain that reaction of hydroxylamine with particular ester groups in a protein derivative or peptide fragment is quantitative. The most sensitive method of estimating hydroxamic acid groups is the one devised by Yasphe et al. 19 and effectively applied to the determination of ester groups in esterified lysozyme2
Reagents Sodium acetate, 5% Sulfanilic acid reagent (dissolve 10 g of sulfanilic acid in 1 liter of 30% acetic acid) Iodine reagent (dissolve 1.3 g I2 in 100 ml of glacial acetic acid) Sodium thiosulfate, 2.5% a-Naphthylamine reagent (dissolve 3 g of a-naphthylamine in 1 liter of 30% acetic acid) Hydrochloric acid, 3 N
Procedure. To 1.0 ml of sample containing from 0.05 to 0.1 t~eq of hydroxama*e, add 5 ml of 5% sodium acetate, 1 ml of sulfanilic acid reagent, and 0.5 ml of iodine reagent. Shake the mixture and allow it to stand for 5 minutes. Add 0.5 ml of 2.5% thiosulfate in order to remove excess I2, add 0.5 ml of 3 N HC1, and finally, add 1 ml of naphthylamine reagent. Make up the volume to 10 ml and determine the optical density at 530 nm after 90 minutes. Construct a calibration curve using a standard solution of acetylhydroxamic acid. The optical density for 0.1 ~mole is about 0.35. Estimation o] Esterified Residues by Di]]erence, Using the Lossen Rearrangement The conversion of ester groups in protein derivatives to hydroxamic acid groups, as indicated above, allows the identification of the residues 18F. Bergmann and R. Segal, Biochem. J. 62, 542 (1956). 1~j. Yasphe, Y. S. Halpern, and N. Grossowicz, Anal. Chem. 32, 518 (1960).
612
MODIFICATION REACTIONS
[SSl
as aspartic or glutamic esters by means of the Lossen rearrangement.2°-23 The hydroxamic acid groups are reacted with 2,4-dinitrofluorobenzene, the rearrangement is brought about by treatment with 0.1 N NaOH, the modified protein is hydrolyzed with 6 N HC1, and a sample is analyzed on the automatic amino acid analyzer. Those residues of aspartic acid which had been esterified in the protein derivative appear in the analysis as 2,3-diaminopropionic acid, and those residues of esterified glutamic acid appear as 2,4-diaminobutyric acid. Details of the procedure may be found in Volume 11 [74], pp. 610-611.
Estimation o] Esterified Residues by Di]]erence, U~ing the CDI Method ]or Free Carboxyl Groups A method for determination of the total content of free carboxyl groups in a protein has been developed by Hoare and Koshland 24 (see this volume [56]). The carboxyl groups are activated in the presence of an excess of glycine methyl ester by the use of a water-soluble carbodiimide in a denaturing solvent, such as 8 M urea or 6 M guanidinium chloride. After recovery of the modified protein, the incorporation of glycine may be determined by amino acid analysis. When this method is applied to a sample of esterified protein, the decrease in glycine incorporation compared to the amount obtained with native protein will give an estimate of the total number of esterified residues, s,9
Total Enzymatic Hydrolysis and Estimation o] Aspartic or Glutamic Esters Methods are now known by which esterified aspartic or glutamic acid residues may be liberated from certain modified proteins and identified by conventional chromatographic analysis.17,~5 It appears that several proteases attack the bonds on either side of the esterified residues at least as readily as the bonds to aspartic and glutamic acid residues themselves. For purposes of comparison, samples of ethyl or methyl esters of the acidic amino acids may be obtained commercially. However, two useful laboratory methods are given below, along with the procedure for the synthesis of the ~/-carboxymethyl ester of glutamic acid. A sample of ribonuclease T1 that had been inhibited by esterification with [14C]iodoacetate was fragmented by treatment with subtilisin BPN' O. O. Blumenfeld and P. M. Gallop, Biochemistry I, 947 (1962). 21E. Gross, and J. L. Morell, Y. Biol. Chem. 241, 3638 (1966). ~2L. Visser, D. S. Sigman, and E. R. Blout, Biochemistry 1O, 735 (1971). ~SF. C. Hartman, J. Amer. Chem. Soc. 92, 2170 (1970). ~D. G. Hoare and D. E. Koshland, Jr., J. Biol. Chem. 242, 2447 (1967). = S. M. Parsons and M. A. Raftery, Biochemistry 8, 4199 (1969).
