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A modification of the Ellman procedure for the estimation of protein sulfhydryl groups
ARCHIVES
OF
BIOCHEMISTRY
AND
A Modification
of the of
118,
BIOPHYSICS
ElIman
Protein
Metabolism Laboratory, and the Institute for
Procedure
November
for
the
Estimation
Groups’
H. BAUM,2
Veterans Administration Enzyme Research, University Received
(1967)
Sulfhydryl
P. H. W. BUTTERWORTH, The Lipid Chemistry
716-723
AND
J. W. PORTER
Hospital; the Department of Wisconsin, Madison,
of Physiological Wisconsin 5sYO6
30, 1966
The reaction of the Ellman reagent [5,5’-dithiobis-(2-nitrobenzoic acid)] with a protein sulfhydryl group yields 1 mole of thiophenylated protein and 1 mole of thiophenylate anion, when the reaction is carried out above pH 7.0. The quantity of thiophenylate anion liberated in this reaction is then determined by light absorption measurement at 412 rnp. In the present paper, this technique is modified so that the thiophenylated protein is isolated from the reaction mixture. The amount of thionitrobensoic acid bound to the protein is then determined. Using this principle, sulfhydryl analyses have been successfully made on (1) a sulfhydryl-containing protein in the presence of a reduced thiol; (9) heme proteins; (5) an insoluble protein; and (4) a protein rendered insoluble by denaturation. The classical Ellman procedure cannot be used for assays of free sulfhydryl groups on any of these proteins.
The Ellman reagent (1) [5,5’-dithiobis-(2nitrobenzoic acid)] has been a useful tool for assays of free sulfhydryl groups in many proteins. The reaction of this reagent with protein sulfhydryl groups is shown below:
The conventional assay for SH groups in proteins is carried out by incubating the protein with DTNB at pH 8.0. The quantity of liberated t’hionitrophenylate anion is then determined spectrophotometrically at 412 rnp (&,,,I = 13,600) (1). However, the direct application of this technique has several drawbacks which greatly limit its usefulness for assays of sulfhydryl groups in many proteins. The three most serious disadvantages of the direct Ellman procedure (1) which we have encountered are the following: (1) There is a tendency for the liberated thionitrophenylate to autoxidize, particularly if prolonged incubations under denaturing conditions are employed to titrate masked sulfhydryl groups. (2) This method cannot be used with colored proteins, especially hemoproteins where the Soret band overlaps the 412 rnp absorption maximum of the thiophenylate
-+ -sq-;
+
DTNB’
“=‘o TNB-protein * This work was supported in part by the National Institute of General Medical Science program project, grant GM-12,847 (USPHS), and by research grant A-1383 from the National Institute of Arthritis and Metabolic Diseases of the National Institutes of Health (USPHS). 2 On leave of absence from the Royal Free Hospital School of Medicine, London W.C. 1, England. 3 Abbreviations used: DTNB, 5,5’-dithiobis-
(2-nitrobenzoic acid) ; TNB-protein, the mixed disulfide formed between protein sulfhydryl groups and thionitrobenzoic acid; CoA, coenzyme A; FAS, fatty acid synthetase; EDTA, ethylenediamine tetraacetic acid (d&odium salt); DTT, dithiothreitol; BSA, bovine serum albumin. 716
MODIFIED
TITRATION
OF SULFHYDRYL
anion. (3) Reducing agents (reduced thiols) which are necessary to activate or stabilize mstny enzymes must be removed prior bo sulfhydryl titration. This is inconvenient and often undesirable if a realistic result is to be obtained. For example, removal of the reducing agent may give rise to nonspecific inter- and intramolecular disulfide bond formation. We have found that these and other limitat’ions of the conventional DTNB assay can be overcome by isolation of the TXBprotein from the incubation mixture, followed by a subsequent assay of the amount of the thiophenylate moiety bound t’o theprotein. EXPERIMENTAL
PROCEDURE
i&falerials. DTNB was purchased from the Aldrich Chemical Company, and it was dissolved in 0.1 M potassium phosphate buffer, pH 7.5, (4.0 mg/ml) immediately before use. Dithiothreitol was obtained from the California Corporation for Biochemical Research. Sodium t,aurocholate was prepared by t,he method of Norman (2). Guanidine hydrochloride, secured from the Eastman Kodak Company, was decolorized with activated charcoal in boiling ethanol, and then recrystallized from ethanol-ether before use. Maleic acid was obtained from the Sigma Chemical Company; sodium mersalyl was purchased from K & K Laboratories; and m3Hg-chloromerodrin (specific activity 500 rC/mg) was supplied by Nuclear Consultants. Antimycin, under t)he t,rade name of Blastmycin, was obtained from the Kanegafuchi Chemical Industries Limited, Osaka, Japan. Sephadex (G-25, G-50, and G-100) was purchased from the Pharmacia Company, Upsala, Sweden; and Bio-Gel P-2 was supplied by Biorad Laboratories. Co.4 was purchased from Pabst Laboratories; and insulin was obtained from the Eli Lilly Company. Chymotrypsin was secured from the Worthington Biochemical Corporation; two samples of bovine serum albumin were obtained, one from the Sigma Chemical Company and the other from Pentex Incorporated. Pigeon liver fatty acid synthetase was prepared by t,he method of Hsu et al. (3) as modified by Butterworth et al. (4). Complex III of the mit,ochondrial electron transfer chain (reduced coenzyme &-cytochrome c reductase) was prepared by the method of Rieske (5) and “core protein” was isolated according to the method of Silman et ccl. (6). Sulfhydryl esfimalion. Two modifications of the Ellman method (1) for t,he direct assay of protein sulfhydryl gronps are described. Only the general
