A spectrophotometric method for quantitation of carboxyl group modification of proteins using Woodward's Reagent K

ANALYTICAL

BIOCHEMISTRY

151,327-333

(1985)

A Spectrophotometric Method for Quantitation of Carboxyl Group Modification of Proteins Using Woodward’s Reagent K UMA

SINHA AND JOHN M. BREWER

Department of Biochemistry, University of Georgia, Athens, Georgia 30602 Received June 20, 1985 Reaction of proteins with Woodward’s Reagent K in 0.05 ionic strength Tris-HCl, pH 7.8, followed by removal of excessreagent by chromatography on Sephadex G-25 in the same buffer, results in covalently attached chromophores with an absorption maximum at 340 nm and an extinction coefficient of 7000 M-’ cm-‘. This absorbance can be used to quantitate the reaction of Woodward’s Reagent K with carboxyl groups in proteins, provided sulthydryl groups do not react. The chromophore also enables specific detection and identification of carboxyl-modified peptides upon separation by chromatography or electrophoresis. o 1985 Academic RSS, I~C. JSEY WORDS: Reagent K, carboxyl; spectrophotometric; protein; modification; quantitation.

The qualities an ideal protein modifying reagent should have include ability to react under physiological conditions, specificity of reaction, and ease of quantitation. The most useful method of quantitation involves production of a chromophore with a convenient absorption maximum upon reaction of the reagent with the protein. A number of chromophoric reagents have been developed for reaction with amino, imidazole, or sullhydryl groups in proteins (1). No such reagent is known for carboxyl groups. Current methods of carboxyl group modification involve use of carbodiimides or Woodwards Reagent K and quantitation by incorporation of radiolabel or analysis for amino acid or ethylamine incorporation (2,3). Figure 1 shows the structure of Woodward’s Reagent K and its possible reactions, as described by Petra (4). The reagent was developed for activation of carboxyl groups (5) but can be used as a crosslinking reagent, since amino groups can apparently react subsequently with the enol ester (6) (see below). Hydrolysis of the enol ester yields ethylamine, which can be measured using an amino acid analyzer. The color constant for ethylamine depends on the analyzer program used but is often lower than

those for amino acids (4,7), limiting sensitivity. Also acid hydrolysis and amino acid analysis is time consuming and expensive. We observed that reaction of yeast enolase with Woodward’s Reagent K was accompanied by an increase in the near-ultraviolet absorbance, an increase which persisted after dialysis. Clearly, the reaction involved production of a covalently attached chromophore. We decided to see if this phenomenon could be exploited to facilitate quantitation of carboxy1 modification using this reagent. MATERIALS

AND METHODS

Ultrapure urea was purchased from BectonDickinson Immunodiagnostics. Trizma base (Sigma) was twice recrystallized from ethanol. 2-Phospho-Dglycerate, Reagent K, iodoacetamide, porcine pancreatic trypsin, bovine serum albumin (Fraction V), ovalbumin (grade V), and lysozyme (grade I) were also obtained from Sigma. All other reagents used were either from Sigma or Fisher. The water used was filtered and deionized (Continental Deionized Water Corp.). Yeast enolase was prepared, assayed, deionized, and concentrated to millimolar levels for storage purposes as described before (8). Enolase and 327

0003-2697185 $3.00 Copyright 8 1985 by Academic Press. Inc. All rights of reproduction in any form x?.mwd

328

SINHA AND BREWER

Woodward’s Reagent

K

Ketoketenimine Amide

4 & RxNH’ ‘4 + products

biP,M;:y;;f 3 pro+duct

2

3 s

FIG. 1. Structure of Woodward’s Reagent K and scheme showing possible reactions. The scheme is based on a figure in the article by Petra (4). The Reagent K is believed converted to the ketoketenimine at neutral pH which then either breaks down to the unreactive amide or reacts with organic carboxylates (Q-COO-) to form the enol ester. This is thought to be the chromophore described in the text.

