Determination of mercapto groups in proteins with N-ethylmaleimide

.\XALYTICAL

BIOCHEMISTRY

Determination

JAMES

LESLIE,

From

3, 257-263

(1962)

of Mercapto Groups with N-Ethylmaleimide’ DAVID

the Department

L. WILLIAMS,

in Proteins

AND

oj Chemistry, Oklahoma Stdhater, Okkhorna Received

June

GEORGE

GORIN

State Ur&wsity,

21, 1961

INTRODUCTION

iv-Ethylmaleimide (NEM) has been used in a simple, rapid spectrophotometric determination of cysteine and glutathione (l-3). The reagent has also been used to determine the mercapto groups in some proteins, but its applicability to this purpose has not, been clearly established. Alexander did not find t.he reagent suitable for ovalbumin (3), while the results obtained by Habeeb (4) and by Stark et al. (5) for /3-lactoglobulin were not very precise ; on the other hand, reasonable results were obtained with bovine serum albumin (2) and hemoglobin (6), but the performance of the reagent was not examined in detail. This paper reports the results of a more extensive investigation upon the reaction of NEM with ovalbumin, P-lactoglobulin, and bovine serum albumin. It will be seen that NEM gives acceptable results under proper conditions, provided the proteins are appropriately denatured, and that it may t.herefore be a suitable reagent for the determination of mercapto groups in proteins. METHODS

Materials

The following reagents were obtained from the sources indicated and used without further purification: ovalbumin, Worthington Biochemical Corp., Freehold, N. J.; p-lactoglobulin and bovine serum albumin, Pentex, Inc., Kankakee, Illinois; NEM, Delta Chemical Works, Inc., New York, N. Y.; sodium dodecyl sulfate, Fisher Scientific Co., Fairlawn, N. J. Urea, initially of C.P. grade from Fisher Scientific Co., was purified by treating a saturated solution of it with Amberlite MB-l resin, 1 This work was Health, Department

supported of Health,

by Grant Education,

A-2735 from and Welfare. 257

the

National

Institutes

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filtering, allowing the urea to crystallize, and drying at 50” (7). Guanidine hydrochloride, initially of (‘Aero” (technical) grade from the American Cyanamid Co., Bound Brook, N. J., was purified by dissolving in the minimum amount of hot water, treating the extract with sodium hydroxide, and boiling it to expel aI1 the ammonia, filtering, and cooling to 15” (8). The material which separated was recrystallized five or six times from methanol and had a negligible absorbance at 300 rnp in 6 M solution. All other chemicals were of analytical reagent grade. Procedure

The procedure was essentially that prescribed by Alexander (3). The protein sample was dissolved in 0.1 M phosphate buffer, pH 7.0. A stock solution of NEM was prepared by dissolving approximately 0.120 gm in 100 ml of buffer and filtering to remove undissolved particles; it was discarded after a maximum of 6 hr. Solutions of the denaturants were also prepared in the phosphate buffer, and the pH readjusted to 7.0 with acid or base as necessary. An aliquot portion of the protein solution, 1 ml of NEM solution, and denaturant solution or plain phosphate buffer were mixed to give a total volume of 10 ml. The absorbance of t,he mixture was determined in l-cm cells at 300 rnp with a Beckman DU spectrophotometer. The reference solution was mixed from the same reagents, excepting the NEM solution. A control solution was also mixed from the reagents, excepting the protein solution, and its absorbance was measured from time to time to provide the correction needed for the spontaneous hydrolysis of NEM. rl‘he experiments were carried out at room temperature, 26 f 2”. Calculations

Protein concentrations were determined from the absorbance at 280 mp. The following specific absorbancies, E i?m, and molecular weights were used: ovalbumin, 7.35, 45,060 (9) ; p-lactoglobulin, 9.5, 35,006 (10) ; bovine serum albumin, 6.6, 67,000 (11). The molar absorbancy of NEM at 300 mp was taken to be 620 (3) ; the analysis of a cysteine sample calculated from this value agreed with that determined with ferricyanide (12). RESULTS

NEM has an absorption maximum at 300 rnp, which disappears on reaction with mercapto groups. The rate and extent of reaction were determined from measurements of the change in absorbance at this wavelength. NEM was hydrolyzed at an appreciable rate in the phosphate buffer used as reaction medium, but t,he extent of this reaction was small

DETERMINATION

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in the period of time required for the quantitative mereapto group determinations. Suitable controls provided the appropriate corrections for this hydrolytic reaction and for the absorption of the protein; it has been shown that the products of hydrolysis do not absorb appreciably at. 300 mp (3). The rate of reaction of NEM with ovalbumin in various conditions is represented by Figs. 1 and 2. In the absence of denaturing agents, the

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0

/o

20

JO T/Ad&

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(MNUTCS)