GROUPS
717
procedures and conditions for these modified assays are given. Each modified procedure may be modified furt.her to suit the particular properties of the materials to be assayed. Application to specific sulfhydryl-containing moieties which cannot be titrated by the conventional Ellman procedure (1) are described in detail in the section on Results. Schejne I. iln excess of DTNB is incubated at room temperature w-ith a solution of the material to be assayed and at a pH between 7.0 and 8.0 in a minimum volume (approximately 1 ml). Several assays are carried out at differing incubation t,imes. The reaction mixture is then applied quantitatively to a suitable molecular filter, previously equilibrated with 0.1 M Tris-acetate buffer at pH 7.5, and protein is eluted from the column with the same buffer. The criteria for the choice of molecular filter and the column dimensions are entirely dependent on t,he molecular weight, of the protein to be assayed. Complete separation must be achieved between the TNBprotein and the mixture of excess DTNB, liberated thionitrophenylate, and (in cases where reduced thiols are present in the initial incubation mixture) small molecular weight mercaptan-thiophenyl disulfides. The peak of protein eluted from the column is located by light absorption at 280 rnp. All proteincontaining fractions are bulked and made up to a known volume. An excess of a reducing agent (dithiothreitol) is then added to cleave the mixed disulfide bond. In the case of colorless proteins, the liberated thionitrophenylate anion is assayed spectrophotometrically at 412 mp (1). The amount of t,hionitrophenylate liberated from the protein by this procedure is then related (moles per mole of protein) to the known amount, of protein applied to the column. When colored proteins are assayed, protein is removed from solution by precipitation before assay for thiophenylate. The following method is used. After treat.ment of the thiophenylated protein with reducing agent, sufficient 6Orj, perchloric acid is added to precipitate the protein completely. The precipitate is thenseparated from the solution by cent,rifugation and the supernat,ant solution is withdrawn and placed in a volumetric flask. The precipitate is washed at least twice more with5% perchloric acid and centrifuged, and the supernat,ant solutions are combined. The bulked supernatant solutions are made alkaline with 6 N sodium hydroxide and water is added to volume. Thionitrophenylate anion is assayed spectrophotometrically. As a check of the protein recovery in this procedure, the perchloric acid precipitate is dissolved in 1 N sodium hydroxide. (In cases where heme is present, 0.01 ml of 30yo H,O, is added and the mixture is boiled to de-
718
BUTTERWORTH,
BAUM,
colorize the chromophore.) Biuret reagent (7) is added and the quantity of protein is determined spectrophotometrically at 540 rnp. Routine checks have shown that the amount of protein precipitated by perchloric acid is the same as that applied to the column, i.e., recovery is essentially 100%. Therefore, once the procedure has been established with a particular protein (either colorless or colored), only one of the two protein estimationa may be deemed necessary. In Scheme I, TNB-protein is isolated free from other potential chromophores by gel filtration. Quantitative recoveries from the column are not necessary because any aliquot containing protein can be treated as in the procedure described for colored proteins, i.e., the thionitrophenylate liberated, the protein aliquot (from which the chromophore was liberated) separated by precipitation and both the quantity of protein and chromophore determined. Thus, protein and chromophore can be assayed on the same sample (and on duplicate samples if several aliquots from the bulked column eluate are taken). Scheme II. Some denaturing procedures render TNB-protein unsuitable for gel filtration (Scheme I). The following modification in method has therefore been devised. Protein is incubated with the denaturing reagent (e.g., guanidine hydrochloride) for the required length of time in the presence of a large excess of DTNB. The TNBprotein is then precipitated. The conditions required for the precipitation of the protein vary greatly from one material to another. Often, dilution with distilled water is quite sufficient. The precipitated protein is centrifuged and the supernatant solution is discarded. The precipitate is washed with dilute acid (0.2 N acetic acid) and resedimented until the washings contain no DTNB The presence of this compound can be tested by rendering the supernatant solution strongly alkaline. (The thionitrophenyl disulfide bond is cleaved by strong alkali, liberating the chromophore.) The precipitate is then solubilized. This is often best achieved by use of the reagent which was originally used to denature the protein. The pH is then raised to neutrality with dilute alkali and the TNB-protein disulfide bond is cleaved by the addition of an excess of strong reducing agent (dithiothreitol). Between three and five volumes of water are added and the protein is precipitated with 60% perchloric acid. The precipitate is separated by centrifugation and the supernatant solution is transferred to a volumetric flask. The precipitate is washed at least twice with 5% perchloric acid, recentrifuged, and the supernatant solutions from each wash are bulked in the volumetric flask. The bulked supernatant solutions are made slightly alkaline with 6 N sodium hydroxide,