other protein concentrations were determined spectrophotometrically using published extinction values (9). Concentrations of modified proteins were routinely determined using the dye-binding method of Bradford (10). The reagent was filtered once through Whatman No. 1 filter paper prior to use. Unmodified proteins (enolase, BSA, ovalbumin, lysozyme) were used to set up the respective standard curves, the concentration of each being determined from its absorbance at 280 nm. For dilute solutions of modified ovalbumin, a modified assay was used (11). For the modification of protein carboxyl groups by Reagent K, varying amounts of protein were dissolved in 0.05 ionic strength Tris-HCl , pH 7.8 (“standard Tris buffer”), and desired amounts of Woodward’s Reagent K (stock 100 to 200 tIIM aqueous solutions) added and thoroughly mixed. The reaction was quenched by dialysis or desalting on Sephadex G-25.

Amino acid analyses of protein samples were carried out by the usual method (12). Modified proteins/peptides ( 100-2000 pg), dialyzed versus distilled water and lyophilized, were hydrolyzed in 6 N HCl in sealed, evacuated Pyrex tubes for 24 h at 110°C. Ethylamine hydrochloride (Aldrich) as well as ethylamine (J. T. Baker) stock solutions in 0.1 M HCl were used for preparing appropriate standards for amino acid analysis. Hydrolysis in unevacuated polypropylene centrifuge tubes did not give satisfactory results. The number of cysteine residues in a protein were determined by reacting denatured proteins with Ellman’s reagent, DTNB,’ which is specific for free -SH groups in a protein (13). SCarboxamidomethylation of proteins (to block -SH groups) was carried out as described (1). The absence of any reactive -SH groups in the reduced and carboxamidomethylated protein was confirmed by the DTNB reaction. SDS-PAGE of extensively modified proteins was carried out by a modification of the original procedure ( 14) to check for crosslinking. A 12.5% separating gel was used. Absorbance measurements were carried out using a Bausch & Lomb Spectronic 200 spectrophotometer equipped with a digital readout. Measurements of pH were made with a Model 701A Orion Research Digital Ionalyzer. Amino acid and ethylamine analyses were carried out on a Beckman 119CL analyzer. RESULTS

Specificity of Woodwards Reagent K. Woodward’s original report on the use of isoxazolium salts for peptide synthesis used N-protected amino acids and peptides (15). When free amino acids were reacted with Reagent K, in standard Tris, only cysteine yielded a reaction product absorbing in the near ultraviolet, maximally at 330 nm. Since this indicated a reactivity with -SH groups, 2-mer’ Abbreviations used: DTNB, 5,5’-dithiobis(2-nitrobenzoic acid); SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

SPECTROPHOTOMETRIC

CARBOXYL

captoethanol was reacted with substoichiometric amounts of Reagent K, added in aliquots. The resulting linearity in the increase in absorbance was used to estimate an extinction coefficient of 20,000 M-’ cm-‘, with a maximum at 348 nm. The pH was checked both before and after Reagent K addition to eliminate any possibility of shift in the absorption maximum owing to a change in pH. Concentrations of l- 10 InM N-acetylglytine, N-carboxyphenylalanine, N-acetylalanine, and oxidized glutathione were all treated with 50 PM Reagent K in standard Tris. Each of the resulting products had an absorption centered at 340 nm. This clearly indicated that the enol ester resulting from the reaction of the carboxyl groups with Reagent K had a specific absorption centered at 340 nm. The reaction with the model amino acids/peptides is slower than that with proteins but is over in about 3 min. Clearly, however, Reagent K is not specific for carboxyl groups. In this connection it is significant that neither of the two proteins extensively studied using Reagent K, bovine pancreatic trypsin or bovine carboxypeptidase A have any free sulfhydryl groups (4,15). The absorption spectrum of Reagent Ktreated yeast enolase has an absorption maximum at 340 nm (Fig. 2). No reaction of its single sulfhydryl per subunit was detected (it is not reactive unless the protein is denatured) (16). This lends more support to the observation that the maximum for the enol ester is indeed at 340 nm. The absorption maximum did not shift owing to subsequent treatment with either 8 M urea or 6 M guanidine-HCl. The chromophore is nonfluorescent under any conditions examined by us.