FIG. 1. Reaction of 1.6 x lo-” M ovalbumin with approximately 9.6 x 10e4M NEM: ( x ) native ovalbumin, (0) NEM added before 8 M urea, (0) in 8 M urea for 80 min before addition of NEM, (A) NEM added before 5.8 M guanidine hydrochloride, (V) in 5.8 M guanidine hydrochloride for 30 min before addition of NEM. pH 7.0, 0.1 M phosphate.

decrease in absorption at 300 rnp occurred very slowly, at about the same rate as with the buffer alone (curve IA). In the presence of 5.8 M guanidine hydrochloride, the reaction took place within 5 min, regardless of whether the denaturant had been added at the same time as the NEM or 30 min before (curves 1D and 1E). The amount reacted was 3.8 moles/mole of protein. Similar results were obtained with 0.48% sodium dodecyl sulfate, although the reaction was somewhat slower (Fig. 2). In a set of twelve determinations done with either of these denaturants on amounts of protein varying between 20 and 100 mg the

260

LESLIE,

WILLIAMS,

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0.4 Y

”

Y

0.5

k

0.c

> 4 3

gj T

0.3

0.2

0.l

FIG. 2. Reaction of 1.6 X IO-‘hf NEM : (X ) native ovalbumin, (0) before addition of NEM, (0) NEM pH 7.0, 0.1 M phosphate.

ovalbumin with approximately 9.6 x 10m4 M in 0.48% sodium dodecyl sulfate for 40 min added before 0.48% sodium dodecyl sulfate.

precision obtained was +2%. The same number of mercapto groups was found in the sample by the ferricyanide method (12). In 8.0 M urea, NEM reacted rapidly with protein which had been exposed to the denaturant for 80 min previously, but the amount of NEM consumed was smaller (curve 1B). When the denaturant was added at the same time as the NEM, reaction was quite slow (curve 1C). NEM did not react with native p-lactoglobulin, but the reaction occurred in a few minutes in the presence of 6 M guanidine hydrochloride or 6 M urea. With sodium dodecyl sulfate, 0~54%~ the reaction was a little slower, but complete in about 20 min. In all three cases, 1.9 moles of NEM was consumed per mole of protein. The same result was obtained by amperometric titration with silver ion according to Benesch et al. (7). Bovine serum albumin differed from the other proteins; it reacted with NEM in the native state. The reaction was complete in a few minutes both in the absence of denaturants and in the presence of 4 M

DETERMINATION

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guanidine hydrochloride or 0.54% sodium dodecyl sulfate. The amount of NEM consumed was 0.43 mole/mole of protein in all cases. The reaction with native protein was also measured at pH 6.0, and found to be somewhat slower; about 15 min was required for complete reaction. The same number of mercapto groups was found by the method of Benesch et cd. (7). DISCUSSION The investigators who developed the NEM method for determining simple mercapto compounds expressed c1oubt.s concerning its applicability to t.he mercapto groups of proteins (l-3). The results reported above indicate that they were unduly pessimistic. The mercapto group content found for the sample of bovine serum albumin employed in this work, 0.43 group/molecule, is lower than the value of 0.67 frequently reported (13). However, recent evidence clearly indicates that samples of this protein may contain several components of differing mercapto group content (14, 15), so that one cannot expect to find a definite value for this quantity; t.he agreement which has been reported may be, to some extent at least, accidental. The result found with NEM agrees with that obtained by amperometric titration with silver nitrate (7), a method t,hat has been found generally satisfactory in its application to this protein (13). The fact that t’he same quantity of NEM was consumed by the native and the denatured protein either at pH 6.0 or at pH 7.0 supports the supposition that the value found corresponds to the total amount present in the sample examined. In the sample of ovalbumin analyzed by Alexander (3), the number of mercapto groups found was 3 by the p-mercuribenzoate derivative method of Boyer (10) and 2.2 by use of NEM. Both these values arc lower than the value of 4 usually reported (12) For the sample used in this work, NEM gives the same result as the ferricyanide method of determination, which has been shown to give satisfactory results with this protein (12) _ The ovalbumin must be denatured, and the results show that 6 M guanidine hydrochloride effects the requisit.e denaturation rapidly, 0.5% sodium dodecyl sulfate somewhat more slowly. Urea at 8M concentration is not satisfactory because it is quite slow acting, and in the long period required for denaturation the mercapto groups may be lost by oxidation and reaction with cyanate (5). As far as p-lactoglobulin is concerned, the preponderance of extant evidence favors the value of two mercapto groups per molecule (13, 16, 17) and t.he NEM method gives a result in close agreement, 1.9 groups. Either guanidine hydrochloride or urea in 6 M concentration denatures the protein rapidly and gives a definik result in :t few minutes; with

