ANALYTICAL

BIOCHEMISTRY

A New

Method

67.493-502

for

( 19751

Disulfide

W. L. ANDERSON Department

Analysis

of Peptides

AND D. B. WETLAUFER

oj’ Biockmistry, University oj‘ Mimesotu Minneapolis. Minnesota 55455

Received September 30, 1974: accepted March 7. 1975 A simple, sensitive, reliable method for determining disulfide groups in peptides is presented. The disulfides are cleaved in a brief treatment with strong alkali. Following neutralization with phosphoric acid. thiol resulting from the alkaline cleavage is estimated calorimetrically with 5,5’-dithio-bis(7-nitrobenzoic acid). In the presence of EDTA, the color yield is stable and is linear with the concentration of oxidized glutathione. The stoichiometry with other peptide disulfides appears to be somewhat variable but not so as to interfere with detection of peptide disulfides in chromatographic fractions. The present method compares favorably with two other proposed disulfide analytical methods. The cleavage assay is chromogenic with disulfides, thiols, and with certain blocked thiols but is not chromogenic with methionine and lanthionine.

The isolation and purification of disulfide-containing peptides from an enzymic hydrolysis of a protein has been a problem for many investigators, especially when this involved disulfide analysis on hundreds of chromatographic fractions. There are basically two different approaches that have been used to solve this problem. One approach to the analysis of disulfide depends on the direct analysis of sulfur. Sease rl (11.(1) have shown that the II and IV oxidation states of sulfur will bleach an iodoplatinate solution. This has been used by Fowler and Robins as the basis for a very sensitive and rapid disulfide assay (3). However, sulfur as thiol, disulfide, or thioether will react in this assay. A second approach to the detection of disulfide peptides involves the initial cleavage of the disulfide by either oxidation or reduction. The species formed from this cleavage is then used for further analysis. In the case of the oxidative cleavage, after the oxidation, the peptide sample is rerun on the same ion-exchange column (3) or under the same electrophoretic conditions (4) as used for the initial peptide separation. A change in mobility indicates the cleavage of a disulfide. The reductive disulfide cleavage involves first treating the disulfide with a reducing agent such as dithiothreitol (5) or sodium borohydride (6,7), followed by removal of the excess reducing reagent and, finally, estimation of the resulting thiol. Both the oxidative and reductive cleavage assays are time 493 Copyright @ 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

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WETLAUFER

consuming and therefore not well suited to the analysis of many chromatographic fractions. Karush et al. (8) have described a sensitive, rapid disulfide assay. Their technique involves treating a disulfide with an alkaline solution of fluorescein mercuric acetate. A quenching of fluorescence is observed in disulfide-containing samples. To achieve reproducible results in this fluorometric procedure requires very careful handling of the samples and reagents, which does not easily lend itself to the rapid analysis of many samples. There is evidently a need for a rapid, sensitive, reproducible disulfide assay. The assay should, moreover, require only simple equipment and be readily adaptable to numerous samples or be readily automated. The assay described here meets these criteria. The assay is based on alkaline cleavage of disulfide bonds. The excess alkali is then neutralized and the products of the alkaline reaction detected with DTNB.’ MATERIALS

AND

METHODS

Sources of reagents. GSH, GSSG, pantethine, lanthionine, PCMB sigma product #C4378, and BPA were obtained from Sigma. Cystine, methionine, and glutamic acid were Mann products. NEM and TNBS were obtained from Pierce, DTNB from Aldrich, iodoacetic acid from Eastman, EDTA from Mallinckrodt. All other reagents were of reagent grade or better from various suppliers. Disuljide assay procedure. All experiments described are variations on one basic procedure. A 0.25ml sample of peptide in 0.10 N acetic acid is mixed with an equal volume of 6.00 N sodium hydroxide. The alkaline cleavage is allowed to proceed for an appropriate length of time. The reaction is stopped by the addition of 0.50 ml of 6.00 N (2.0 M) H,PO, which is also 2 x 10W3M in EDTA. This acidified reaction mixture is then mixed thoroughly to insure that there are no local high concentrations of base remaining. Differences in the densities of the alkali solution and the neutralizing acid readily lead to formation of stable density gradients. The pH at this point should be between 6 and 7. The chromophore is developed on the addition of 0.10 ml of DTNB (1.00 mg/ml in 0.02 M sodium acetate pH 5.5). The absorbance at 412 nm is measured in a cuvette with a l.OO-cm light path. Synthesis of S-( N-ethylsLaccinimido)-glututhione. The N-ethylmaleimide adduct of reduced glutathione was synthesized and iso’ Abbreviations employed: GSH and GSSG, reduced and oxidized glutathione: EDTA. ethylenediamine tetracetic acid: DTNB, 5.5’-dithio-bis(2-nitrobenzoic acid); Ellman’s anion: 5-thiolate-?-nitrobenzoate: TNBS. trinitrobenzenesulfonic acid: NEM, Nethylmaleimide; PCMB, p-chloromercuribenzoic acid: BPA. bovine plasma albumin: DTT, dithiothreitol.