AND
PORTER
adjusted to volume, and the thionitrophenylate anion is estimated spectrophotometrically as before. The precipitated and washed protein is dissolved in 1 N sodium hydroxide and the protein is estimated by the biuret method of Gornall et al. (7). (In the case of heme proteins, prior decolorization with Hz02 is carried out as in Scheme I, above.) The principal disadvantages of Scheme II are that it is slow and the initial precipitation of protein from the denaturing reagent (for the removal of excess DTNB) is not always complete. It is advocated that this technique be used only for insoluble proteins and for proteins which are rendered insoluble by treatment with denaturants. However, if the solubilized TNB-protein (free of DTNB) is transferred to a clean centrifuge tube all subsequent steps may be carried out in a strictly quantitative manner, and highly reproducible and reliable results are obtained. Under these circumstances, there is a parallel determination of protein and bound thionitrophenylate on a single aliquot of the isolated TNB-protein. Thus quantitative recovery of TNB-protein is not critical to the accuracy of this procedure. RESULTS
AND
DISCUSSION
REDUCED COENZYME A The modified procedure of assay for sulfhydryl groups has been tested through the isolation and assay of the TNB-derivative of coenzyme A. Coenzyme A was fully reduced by incubation of 3.75 pmoles of the trilithium salt with 20 pmoles of dithiothreitol in 1.5 ml of 0.1 M potassium phosphate buffer, pH 7.5, for 1 hour at 38”. The dithiothreitol concentration was then reduced to 0.001 M by passage of the incubation mixture through a column of Bio-Gel P-2 (1.4 X 12 cm), previously equilibrated with 0.1 M potassium phosphate buffer, pH 7.5 and 0.001 M in dithiothreitol. Reduced coenzyme A, 0.215 pmoles eluted from this column was then incubated with a large excess of DTNB (5 pmoles) at room temperature for 30 minutes. (Coenzyme A concentrations were determined by light absorption at 260 rnp, using a molecular extinction coefficient of 15.4 X lo3 K1 cm-l). The incubation mixture was then applied (quantitatively) to a Bio-Gel P-2 column (1.0 X 23 cm) previously equilibrated with 0.1 M potassium phosphate buffer, pH 7.5.
RIODIFIF,D
TITRATION
OF SULFHYl>RYL
One-ml fractions were collected. The fractions contraining the TKB-derivative of coenzyme A were bulked and made up to a known volume with 0.1 M phosphat’e buffer. Excess solid dithiot’hreitol was added and the liberated chromophore was assayed by a determination of light absorption at 412 rnp. Reduced coenzyme A, 0.21.5 pmole, gave rise to 0.220 pmole of thiophenylate anion. This corresponds t’o a molar ratio of SH: Coenzyme A of 1: 1.
(:HOIW5
i19
1000 g. The supernatant solution was transferred to a lo-ml volumebric flask. The precipitate was washed with 3 ml of 5 % perchloric acid and resedimented at 1000 g. The supernntant solutions were combined and made alkaline with 1.25 ml of 6 K sodium hydroxide. This solution was made up to 10 ml and the chromophore was e&mated spectrophotometrically. The precipitate was dissolved in 1.5 ml of 1 N sodium hydroxide and assayedfor protein by the method of Gornall et al. (7). The resuhs of t)he above duplicate assays PIGEON LIVER FATTP ACID SYKTHETASE are tabulated in Table I. After a lo-minute This enzyme complex is purified in a meincubation with DTNB, 39 moles SH per dium containing 0.001 M dithiothreitol (4). mole fatty arid synthet,ase (450,000 gm) Sulfhydryl analyses have been carried out on the purified complex by (a) Scheme I, in the were Ctrated. Incubation for SO minutes presence and absence of 0.001 M dithio- raised the titer to 58 moles SH per mole of enzyme complex. t’hreitol and (b) Scheme II. In the latter In a second experiment’, the dithiothreitol case, guanidine hydrochloride (4 M) was used present in the medium used to prepare the as the denaturing reagent. The direct Ellman FAS was removed by molecular filtration on titration (1) was used to check the results Sephadex G-50 equilibrated wit’h 0.1 M potasobtained using Scheme I, where thiol was sium phosphate buffer, pH 7.0, and 0.001 v removed by molecular filtration prior to the EDTA. Fatty acid synthetase (8.35 mg in 0.5 sulfhydryl assay. The results of Scheme II ml) prepared in this way was incubated (which should reveal the total sulfhydryl content of the enzyme, i.e., both “fast” and immediately with 1.0 ml Bellmanreagent (1). “slow” reacting sulfhydryl groups) has been The final volume was made up to 2.0 ml with 0.5 ml of 0.1 M phosphate buffer, pH 7..5,and checked by cysteic acid analysis. incubation was carried out at room temperaa. Scheme I. The fatty acid synthetase used in this experiment was prepared in a ture. Aliquot’s, 0.S ml, were removed after 15 medium composed of 0.2 M potassium phos- minut’es and after 70 minut’es, and assayed by Scheme I as above. The results derived phate, pH 6.8, 0.001 M EDTA, and 0.001 M from this assay were checked by assays by dithiothreitol. Two to four mg fatty acid synthetase, in a the direct Ellman method (1) on O.l-ml volume of 0.15 ml, were incubated with 0.5 aliquots t’aken at the same times (15 and 70 ml Ellman reagent (1) at room temperature minutes). Each aliquot was diluted t,o 1.0 ml for 10 minutes and for 80 minutes. The with 0.1 M pot,assium phosphate buffer, pH TKB-protein was separated from excess 7.5, and then assayedspectrophotomet,rically DTNB and other low molecular weight reac- at 412 mp. The results are t’abulated in Table I. It tion products by molecular filtration on a column of Sephadex G-50 (1 X 22 cm) equili- can be seenthat the t,wo techniques [Scheme brated with 0.1 M Tris-acetate buffer, pH I and direct Ellman tit’ration (l)] gave virtu7.5. The peak of TNB-protein eluted from ally identical values, thus confirming t)he each column was collected and then divided validity of the isolation of the TKB-protein as a reliable method of assay for free sulfhyinto two roughly equal fract,ions (approximately 2 ml each). These fractions were dry1 groups in proteins. Removal of the thiol and immediate sulftreated as duplicates. Excess solid dithiohydryl assay did not affect the sulfhydryl threitol was added to the solution of TNBtiter obtained in the assays of this protein protein to release the chromophore. Two (Table I). However, we have found that drops of 60 % perchloric acid were added and the prot’ein precipitate was sedimented at after 6 hours of storage of t)he t,hiol-free fatty
720
BUTTERWORTH,
BAUM, TABLE
SULFH~DRYL Experiment
Buffer medium of protein solutiona
TITRLLTIONS
ON
Assay
technique
THE
AND
PORTER
I
PIGEON LIVER
FATTY
BCID
Additions
1
+DTT
Scheme
I
None
2
-DTT
Scheme
I
None
3
-DTT
Ellman
(1)
None
4
+DTT
Scheme
II
4 M guanidine
hydrochlorided
5
-DTT
Ellman
(1)
4
M guanidine
hydrochlorided
SYNTHET.\SE Incubation SH groups/ time with mole enzyme DTNB” (min) complexC
10 80 15 i0 15 70 G 11 16 3 9 15
39 58 44 55 43 57 66 64 65 59 41 25
a The medium used t,o prepare the fatty acid synthetase contained 0.2 M potassium phosphate, pH 6.8, 0.001 M EDTA, and 0.001 M dithiot’hreitol (+DTT). In experiments where DTT was removed by molecular filtration, the medium was composed of 0.2 M potassium phosphate buffer, pH 6.8, containing 0.001 M EDTA (-DTT). b When a denaturing reagent, was used, it was added immediat,ely after the addition of DTNB. c The molecular weight of the fatty acid synthetase is 450,000. d The final concentration of this compound in the incubation medium.
acid synthetase, the total number of titratable sulfhydryl groups was reduced by about 35 %. The rate at which protein sulfhydryl groups autoxidize in the absence of reduced thiol is no doubt variable. Possible errors which may be incurred in this way can be eliminated by adopting the procedure described here (SchemeI), which doesnot depend on the removal of the reducing agent prior to titration. b. SchemeII. In order to titrate both “fast” and “slow” reacting sulfhydryl groups, the fatty acid synthetase was treated with DTNB in the presenceof 4 M guanidine hydrochloride. Dilution of the incubation mixture with water rendered the protein insoluble and facilitated the isolation of the TNB-protein by Scheme II. A sample of fatty acid synthetase (4.5 mg) in a volume of 0.15 ml of 0.2 M potassium phosphate buffer (0.001 M in EDTA and 0.001 M in dithiothreitol) was incubated with 0.4 ml Ellman reagent (1) and 1.2 ml of guanidine hydrochloride (6 M). The final concentration of guanidine hydrochloride was 4 M. Incubation was carried out at room temperature for 6, 11, and 16 minutes. After incubation, the volume of each sample was
made to 8 ml with water. The solution became turbid, and the protein precipitated within a few minutes. The precipitate was sedimented and washed free of excessDTNB and other potential chromophores with 0.2 N acetic acid. The solubilization of the TNBprotein (now free of DTNB) was achieved by addition of 1 ml of 6 M guanidine hydrochloride. The subsequent release of the thionitrophenylate anion, its assay, and the estimation of protein followed the procedure described previously for Scheme II. The results of this experiment are recorded in Table I (experiment 4). It can be seen that about 65 sulfhydryl groups were titrated after a relatively short incubation of the fatty acid synthetase with DTNB in the presence of 4 M guanidine hydrochloride. Varying the incubation time did not affect the sulfhydryl titer in this medium. Parallel assays carried out by the direct titration technique of Ellman (1) using fatty acid synthetase in the presenceof 4 M guanidine hydrochloride (but in the absence of DTT) showed that the sulfhydryl titer steadily declined with time (Table I, experiment 5). The precise causeof lossof chromophore is obscure, but we have consistently
MODIFIED
TITRATION
OF
recorded the same result during sulfhydryl titration of other proteins using the direct Ellman method (1) in the presence of guanidine hydrochloride. By adoption of the modification of the standard technique which we have described, this effect is eliminated. Confirmat,ion of the value for total sulfhydryl content of the fatty acid synthetase derived from Scheme II has been obtained by cysteic acid analysis of the enzyme complex using the technique of Xoore (8). A value of 64 moles of cysteic acid per 450,000 gm of protein was obtained on two separate analyses. COMPLEX
III
OF
TRANSFER
THE ELECTRON CHAIN