262

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0.5% sodium dodecyl sulfate a somewhat longer reaction time is required. Smyth et al. (18) and Riggs (19) have found that NEM reacts with amino acids other than cysteine, but the reaction is comparatively slow even at 0.1 M concentration of free amino acid. It may accordingly be expected that this reaction would not interfere with determinations like those under discussion, which can be completed in a few minutes; a preliminary report by Riehm and Speck (20) supports this expectation. These results, as well as those obtained for hemoglobin by Cole et al. (6), show that NEM gives acceptable results for the mercapto group content of four well-characterized proteins. Denaturation was necessary in three of the cases, and it would doubtless be required in many others. This introduces an additional factor that must be considered, but a similar limitation applies to other mercapto group reagents and cannot be considered a shortcoming peculiar to NEM. Many reagents and procedures have been proposed for the determination of mercapto groups in proteins (13,21) and a detailed comparison with each cannot be attempted in the available space; it may be remarked in general that this very abundance of alternatives indicates no one method is fully satisfactory. The methods most used in the recent past have been the p-mercuribenzoate derivative method of Boyer (10) and amperometric titration with silver ion (13). Boyer’s method is more sensitive but is based on spectrophotometrie measurements at 250-5 rnp, a wavelength region in which many substances interfere very seriously and in which spectrophotometric accuracy begins to suffer from various causes (use of wide slits, light scattering, etc.). As far as the argentimetric titration is concerned, so many doubts have been raised about its stoichiometric accuracy that it cannot be considered to be generally reliable, although it appears to be so for ,&lactoglobulin and bovine serum albumin (13). These considerations suggest that NEM may be of general utility for determining mercapto groups in proteins and that it offers important advantages when maximum sensitivity is not the determining requisite. SUMMARY

N-Ethylmaleimide has been found suitable for the determination of mercapto groups in proteins. Its application to ovalbumin, p-lactoglobulin, and bovine serum albumin has given results in accordance with those of other analytical methods. The first two proteins must be denatured, and different denaturants vary in their action. Since the reactivity of mercapto groups in proteins is determined by many factors, more than one reagent and a single set of conditions should in general be used.

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REFERENCES 1. 2. 3. 4. 5. 6.

GREGORY, J. D., J. Am. Chem. Sot. 77, 3922 (1955). ROBERTS, E., AND ROUSER, G., AnaZ. Chem. 30, 1291 (1958). ALEXANDER, N. M., Anal. Chem. 30, 1292 (1958). HABEEB, A. F. S. A., Can. J. Biochem. Physiol. 38, 269 (1960). STARK, G. R., STEIN, W. H., .~ND MOORE S., J. Viol. Chem. 235, COLE, R. D., STEIN, W. H., AND MOORE, S., J. Biol. Chem. 233, BENESCH, R. E., LARDY, H. A., AND BENESCH, R., J. Biol. Chem.

7. 8. American

3177 (1960). 1359 (1958). 216. 663 (1955).

Cyanamid Company, Technical Bulletin. L. W., NUENKE, B. J., AND STR.~YHORK, W. D., J. Biol. Chem. 228, 835 (1957). 10. BOYER, P. D., J. Am. Chem. Sot. 76, 4331 (1954). 11. COHN, E. J., HUGHES, W. L., JR., AND WEARE, J. H., J. Am. Chem. Sot. 69, 1753 (1947). 12 KATTAL, J. M., AND GORIN, G., Arch. Biochem. Biophvs. 82, 319 (1959). 13. CECIL, R., AND MCPHEE, J. R., Adv. Protein Chem. 14, 255 (1959). 14. HUGHES, W. L., JR., J. Am, Chem. Sot. 69, 1836 (1947). 15. HARTLEY, R. W., PETERSON, E. A., ROSENBERG, L., AND SOBER, H. A., Abstracts, 139th National Meeting, American Chemical Society, St. Louis, MO., March, 1961. 16. FRAENKEL-CONRAT, J., COOK, B. B., AND MORGAN, A. F., Arch. Biochem. Biophys. 35, 157 (1952). 17. HUTTON, J. T., .%ND PATTON, S., J. Dairy Sci. 35, 699 (1952). 18. SMTTH, D. G., NAGAMATSU, H., AND FRUTON, J. S., J. Am. Chem. Sot. 82, 4600 9. CUNNINGH.~M,

(1960).

19. RIGGS,

A., J. Biol. Chem. 236, 1948 (1961). J. P., AND SPECK, J. C., Abstracts, 140th National Meeting, American Chemical Society, Chicago, Ill., September, 1961. 21. CHINARD, F. P., AND HELLERMAN, L., in “Methods of Biochemical Analysis” (D. Glick, ed.), pp. l-26. Interscience Publishers, New York, 1954. 20. RIEHM,