ANALYSIS

OF

DISULFIDE

PEPTIDES

495

lated by the method of Lee and Samuels (9). The isolated material gave a positive reaction with TNBS, no reaction with DTNB and a positive test for the S-(IV-ethylsuccinimido) group (10). Only one spot was observed on cellulose thin layer chromatography in the n-butanol: acetic : water (7 : 5 : 2) and the n-butanol : formic acid : water (7 : 3 : 3) solvent systems. In both solvent systems the Rf value of the spot does not correspond to either reduced or oxidized glutathione. Preparation of S-carboxymethylglutathione. S-carboxymethylglutathione was synthesized by making an equimolar mixture of reduced glutathione and iodoacetic acid in a 0.10 N ammonium acetate buffer, pH 6.0. The pH of this mixture was maintained at 6.0 for 3 hr with the addition of ammonium hydroxide. The reaction mixture was concentrated by lyophilization. The concentrated reaction mixture was applied to a 0.9 x 30-cm column of Aminex AG 1 X 3, equilibrated and eluted with 0.05 N ammonium acetate, pH 6.0. The first material eluted was further purified by preparative cellulose thin layer chromatography in the n-butanol : acetic acid : water (7 : 5 : 2) system. This material was TNBS positive, it did not react with DTNB and showed only one spot on thin layer chromatography in the n-butanol:formic acid: water (7 : 3 : 3) system. The Rf of this spot did not correspond to that of either reduced or oxidized glutathione. Preparation of S-(p-mercuribenzoate)-glutathione. S-(p-mercuribenzoate)-glutathione was synthesized by making an equimolar mixture of reduced glutathione and PCMB at pH 6.0 in 0.10 N ammonium acetate. After 5 hr, the reaction mixture was concentrated by lyophilization. The concentrated reaction mixture was applied to a 1 X 60-cm column of Sephadex G-l 5 equilibrated with and eluted with 0.10 N acetic acid. The major gel-filtration peak was further purified by preparative thin layer chromatography on cellulose with the n-butanol : acetic acid : water (7 : 5 : 2) solvent system. The isolated material was TNBS positive and DTNB negative. Determination of sample concentrations. The concentration of all samples was determined by dry weight and, where possible, by the TNBS reaction. The following procedure was used for all TNBS determinations. A 0.25-m] aliquot of the sample solution was mixed with 1.00 ml of 4% NaHCO,. To this mixture 0.50 ml of 0.1% TNBS in water was added. The reaction mixture, in sealed tubes, was heated in the dark at 40 “C for 30 min. After the reaction period, 0.50 ml of I .O N HCl was added and the absorbance at 340 nm in a 1.OO-cm cuvette measured. Concentrations were determined by comparison to standard curves of oxidized glutathione and L-glutamic acid. Peptic digestion. BPA was dissolved in 5% formic acid at 5 mg/ml. Pepsin was then added at an enzyme to substrate ratio of 1 : 100 by

496

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WETLAUFER

weight. After 18 hr of digestion another aliquot of pepsin was added. The reaction was terminated after 24 hr by lyophilization. The lyophilized product was stored dry at 0-5°C. Column chromatography of BPA peptic digest. Column chromatographic separation of the peptic peptides of BPA was carried out on a 1.5 X 30-cm column of Whatman carboxymethyl cellulose. The carboxymethyl cellulose was washed and equilibrated with 0.10 N ammonium acetate pH 3.0. BPA peptic peptides were dissolved in and applied to the column in the same ammonium acetate buffer. The peptides were eluted with a 1,000 ml ammonium acetate gradient from 0.3 N, pH 3.0, to 0.3 N, pH 6.0, and lO.O-ml fractions collected. Three different methods were used to locate disulfide- and thiol-containing peptides in the column eluate: The dithiothreitol-arsenite method (5), DTT absorbance method (18), and the standard alkaline cleavage assay of the present paper. RESULTS