[55]
ESTERIFICATION
613
(Nagarse) for 3 hours at pH 8 and 37 °. A radioactive peptide was isolated by paper chromatography. The peptide consisted of the sequence of residues 57-62, Try-Glu-Trp-Pro-Ile-Leu. A sample of the peptide was hydrolyzed with aminopeptidase M for 24 hours at pH 7 and 37 °. Analysis of the hydrolyzate on the automatic amino acid analyzer (0.9 X 60-cm column) showed the presence of tyrosine, isoleucine, leucine, small amounts of tryptophan and proline, and a new ninhydrin-positive peak which emerged at 4 3 _ 3 ml, just ahead of the position for S-carboxymethylcysteine. The new component was identical in its properties with a synthetic sample of the ~,-carboxymethyl ester of glutamic acid. The stable, monoethyl ester of lyozyme inhibited by Meerwein's reagent has been hydrolyzed with subtilisin (Carlsberg) for 4 hours at pH 7 and 25 °, followed by aminopeptidase M for 20 hours at pH 7.0 and 25 °. An aliquot of the hydrolyzate was subiected to amino acid analysis using 0.3M lithium citrate buffer, pH 2.80 (Spinco Technical Bulletin A-TB-044), at 25 ° in a Beckman-Spinco Model 120B analyzer. Compared with the analysis of native lysozyme, the derivative gave one less aspartic acid residue and a new peak appeared in the chromatogram between the positions of glutamic acid (plus glutamine) and proline. The position of this peak corresponded to that found for an authentic sample of fl-ethyl aspartate. This compound is completely resolved from peaks of the common amino acids by using the lithium buffer system. A chymotryptic peptide was isolated from a sample of the monoethyl ester of lysozyme. This peptide corresponded to fragment C-15 obtained by the action of chymotrypsin on native enzyme and was composed of residues 45-53, Arg-Asn-Thr-Asp-Gly-Ser-Thr-Asp-Tyr. Treatment of the esterified peptide with carboxypeptidase A resulted in the rapid liberation of the COOH-terminal tyrosine residue, followed by a somewhat slower liberation of fl-ethyl aspartate from the penultimate position. This result is in contrast to the slow liberation of the penultimate aspartic acid residue from the peptide obtained from native protein. The rate of formation of free amino acids was determined by amino acid analysis with the automatic analyzer. Synthesis o] the ~,-Carboxymethyl Ester o] Glutamic Acid. 17 Suspend 1.0 g of L-glutamic acid and 20 g of sodium glycolate in 60 ml of dioxane in a round-bottom flask. Chill the mixture in an ice bath and add 10 ml of thionyl chloride. Stopper the flask and allow it to stand for 5 hours at 25 °. An additional 5 ml of thionyl chloride are then added, followed 6 hours later by 25 ml of thionyl chloride and 5 g of sodium glycolate. After the mixture has stood overnight, the dioxane is removed in a rotary evaporator and about 100 ml of ethyl ether is added. The precipitate is collected and washed with ether. About one-sixth of the product
614
MODIFICATION REACTIONS
[55]
is purified on a column (4 }( 19 era) of Dowex 50-X2 (200-400 mesh) using 0.1 M pyridinium acetate buffer, pH 3.2, as eluent at a flow rate of 20 ml per hour. The elution of the product is followed by analyses for amino acid with ninhydrin and for glycolic acid with chromotropic acid reagent (see Vol. 11 [74] p. 615). The peak fractions which give positive tests for both amino acid and glycolic acid are pooled and evaporated to dryness in a rotary evaporator at 40 °. The residue is transferred to a test tube with 1 ml of water, and about 5 volumes of acetone is added. The precipitate is collected, and the material is crystallized from a small amount of water and acetone at 0 °. The product is collected by centrifugation, washed with a little acetone and ether, and stored in a vacuum desiccator over sulfuric acid. Synthesis of fl-Ethyl Aspartate Hydrochloride3 ~ Reflux a mixture of 5.0 g of DL-aspartic acid hydrochloride and 1.1 g of anhydrous HC1 in 15 ml of absolute ethanol for 15 minutes. Add dry ether to the warm solution until a slight turbidity develops, and allow the product to crystallize at room temperature. The crystals are collected on a filter, washed with dry ether, and stored in a vacuum desiccator. Yield, 2.8 g, mp 174-177 °. Synthesis of ~,-Ethyl L-Glutamate. 2~ This compound may be readily synthesized by esterification of poly-L-glutamic acid with triethyloxonium fluoroborate according to the procedure described above for the esterification of lysozyme. The polymer is then hydrolyzed with subtilisin and aminopeptidase M to yield a solution containing essentially only L-glntamic acid and v-ethyl L-glutamate in a ratio of 1:3. An aliquot of this solution may be used as a convenient reference for the amino acid analysis of enzymatic hydrolyzates of modified protein.