Scheme I has been used to invest’igate certain aspects of the structural organization of Complex III of the electron kansfer chain. Complex III (reduced coenzyme Q-cytochrome c reductase) contains two equivalent’s of cytochrome b and one of cytochrome cl per particle weight of 300,000 (9). By virtue of its heme content, the direct determination of sulfhydryl groups by the Ellman procedure (1) is impossible. Moreover, in t’he presenceof mercurials, the complex is rapidly cleaved with the separation of insoluble components and the destruction of the antimycin A-binding site (6). Therefore the determination of the number of titratable sulfhydryl groups in the complex under various conditions cannot satisfactorily be carried out by the standard techniques involving mercurial reagents. On the other hand, titration with DTNB using Scheme I (above) proved very satisfactory. Table II illustrates a number of interesting features revealed by such titrations. It. can be seenfrom the dat,a presented in Table II that,, in the caseof the uninhibit’ed, oxidized enzyme, 8 sulfhydryl groups were readily titratable (1 hour incubation). After a l-hour incubation with DTNB the TNBcomplex was still fully active enzymically. Only after 4 hours of incubat’ion was this titration significantly increased. Under these circumstances there was evidence of cleavage of the complex. Some confirmation of the validity of these titrations was obtained by following t,hc loss of enzymic activity on
SUI,FHYl)RYL
(:R0l*PS
721
TABLE AVAILABILITY PLEX
III
TO
UNDER
GROUPS
TITRSTION
VARIOUS
WITH
(lo?&) (lOyO) (10%)
OF
COM-
DTNB
COKDITIONS.
Time of incubation at 25’ (hours)
Additiona
None None None None Taurocholate Taurocholate Taurocholate
II
OF SULFHYDRYL
0.17 1.0 2.0 4.0 0.75 2.0 18.0
Sulfhydryl equivalents
mole cytochrome
per
OxidizedC
AntimycirP
6 8 8 13 19 23 23
6 8
c,*
9 9 11 12
a All incubation mixtures contained, in 0.6 ml, 4 mg of Complex III protein, 2 Nmoles of DTNB, and 0.02 M Tris-acetate, pII 8.0. The other components added to the incubation media were present in the final concentrations as indicated. * After incubation at room temperature for the time indicated, samples were introduced onto a column of Sephadex G-25 (medium grade) equilibrated with 0.1 M Tris-acetate, pH 7.Ti. Protein was eluted with the same buffer (i.e., Scheme I was applied). The fraction containing the TNB-protein was collected and the amount of bound thiophenylate was estimated according t,o the procedure described for colored proteins in the section on Experimental Procedure. c Preparations of the complex were oxidized with a trace of 0.01 M potassium ferricyanide. d Antimycin A (10 equiv per mole of cyt,ochrome ~1) was added as a solution in ethanol (10 DIM) to these samples (following oxidation with a trace of 0.01 M potassium ferricyanide).
t#itration with mersalyl (10). The addition of S equivalents of mersalyl did not affect the activit)y of the complex even upon prolonged incubation; but 12 equivalents of mersalyl caused complete inactivation, which could be reversed by treat’ment with mercaptans (10). Table II also shows that in the presence of high concentrations of detergent, a total of 23 sulfhydryl groups per complex could be titrated within 2 hours, the titration remaining constant on further prolonged incubation. It can also be seenthat, treatment of the complex with the specific inhibitor antimycin A did not affect the number of readily titratable sulfhydryl groups, but very significantly inhibited t’he titration of further
72’1
BUTTERWORTH,
BAUM,
groups in the presenceof concentrated detergent. The significance of this effect of antimycin A in apparently increasing the conformational stability of the complex is discussed elsewhere (10). Nonetheless, it is relevant to point out that only by the use of the modified titration procedure described in this communication was it possible to obtain t,hesedata. “CORE
PROTEIN”
A new major protein component of Complex III of the electron transfer chain has recently been isolated and characterized (6). It is a colorless, insoluble protein with a molecular weight of about 47,000. In view of the finding that separation of this component from the complex was accomplished by the use of any of a number of sulfhydryl group reagents, it was of interest to determine the sulfhydryl content of the protein. This posed experiment)al problems for two reasons. The first was that the protein as isolated contained bound mersalyl (there was also some evidence of disulfide bond formation during isolation). The second was the insolubility of ‘(core protein.” These difficulties were overcome by using a suitable adaptation of Scheme II as outlined below. A suspension of “core protein” (about 5 mg) in 3 ml of 0.01 M dithiothreitol was incubated overnight with stirring. The reduced protein was washed three times with 12-ml portions of water and was then dissolved in 0.4 ml of 6 M guanidine hydrochloride. To this solution was added 0.4 ml of a 0.01 M solution of DTNB in 0.1 M potassium phosphate, pH 7.5. The mixture was incubated for 1 hour at 25” and t’hen diluted to 10 ml with water, at which stage the mixture be-came turbid. The suspensionwas then adjusted to pH 5.0 with dilute acetic acid and the TNB-protein was separated by centrifugation. The subsequent procedure : washand ing, release of thionitrophenylate, precipitation and estimation of protein, was as described in the section on Experimental Procedure. By this procedure, a value of 150 rnl.cequiv of sulfhydryl groups per milligram of protein was obtained, corresponding to about 7 sulfhydryl groups per molecule of protein (molecular weight 47,000).