MODIFICATION

QUANTITATION

AA

250

300

350

400

A, "n-l FIG. 2. Absorption spectrum of native yeast enolase before and after reaction with Woodward’s Reagent K. Yeast apoenolase (0.2 ml, 50 am) in 0.05 ionic strength TrisHCI, pH 7.8, was mixed with 10 pl of 0.1 M Woodward’s Reagent K in water, and then chromatographed on Sephadex G-25. The spectrum is of the fraction at the void volume. The spectrum was obtained using the Tris buffer as reference (open circles). The line defined by the closed circles is the spectrum of the same concentration of unmodified enzyme in the same buffer.

number of free sulfhydryl groups have to be considered. Yeast enolase was reacted with substoichiometric amounts of Reagent K. Estimation of free sullhydryl groups by the DTNB reaction showed that the number did not change, indicating that the 340-nm absorbance was entirely owing to carboxyl group modification. The extinction coefficient could be determined if all the Reagent K reacted with the protein. Sephadex G-25 chromatography, which was normally employed to remove excess reagent, Extinction coejicient of enol ester chromo- was used to check for Reagent K which had phore. These observations led to the premise not reacted with the protein; the reagent after that it is possible to determine the extent of a mixing with buffer alone absorbs at 3 19 nm given protein’s reaction with Reagent K spec- in standard Tris, and the lack of A319in the trophotometrically, provided it could be dem- included volume assured complete reaction onstrated that the A+,,, correlated with the (Table 1). number of groups modified. In order to deThe extinction coefficient of the modified termine the value of the extinction coefficient, carboxyl side chains in bovine serum albumin the number of carboxyl side chains and the and ovalbumin had to be determined in a

330

SINHA AND BREWER TABLE

1

volved (a) blocking of sullhydryl groups by Scarboxamidomethylation; (b) exhaustive reaction of the protein with repeated additions of Reagent K, carried out under denaturing (A) Substoichiometric addition conditions in 6 M urea; (c) chromatography of Woodward’s Reagent K: on Sephadex G-25 in standard Tris with 6 M Apoenolase Reagent K E340 urea; (d) measuring A340 of the modified pro(M-l cm-‘) mf) (mM) A341 tein; and (e) estimation of protein by the 0.25 0.1 0.685 6850 Bradford method (10). The above procedure 6920 was carried out with bovine serum albumin, 0.26 0.1 0.692 0.28 0.1 0.633 6330 ovalbumin, yeast enolase, and lysozyme. Step 7710 (a) was omitted for lysozyme since it does not 0.28 0.1 0.771 0.30 0.1 0.724 7240 contain any free sulfhydryl groups (4 S-S Mean = 7010 only) ( 17). SD = 509 Reagent K is very acidic in aqueous solution (the pH of the stock 100 IIIM solution is 2.5(B) Exhausive reaction with 3.0). If concentrated Reagent K solution is Woodward’s Reagent K: added to concentrated protein solutions, proE340 tein precipitation due to crosslinking/dena(M-’ Cm-‘) Protein turation occurs. It was desirable to reduce the Bovine serum albumin 7127 possibility of crosslinking, so the exhaustive 7394 reaction had to be carried out by adding a 7200 small volume (0.1-0.2 ml) of the stock reagent Ovalbumin 6534 solution to 5 mg protein dissolved in 20 ml of 7293 buffer plus 6 M urea, concentrated by ultrafil5938 tration in an Amicon cell (PM- 10 membrane), 6758 Lysozyme and then applied to a G-25 column for de6782 salting. To get exhaustively modified protein, 6861 the sequence of steps were repeated until the 6930 Yeast enolase ratio of absorbance at 340 nm to protein con7280 centration (as measured by the Bradford assay) 6890 was constant. The average fraction of carboxyl Mean = 6916 groups modified as estimated from the ethylSD = 401 amine contents was 59% of theoretical for ovalbumin, 76% for lysozyme, 112% for enoNote. The experiments in (A) involved addition of 50 lase, and 117% for bovine serum albumin, with pl of 2 mM Reagent K to 1.0 ml of yeast apoenolase, an average standard deviation of 4.3%. mixing, and chromatography on Sephadex G-25, all in standard Tris. Each determination in (B) involves a sepExhaustively modified protein samples were arate preparation of modified protein. dialyzed against several changes of water for 24 h and lyophilized, and acid hydrolysis was more indirect manner. The reason for this was carried out. Ethylamine could be more prethe concurrent modification of the free cisely quantitated on the amino acid analyzer sulfhydryl residues in these proteins. The dif- using exhaustively modified proteins because of the relatively large number of carboxyl ficulty of quantitating ethylamine by amino acid analysis given the very low color constant groups modified. The lysine and arginine for ethylamine in the system employed here peaks were used to estimate the number of (one-fortieth of that of arginine) necessitated nanomoles of protein actually transferred. (We exhaustive modification of the carboxyl groups could not be certain how the carboxyl group in these proteins. The general procedure in- modification affected the Bradford assay.) EXTINCTION COEFFICIENT DETERMINATION PROTEIN-REAGENT K CHROMOPHORE