The dependence of alkaline cleavage rate on base concentration is shown in the first figure. From this figure it is evident that making an oxidized glutathione solution 3.0 N in sodium hydroxide causes a rapid change in the disulfide to form some alkali-stable compound(s) that react with DTNB. The duration of alkali stability of the cleavage products is unknown: however, the concentration of DTNB-reactive compounds is unaltered after 4.0 hr in 3.0 N sodium hydroxide at 25°C. Lower base concentrations result slower cleavage rates. No detectable cleavage was observed at pH values below 12.

I

0

I

IO TIME

I

I

30 (min)

I

I

I

I

50

FIG. 1. Color yield with DTNB after incubation of GSSG in NaOH for varying times. The standard assay procedure, as described in Materials and Methods, was employed, with the following variations. The uppermost curve (0) employed 6.0 N NaOH, followed by 6 N H,PO, after the indicated reaction time; The middle curve (A) employed 1.5 N NaOH, followed by 1.5 N H,PO,; (W) indicates the addition of 0.50 N NaOH, followed by 0.50 N H,,PO,.

ANALYSIS

0

OF DISULFIDE

20 TIME

60 (min.)

PEPTIDES

497

100

FIG. 2. Color developed with DTNB at pH 6.3, after varying times of prior reaction in 3 N NaOH, for several disulfides: (0). GSSG; (m), pantethine; (A), cystine; (0), DTNB. The maximum absorbance is that observed for a GSSG solution of the same molar concentration. The standard assay procedure was employed.

The rate of cleavage of different model disulfides, in 3.0 N sodium hydroxide, is seen in Fig. 2. It is clear from this figure that the most rapid cleavage of disulfide takes place when the cystine residue is involved in peptide bonds, as in oxidized glutathione. Various disulfide peptides from hen egg lysozyme were cleaved at a rate very similar to that shown for oxidized glutathione. All the disulfides tested were cleaved under these conditions; however, the rate of cleavage for nonpeptide disulfides was slower than for glutathione. The possible interference by the common sulfur-containing amino acids with the alkaline cleavage assay is tested in Fig. 3. The amino 00 80 60 40 20 0 0

20

60 T I M E (min.)

100

FIG. 3. The effect of sodium hydroxide reaction time on the color developed after the addition of the DTNB reagent for various thiol-blocked GSH derivatives: (a), S-(N-ethylsuccinimido)-glutathione: (A). S-carboxymethylglutathione; (B), S-(p-mercuribenzoate)glutathione, and the sulfur amino acids: (X), methionine; (O), lanthionine. Maximum absorbance is defined as that observed for a GSH solution of the same molar concentration. The standard assay procedure was employed.

498

ANDERSON

AND WETLAUFER

0.5

I.0

CONCENTRATION

1.5

2.0

2.5 XlO-4hj

FIG. 4. Alkaline cleavage standard curve for GSSG (0) and GSH (M).

acids methionine and lanthionine do not yield color with this assay. The amino acid cysteine does interfere, but it is common practice to first block the thiol of this amino acid before attempting proteolytic hydrolysis of a protein. The cleavage of some of these thiol-blocked compounds is also shown in Fig. 3. As can be seen, thiols blocked with NEM were cleaved slowly under these conditions. The carboxymethyl derivative appears unreactive under the assay conditions, while the mercuribenzoate derivative readily reacts. Two different methods were used to determine the stoichiometry of the cleavage reaction. Calculations using E = 13,600 M-l cm-’ (17) for the Ellman’s anion show that for oxidized glutathione each disulfide yields 1.34 apparent thiols. The stoichiometry can also be determined experimentally by comparing the slopes of standard curves for both reduced and oxidized glutathione. Figure 4 is a sample of this comparison Using this method, we find that one disulfide yields 1.33 apparent thiols. There is a possibility, however, that in the 3.0 N sodium hydroxide, desulfuration of reduced glutathione could be continuing, yielding erroneous results. In order to test this possibility, a standard curve of reduced glutathione was prepared by first mixing the acid and base, followed by the addition of the sample. Standard procedures were used from this point. Both methods yield the same standard curve, thus removing this objection. The standard curve (Fig. 4) shows a linear relation between color yield and GSSG concentration. In the limited testing that we have done with other peptide disulfides, linearity was obtained, but the stoichiometry (slope on Fig. 4) has been somewhat variable. Possible reasons for this are mentioned in the Discussion section. However, no analytical method is presently available for this broad range of disulfides which has a demonstrated invariant stoichiometry. This conclusion is supported