AND
PORTER
Confirmation of t’his value was obtained by preparing “core protein” using 203Hgchloromerodrin instead of mersalyl as the cleaving agent4. In this case, a somewhat lower tit’ration was obt,ained: 130 rnp equiv of mercury bound per milligram of protein. However, since there was some evidence of disulfide bond formation during t#heisolation of ‘(core protein” and of the reduction of such bonds when “core protein” was treated with dithiothreito14, his relatively small discrepancy could be accounted for. PROTEIN
DISULFIDE
BONDS
The rationale of the procedures discussed in this communication depends upon the presumption that, under the conditions of incubation described, there is no disulfide exchange between DTNB and protein disulfide bonds. That is to say, it is assumed that the method is specific for free sulfhydryl groups. This presumption was tested by applying Scheme I to insulin and to chymotrypsin, species which contain no free sulfhydryl groups but which do possessintramolecular disulfide bonds. In neither case, even upon prolonged incubation with an excess of DTNB, was any chromophore bound to the protein. One example which we have encountered does, however, indicate that there are circumstanceswhen disulfide exchange can give rise to anomalous results. A number of samples of bovine serum albumin were examined both by the direct Ellman procedure (1) and by SchemesI and II. As is common experience, there was somevariability of the direct sulfhydryl titer from sample to sample. In all cases, however, the modified procedures gave consistently higher titers, as many as two extra sulfhydryl groups per molecule of albumin being titrated. The most reasonable interpretation of this finding is that preparations of BSA contain anomalous disulfide bonds which do undergo disulfide exchange with DTNB. This interpretation is in keeping with the observation (11) that BSA samplesare heterogeneousand contain components which are mixed disulfides between 4 Silman, H. I., Rieske, J. S., Lipton, and Baum, H. (unpublished data).
S. H.,
MOI)IFIED
TITRATION
OF
mercaptalbumin (or a modified form of mercapt,albumin) and cyst&e or glutatjhione. Alt,hough such an explanation of t’he anomalous titration obtained with BSA is atkactive, it has not been rigorously tested. With the reservation, therefore, that occasional examples of disulfide exchange with at,ypical protein disulfide bonds might be encountered, it is believed that the general principle of the isolat,ion and estimation of TiYB-protein derivatives allows for an extremely versatile and useful approach to the problem of the determination of protein sulfhydryl groups
8~LFHYI)RYL
1. ELLM~N,
G. L.,
bch.
Biochem.
Biophys.
82,
70 (1959). 2. NoRnwN, A., Llrkzv. Kwzi 8, 331 (1955). 3. Hsu, 1~. Y., W.USON, G., .IND PORTER, ,J. W., J. Biol. Chem. 240, 3637 (1965). 4. BUT.PERWORTH, I'. H. W., C+U~HH.UT, R. B.,
5.
6.
7. The authors are grateful for the advice and interest of Dr. 1)avid E. Green in the development of this work. We also wish to express ollr appreciation to Dr. A. G. Haavik for his assistance in this work and for his help in the preparat,ion of the manuscript. We thank I)r. H. I. Silman for permission to cite his nnpuhlished work, and for a generous gift of “core protein.” One of 17s (H. B.) is grateful to the Wellcome Trustees for the award of a Wellcome Research Travel Grant.
2x3
CROUPS
8. 9.
10.
Il.
B.IUM, H., OLSOX, E. B., &bRGOLIS, S. A., .\NL) I'ORTER, J. W., Arch. Biochem. Hiophys. 116, 453 (19G6). KIESKE, J. S., in “Methods in Enzymology” (R. W. Estabrook and &I. E. Pullman eds.), \-01. X. Academic Press, New York (1967). SILErl.\N, ti. I.. k.\UM, I%., .kND I,IPTOE, 8. H., Federution Proc. (1967). (In press). GORN.U,L, A. G., B2\~uz~w~~t, C. J., MD ~)avr~,~~.M.,J.Riol. Chem. 1’77,751 (1949). MOORE, S., J. Hiol. Chem. 238, 235 (1963). TBIGOLOFF, -4., Y.~NG, P. C., WHARTON,D. C., .~XD RIESKE, J. S., Riochim. Biophys. Acta 96, 1 (1965). B.\uM, II., RIESKE, J. S., HILM.IN, H. I., .IND LIPTON, s. II., PrOC.1Z'all. ACUd. &i. (1967). (In press). AKDERSSON, L., Uiochim. Biophys. Acta 117, 115 (1966).