OF

SPECTROPHOTOMETRIC

CARBOXYL

MODIFICATION

QLJANTITATION

331

6.q .’ / 1 From the known amino acid composition and the 340-nm absorbances of the samples, the extinction coefficient of the reaction products of Woodward’s Reagent K with carboxyl groups was calculated (Table 1). As an example, in a bovine serum albumin sample, 664 nmol of ethylamine was observed on the analyzer, along with 135 nmol of arginine. Since bovine serum albumin has 23 arginines per molecule ( 17), 5.9 nmol protein EtNl-tZ (nmoles) was actually hydrolyzed. Ethylamine liberation was 113 nmol of ethylamine/nmol of FIG. 3. Correlation between absorbance at 340 nm of protein. As40for 1 mg/ml of the modified pro- various samples of bovine serum albumin modified to tein was 12.7. which led to an extinction coef- various extents with Woodward’s Reagent K and ethylamine liberated during acid hydrolysis. The ordinate gives ficient of 7394 M-’ cm-’ for modified carboxyl total absorbance (absorbance at 340 rim/ml X ml) of vargroups. The predicted value from the amino ious samples of S-carboxamidomethylated bovine serum acid composition (assuming no deamidation albumin which had been reacted to various extents in 0.05 ionic strength Tris-HCl, pH 7.8, and 6 M urea using had occurred), was 99 nmol/nmol protein ( 17). We have independent evidence that some Woodward’s Reagent K. The absorbances were measured in that solvent. and then known volumes of known abdeamidation occurs in at least one protein sorbance at 340 nm were dialyzed against several changes (enolase), so the higher values of ethylamine of water and lyophilized and hydrolyzed. Abscissa, number are probably influenced by deamidation. of nanomoles of ethylamine obtained after acid hydrolysis. In order to verify that the peak observed in Dashed line, dependence expected if the extinction coefacid hydrolyzates was actually due to the eth- ficient of the chromophore at 340 nm is 7000 Mm’ cm-‘. ylamine liberated, 100 nmol of ethylamine was Solid line, observed dependence. added to aliquots of acid-hydrolyzed protein samples before applying them to the anlayzer. This internal standard increased the peak area Woodward’s Reagent K may reduce the color due to ethylamine by the predicted amount. developed with Bradford’s Reagent, thus inAlso, several separately modified samples of creasing the apparent stoichiometry of reacbovine serum albumin were desalted and pre- tion. While the reduction is small with the pared for acid hydrolysis as described before. proteins examined, it can become significant The number of nanomoles of ethylamine lib- in the case of extensive modification. Also, the erated is then expected to be proportional to reduction varies with the protein and is not the observed A&milligram of protein. This necessarily linear with extent of modification was indeed found to be the case (Fig. 3). The (some data not shown). We can determine the scatter in the data is essentially all due to ex- magnitude of this effect by comparing the raperimental error in the ethylamine determitio, A34,,/milligram of protein, as determined nation. The slope of the line suggests an ex- using the Bradford assay and obtained using tinction coefficient in good agreement with amino acid analysis. For the exhaustively other estimates, considering the possibility of modified proteins, it was found that modifilosses of protein from transfers. The data cation reduces the enolase A&milligram of overall indicate that Woodward’s Reagent K protein by 19%, serum albumin by 7%, lysoreacts with carboxyl groups in proteins, pro- zyme by 6%, and ovalbumin (using the modducing a chromophore with an extinction ified Bradford assay) (11) by 4%. In general, coefficient of 7000 +/- 500 M-t cm-’ under the effect of modification by Woodward’s Rethese conditions. agent K on reaction with Bradford’s Reagent Efect of modiJication on apparent protein can be measured by preparing a set of samples concentration. Modification of proteins with of identical protein concentration but varying