Stabigty of Ester Linkages during Fragmentation and Isolation o] Peptides Because the stability of the ester linkage in different enzyme derivatives has been shown to vary considerably depending upon the nature of the enzyme and the type of esterifying reagent, no general procedures for the fragmentation of these derivatives can be given. Nevertheless, a summary of successful methods may be useful' to other efforts directed toward the location of the site of esterification in an enzyme derivative. The ester linkage in pepsin inhibited with diazo compounds appears to be labile to incubation at pH values above 72 ,13 The linkage becomes even more labile upon denaturation of the protein. Therefore, the derivative has been denatured either with acetone at low pH or by treatment at pH values around 7 for only 2 hours at 25 °. The denatured protein can then be fragmented by digestion with native pepsin at pH values
[55 ]
ESTERIFICATION
615
near 3 with no cleavage of ester bonds. Esterified peptides have been isolated by gel filtration, paper electrophoresis, and paper chromatography, using acidic buffers, several containing pyridine and acetic acid. The lability of the ester bond was put to some use by adapting the cleavage to the method of "diagonal electrophoresis." The mobility of the esterified peptide was changed between two electrophoretic runs by treating the paper with an aqueous solution saturated with triethylamine2 The ester linkage in several other enzyme derivatives is more normal in stability. The linkage in lysozyme ethylated at Asp-52 is stable to the conditions used for oxidation of cystine by performic acid at --10% The oxidized derivative is stable to incubation for 9 hours at pH 7.2 and 25 °. Therefore, enzymatic digestion with trypsin, chymotrypsin, and carboxypeptidase k could be usefully applied? ~ The ester bond at Glu-6 in elastase reacted with a bromoketone is stable to cyanogen bromide cleavage in 70% formic acid, followed by chromatography on SE-Sephadex using a pH gradient from 4.5 to about 7.0.22 In the case of ribonuelease T1, the ester bond in y-carboxymethylGlu-58 was found to be cleaved to the extent of 30% during reduction and carboxymethylation of the protein at pH 8.5, using mercaptoethanol and iodoacetate in 8 M urea over a period of 20.5 hours. The ester bond was stable during digestion with substilisin for 3 hours at pH 8.0.17 Addendum
2G
In a study of the esterification of bovine insulin by treatment with anhydrous methanol-HC1 (0.1M), a specifie N-to-O aeyl shift was found to occur at the bond between Tyr-B26 and Thr-B27. The equilibrium mixture at 25 ° contained about 60% of the protein with an aeyl ester linkage to the oxygen of the threonine residue. This derivative could be isolated by chromatography in 7 M urea at pH 4.75. Deamination with nitrous acid and treatment with 0.1 M sodium carbonate resulted in a specific cleavage of the ester bond between the tyrosine and threonine residues. On the other hand, treatment of the derivative in aqueous solution at pH values above 2.2 resulted in an irreversible O-to-N acyl shift, giving a derivative of the native structure with all six carboxyl groups esterified.
D. Levy and F. g . Carpenter, Biochemistry 9, 3215 (1970).