OF
BIOCHEMISTRY
AND
A Modification
of the of
118,
BIOPHYSICS
ElIman
Protein
Metabolism Laboratory, and the Institute for
Procedure
November
for
the
Estimation
Groups’
H. BAUM,2
Veterans Administration Enzyme Research, University Received
(1967)
Sulfhydryl
P. H. W. BUTTERWORTH, The Lipid Chemistry
716-723
AND
J. W. PORTER
Hospital; the Department of Wisconsin, Madison,
of Physiological Wisconsin 5sYO6
30, 1966
The reaction of the Ellman reagent [5,5’-dithiobis-(2-nitrobenzoic acid)] with a protein sulfhydryl group yields 1 mole of thiophenylated protein and 1 mole of thiophenylate anion, when the reaction is carried out above pH 7.0. The quantity of thiophenylate anion liberated in this reaction is then determined by light absorption measurement at 412 rnp. In the present paper, this technique is modified so that the thiophenylated protein is isolated from the reaction mixture. The amount of thionitrobensoic acid bound to the protein is then determined. Using this principle, sulfhydryl analyses have been successfully made on (1) a sulfhydryl-containing protein in the presence of a reduced thiol; (9) heme proteins; (5) an insoluble protein; and (4) a protein rendered insoluble by denaturation. The classical Ellman procedure cannot be used for assays of free sulfhydryl groups on any of these proteins.
The Ellman reagent (1) [5,5’-dithiobis-(2nitrobenzoic acid)] has been a useful tool for assays of free sulfhydryl groups in many proteins. The reaction of this reagent with protein sulfhydryl groups is shown below:
The conventional assay for SH groups in proteins is carried out by incubating the protein with DTNB at pH 8.0. The quantity of liberated t’hionitrophenylate anion is then determined spectrophotometrically at 412 rnp (&,,,I = 13,600) (1). However, the direct application of this technique has several drawbacks which greatly limit its usefulness for assays of sulfhydryl groups in many proteins. The three most serious disadvantages of the direct Ellman procedure (1) which we have encountered are the following: (1) There is a tendency for the liberated thionitrophenylate to autoxidize, particularly if prolonged incubations under denaturing conditions are employed to titrate masked sulfhydryl groups. (2) This method cannot be used with colored proteins, especially hemoproteins where the Soret band overlaps the 412 rnp absorption maximum of the thiophenylate
-+ -sq-;
+
DTNB’
“=‘o TNB-protein * This work was supported in part by the National Institute of General Medical Science program project, grant GM-12,847 (USPHS), and by research grant A-1383 from the National Institute of Arthritis and Metabolic Diseases of the National Institutes of Health (USPHS). 2 On leave of absence from the Royal Free Hospital School of Medicine, London W.C. 1, England. 3 Abbreviations used: DTNB, 5,5’-dithiobis-
(2-nitrobenzoic acid) ; TNB-protein, the mixed disulfide formed between protein sulfhydryl groups and thionitrobenzoic acid; CoA, coenzyme A; FAS, fatty acid synthetase; EDTA, ethylenediamine tetraacetic acid (d&odium salt); DTT, dithiothreitol; BSA, bovine serum albumin. 716
MODIFIED
TITRATION
OF SULFHYDRYL
anion. (3) Reducing agents (reduced thiols) which are necessary to activate or stabilize mstny enzymes must be removed prior bo sulfhydryl titration. This is inconvenient and often undesirable if a realistic result is to be obtained. For example, removal of the reducing agent may give rise to nonspecific inter- and intramolecular disulfide bond formation. We have found that these and other limitat’ions of the conventional DTNB assay can be overcome by isolation of the TXBprotein from the incubation mixture, followed by a subsequent assay of the amount of the thiophenylate moiety bound t’o theprotein. EXPERIMENTAL
PROCEDURE
i&falerials. DTNB was purchased from the Aldrich Chemical Company, and it was dissolved in 0.1 M potassium phosphate buffer, pH 7.5, (4.0 mg/ml) immediately before use. Dithiothreitol was obtained from the California Corporation for Biochemical Research. Sodium t,aurocholate was prepared by t,he method of Norman (2). Guanidine hydrochloride, secured from the Eastman Kodak Company, was decolorized with activated charcoal in boiling ethanol, and then recrystallized from ethanol-ether before use. Maleic acid was obtained from the Sigma Chemical Company; sodium mersalyl was purchased from K & K Laboratories; and m3Hg-chloromerodrin (specific activity 500 rC/mg) was supplied by Nuclear Consultants. Antimycin, under t)he t,rade name of Blastmycin, was obtained from the Kanegafuchi Chemical Industries Limited, Osaka, Japan. Sephadex (G-25, G-50, and G-100) was purchased from the Pharmacia Company, Upsala, Sweden; and Bio-Gel P-2 was supplied by Biorad Laboratories. Co.4 was purchased from Pabst Laboratories; and insulin was obtained from the Eli Lilly Company. Chymotrypsin was secured from the Worthington Biochemical Corporation; two samples of bovine serum albumin were obtained, one from the Sigma Chemical Company and the other from Pentex Incorporated. Pigeon liver fatty acid synthetase was prepared by t,he method of Hsu et al. (3) as modified by Butterworth et al. (4). Complex III of the mit,ochondrial electron transfer chain (reduced coenzyme &-cytochrome c reductase) was prepared by the method of Rieske (5) and “core protein” was isolated according to the method of Silman et ccl. (6). Sulfhydryl esfimalion. Two modifications of the Ellman method (1) for t,he direct assay of protein sulfhydryl gronps are described. Only the general