332

SINHA

AND

Woodward’s Reagent K concentrations, removing identical aliquots, and measuring As95 with Bradford’s Reagent. Reagent K added to buffer only serves as a control. The extent of the modification produced by those Woodward’s Reagent K concentrations is obtained separately after chromatography. Crosslinking ofproteins by Reagent K. Since contradictory results have been reported by different groups investigating the crosslinking of proteins by Reagent K (6,15), this possible drawback of the procedure as a chemical modifier of protein carboxyl groups was looked into. Exhaustively modified bovine serum albumin, ovalbumin, and yeast enolase were electrophoresed on SDS-PAGE, along with molecular weight standards. All three modified proteins migrated with mobilities corresponding to their respective molecular weights, with only trace amounts or indetectable levels of intermolecular crosslinked proteins being observed on onverloaded gels (not shown). This seems to indicate that crosslinking of proteins is of minor consequence under these conditions, being detectable (if at all) only when exhaustively modified samples are analyzed. Efect of Tris bufers: Stability of chromophore. The enol esters produced by the reaction of Reagent K with carboxyl side chains are capable of further reaction with suitable nucleophiles (5) (Fig. 1). All the modifications described in this study were routinely carried out in standard Tris (0.05 ionic strength TrisHCl, pH 7.8, which is 0.075 M in Tris), so effects of this buffer on the stability of the chromophore were examined. Modification of enolase by Reagent K was carried out in a series of Tris-HCl buffers, pH 7.8, with the Tris concentration varying between 0.2-0.5 M, and excess reagent rapidly removed by gel filtration. Spectra of protein samples were then recorded at various time intervals. The loss of AJ~ was faster at intermediate Tris concentrations-0.3 and 0.4 M-than at either 0.2 or 0.5 M Tris (not shown). However, the reactions are relatively slow, 20-30% maximal loss extending over 7 days. Under the conditions we employ for quantitation of the reac-

BREWER

tion, the chromophore must be considered sufficiently stable. We have found in fact that modified yeast enolase can be digested with trypsin and the peptides separated using a HPLC system at pH 3. By monitoring the effluent at 340 nm some of the modified peptides can be identified and collected for repurification and analysis (to be published). DISCUSSION