ANALYSIS

OF DISULFIDE

‘.O I

0.030

0.0 0

499

PEPTIDES

400

200

600

ELUTION

VOLUME

0000

800 (ml)

FIG. 5. Chromatographic separation of BPA peptide mixture analyzed by the disultide methods of Zahler and Cleland (5). - - -. Iyer and Klee f 18), . . , and the alkaline cleavage method of the present paper, -. The sample size for the Iyer and Klee method was twice that of the other two methods.

and amplified by a comparison of the present method with that of Zahler and Cleland (5) and that of Iyer and Klee (18). In Fig. 5 the three methods are compared on a set of chromatographic fractions of a peptic digestion of bovine plasma albumin. The method of Iyer and Klee is seen to have a sensitivity about l/ 100 that of the other two methods, and also requires a larger sample. The Zahler and Cleland method fails to detect the disulfide peptides eking between 200 and 400 ml, perhaps because these peptides contain vicinal disulfides (5). The recently reported covalent structure of plasma albumin shows numerous Cys-Cys sequences ( 19). The stability of the final color is an important consideration for any assay that must be performed on many samples. The color developed in

aeaoI 0

10

20 TIME

30

40

50

60

(min.)

FIG. 6. The stability of the color developed with DTNB. The abscissa is the time after the addition of DTNB. The ordinate is the percent absorbance relative to the l.O-min reading. Note that the full ordinate scale is from SO-100%. The standard alkaline cleavage assay procedure was used on a ? X lOm4 M GSSG solution: (a), 6.0 N H,,PO, also contained 2 x 10m3M EDTA; 101, 6.0 N H,PO, contained no EDTA.

500

ANDERSON

AND WETLAUFER mllmin

23

NaOH

DEBUBBLER TO WASTE

105.0089 COLORIMETER OEBUBBLER

Dl

HO I

0086

.32

.16 DTNB 50 Air .42 Waste

FIG. 7. Manifold for the Technicon AutoAnalyzer used for the alkaline cleavage disulfide assay. The analysis is carried out at room temperature. The reagents are as follows: NaOH, 6.0 N; H3P0,, 6.0 N: DTNB, 0.50 mg/ml in 0.02 N sodium acetate buffer, pH 5.5. Samples are introduced by a Technicon Sampler II; 10% acetic acid washes are used between samples. All glass coils and fittings are shown in the nomenclature of Technicon Instrument Corp., Tarrytown, NY 10591.

this assay is due to the Ellman’s thiolate anion. Characteristically, this thiol is easily oxidized to disulfide by molecular oxygen, especially in the presence of trace concentrations of Cu (II). Low concentrations of EDTA and low pH should help stabilize the resulting color by inhibiting metal-ion-catalyzed air oxidation of thiols in the system. The effect of millimolar concentrations of EDTA in the final solution is shown in Fig. 6. As can be seen, the presence of EDTA provides an effective means of stabilizing the color, allowing the analysis of several samples in one block of time. In addition to EDTA stabilization, the relatively low pH also inhibits oxidation of peptide thiols. In many instances when there are numerous samples to analyze, it is desirable to have the assay automated. This alkaline cleavage disulfide assay lends itself very easily to automation. Figure 7 is a schematic flow diagram for the automation of this assay using Technicon AutoAnalyzer components. The analyzer details are given in the figure legend. A simple modification to the standard peptide analyzer is also possible which will allow monitoring both peptide and disulfide. In the standard peptide analyzer, after the alkaline hydrolysis of the peptide, the sample stream is neutralized. At this point, the stream can be divided, one part mixed with ninhydrin to detect peptides and the other part with DTNB to locate disulfides. DISCUSSION

This assay for disulfides seems to meet the criteria of being rapid, easy, sensitive, and reproducible. Disulfide peptides are cleaved rapidly