The apparent absence of reaction with free amino acids could be because the enol esters formed with the carboxyl groups are excellent acylating agents and therefore very susceptible to nucleophilic attack (Fig. 1). Free amino groups could react to form an amide with the enol ester, effectively reversing the reaction. Woodward et al. (5) used N-blocked amino acids. Woodward’s Reagent K-modified carboxypeptidase A in water showed a new broad absorption peak with a maximum at 340 nm (4) but this was never examined further. The group absorbing at 340 nm is probably the enol ester. Its absorption maximum is apparently solvent dependent. For a-acetoxy ZVmethylcinnamide in methylene chloride, the absorption maximum is at 267 nm (18). Bodlaender et al. ( 15) and Petra (4) used the 250: 280~nm ratio as a qualitative measure of chemical modification (Fig. 2). The sensitivity of the method described herein is greater than use of amino acid analysis. A l-ml sample of As400.070 contains 10 nmol of chromophore. This can be measured with a precision of 3% or less depending on the spectrophotometer. At the 12.5~nmol level of amino acids, the precision claimed for the 90-min single-column analysis ( 118- 119 CLAN-001) is 10%. Even at equal sensitivity of detection, the spectrophotometric method is at least three times as sensitive. With a color constant for ethylamine that is one-fortieth of that for arginine, the spectrophotometric method is at least 120-fold more sensitive. All procedures based on ethylamine measurement have used modified analyzer pro-

SPECTROPHOTOMETRIC

CARBOXYL

grams to obtain better separation of the ethylamine peak. Saini and Van Etten (7) reported equal color constants for ethylamine and arginine, while Petra (4) reported an ethylamine color constant that was one-third that of arginine. In all cases using protein hydrolyzates (4,7), a minimum of 0.3 to 3 mg of protein had to be hydrolyzed. The spectral method is capable of precise quantitation using nanomoles of protein. This is advantageous when working with small quantities of proteins. Also, the nondestructiveness and rapidity of the method further enhance its utility over the method based on use of the analyzer. ACKNOWLEDGMENTS The authors thank Dr. James Travis for the generous use of his amino acid analyzer and Dr. Harry D. Peck, Jr. for support.

REFERENCES I. Means, G. E., and Feeney, R. E. (1971) Chemical Modification of Proteins, Holden-Day, San Francisco. 2. Arana, J. L., and Vallejos, R. H. (198 I) FEBS Lett. 123, 103-106.

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3. Dinur, D., Kantrowitz, E. R., and Hajdu, J. (1981) B&hem. Biophys. Rex Commun. 100,785-792. 4. Petra, P. H. ( 197 1) Biochemistry 10,3 163-3 170. 5. Woodward, R. B., Olofson, R. A., and Mayer, H. (1961) J. Amer. Chem. Sot. 83, 1010-1012. 6. Patel, R. P., and Price, S. (1967) Biopolymers 5,583585. 7. Saini, M. S., and Van Etten, R. L. (1979) Biochim. Biophys. Acta S68,370-376. 8. Brewer, J. M., Carreira, L. A., Collins, K. M., Duvall, M. C., Cohen, C., and DerVartanian, D. V. (1983) J. Inorg. B&hem. 19,255-261. 9. Brewer, J. M., Pesce,A. J., and Ashworth, R. B. (1974) Experimental Techniques in Biochemistry, Prentice-Hall, Englewood Cliffs, N. J. 10. Bradford, M. M. (1976) Anal. B&hem. 72,248-254. 11. Read, S. M., and Northcote, D. H. (1981) Anal. Biochem. 116, 53-64. 12. Klungsoyr, M., Sirny, R. J., and Elvejehm, C. A. (1951) J. Biol. Chem. 189, 557-569. 13. Torchinsky, Y. M. (1981) Sulfur in Proteins, Pergamon, New York. 14. Laemmli, U. K. (1970) Nature (London) 227, 680685. 15. Bodlaender, P., Feinstein, G., and Shaw, E. (1969) Biochemistry 8,4941-4948. 16. Oh, S. K., Travis, J., and Brewer, J. M. (1973) Biochim. Biophys. Acta 310,42 l-429. 17. Dayhoff, M. 0. (I 972) Atlas of Protein Sequence and Structure, Vol. 5, Natl. Biomed. Res. Found., Washington, D. C. 18. Woodward, R. B., and Olofson, R. A. (1966) Tetrahedron Suppl. 7,4 15-440.