ANALYSIS

OF

DISULFIDE

PEPTIDES

501

to form stable thiols. Other common disulfides are cleaved only slowly, which means that this assay should not be used without modification as a general disulfide assay; instead it should be limited to peptides. Disulfides in proteins can be detected using this assay; however, they are often cleaved only slowly under the assay conditions unless there is a denaturant present. The only amino acid that interferes with this assay is cysteine. This interference of cysteine can be minimized by the judicious use of -SH blocking reagents. A problem with this assay is in the stoichiometry of the cleavage reaction. There is much literature concerning the reaction of alkali with disulfide bonds (11,12). Apparently the high concentration of base initiates desulfuration of the peptide forming some semistable species. A recent report by Donovan and White (13) summarizes two possible mechanisms for this initial alkaline cleavage. One mechanism is an elimination reaction: RCH,CH,SSR

+ OH- + RCH=CH?

+ -SSR + H,O

In this scheme one disulfide should yield one perthiolate anion. The perthiolate may go on to other reactions. The second mechanism is a nucleophilic attack of hydroxyl ion directly on the disulfide bond: RSSR + 20H-

+ RSO-

+ H,O.

In this mechanism the resulting sulfinic acid can dismutate to another thiol and a sulfenic acid, resulting in an apparent stoichiometry of one disulfide yielding 1.5 thiols. Our observed stoichiometry of GSSG yielding 1.3 thiols may be the result of competition between the above reactions. Whatever the reason for nonintegral stoichiometry, the stoichiometry is reproducible over a broad concentration range of GSSG. Limited trials with other disulfide peptides have shown a stoichiometry of 1.2 r+ 0.3 -SH per peptide disulfide. Thus a completely reproducible stoichiometry is not presently available for peptide disulfides, and the matter merits further investigation. However, this does not seriously detract from the utility of the method. It provides a practical method for locating disulfide-containing peptides in column chromatographic separations. The method can also be used for the quantitative analysis of many of these disulfide fractions after the particular stoichiometry has been established. This assay has been used successfully in our laboratory for the past two years to detect disulfide-containing peptides in column chromatographic separations (14-l 6) and is now our method of choice. ACKNOWLEDGMENT This

work

was

supported

by

USPHS

Grant

No.

5 ROI

GM18814.

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WETLAUFER

REFERENCES 1. Sease, T. L., Lee, T., Holman, G.. Swift, E. H., and Neinmann, C. (1948) Anal. c-hem. 20, 43 I. 2. Fowler, B., and Robins, A. J. ( 1972) .I. Chronzatogr. 72, 105. 3. Spackman, D. H., Stein, W. H., and Moore, S. (1960) J. Biol. Chem. 235, 648. 4. Brown, J. R., and Hartley. B. S. (1963) Biochem. J. 89, 59P: (1966) 101, 214. 5. Zahler, W. L. and Cleland, W. W. (1968) J. Biol. Chem. 243, 716. 6. Habeeb, A. F. S. A. (1973) And. Bioclwm. 56, 60. 7. Ristow, S. S. (1972) Ph.D. thesis. p. 67. University of Minnesota, Minneapolis. 8. Karush, F., Klinman, N. R., and Marks, R. (1964) Anal. Biochem. 9, 100. 9. Lee, C. C., and Samuels, E. R. (1961) Can. J. C’hern. 39, 1157. 10. Benesch, R., Benesch, R. E., Gutcho, M., and Laufer, L. (1956) Scierrce 123, 981. I 1. Cecil, R., and McPhee. J. R. (1959) Advan. Protein Chem. 14, 255. 12. Cecil, R. (1963) in The Proteins (Neurath. H., ed.), 2nd ed.. Vol. 1, p. 379, Academic Press, New York. 13. Donovan. J. W., and White, T. M. (1971) Biochemistry 10, 32. 14. Pick, P. (1974) Ph.D. thesis, University of Minnesota, Minneapolis. 15. Johnson, E. R. (1974) Ph.D. thesis. University of Minnesota. Minneapolis. 16. Anderson. W. L. (1974) Ph.D. thesis. University of Minnesota, Minneapolis. 17. Ellman, G. L. ( 1959) Arch. Biochem. Biophys. 82. 70. 18. Iyer, K. S., and Klee, W. A. (1973) .I. Eiol. Chem. 248, 707. 19. Brown, J. R. (1974) Fed. Proc. 33, 941 (Abstract).