GROUPS
717
procedures and conditions for these modified assays are given. Each modified procedure may be modified furt.her to suit the particular properties of the materials to be assayed. Application to specific sulfhydryl-containing moieties which cannot be titrated by the conventional Ellman procedure (1) are described in detail in the section on Results. Schejne I. iln excess of DTNB is incubated at room temperature w-ith a solution of the material to be assayed and at a pH between 7.0 and 8.0 in a minimum volume (approximately 1 ml). Several assays are carried out at differing incubation t,imes. The reaction mixture is then applied quantitatively to a suitable molecular filter, previously equilibrated with 0.1 M Tris-acetate buffer at pH 7.5, and protein is eluted from the column with the same buffer. The criteria for the choice of molecular filter and the column dimensions are entirely dependent on t,he molecular weight, of the protein to be assayed. Complete separation must be achieved between the TNBprotein and the mixture of excess DTNB, liberated thionitrophenylate, and (in cases where reduced thiols are present in the initial incubation mixture) small molecular weight mercaptan-thiophenyl disulfides. The peak of protein eluted from the column is located by light absorption at 280 rnp. All proteincontaining fractions are bulked and made up to a known volume. An excess of a reducing agent (dithiothreitol) is then added to cleave the mixed disulfide bond. In the case of colorless proteins, the liberated thionitrophenylate anion is assayed spectrophotometrically at 412 mp (1). The amount of t,hionitrophenylate liberated from the protein by this procedure is then related (moles per mole of protein) to the known amount, of protein applied to the column. When colored proteins are assayed, protein is removed from solution by precipitation before assay for thiophenylate. The following method is used. After treat.ment of the thiophenylated protein with reducing agent, sufficient 6Orj, perchloric acid is added to precipitate the protein completely. The precipitate is thenseparated from the solution by cent,rifugation and the supernat,ant solution is withdrawn and placed in a volumetric flask. The precipitate is washed at least twice more with5% perchloric acid and centrifuged, and the supernat,ant solutions are combined. The bulked supernatant solutions are made alkaline with 6 N sodium hydroxide and water is added to volume. Thionitrophenylate anion is assayed spectrophotometrically. As a check of the protein recovery in this procedure, the perchloric acid precipitate is dissolved in 1 N sodium hydroxide. (In cases where heme is present, 0.01 ml of 30yo H,O, is added and the mixture is boiled to de-
718
BUTTERWORTH,
BAUM,
colorize the chromophore.) Biuret reagent (7) is added and the quantity of protein is determined spectrophotometrically at 540 rnp. Routine checks have shown that the amount of protein precipitated by perchloric acid is the same as that applied to the column, i.e., recovery is essentially 100%. Therefore, once the procedure has been established with a particular protein (either colorless or colored), only one of the two protein estimationa may be deemed necessary. In Scheme I, TNB-protein is isolated free from other potential chromophores by gel filtration. Quantitative recoveries from the column are not necessary because any aliquot containing protein can be treated as in the procedure described for colored proteins, i.e., the thionitrophenylate liberated, the protein aliquot (from which the chromophore was liberated) separated by precipitation and both the quantity of protein and chromophore determined. Thus, protein and chromophore can be assayed on the same sample (and on duplicate samples if several aliquots from the bulked column eluate are taken). Scheme II. Some denaturing procedures render TNB-protein unsuitable for gel filtration (Scheme I). The following modification in method has therefore been devised. Protein is incubated with the denaturing reagent (e.g., guanidine hydrochloride) for the required length of time in the presence of a large excess of DTNB. The TNBprotein is then precipitated. The conditions required for the precipitation of the protein vary greatly from one material to another. Often, dilution with distilled water is quite sufficient. The precipitated protein is centrifuged and the supernatant solution is discarded. The precipitate is washed with dilute acid (0.2 N acetic acid) and resedimented until the washings contain no DTNB The presence of this compound can be tested by rendering the supernatant solution strongly alkaline. (The thionitrophenyl disulfide bond is cleaved by strong alkali, liberating the chromophore.) The precipitate is then solubilized. This is often best achieved by use of the reagent which was originally used to denature the protein. The pH is then raised to neutrality with dilute alkali and the TNB-protein disulfide bond is cleaved by the addition of an excess of strong reducing agent (dithiothreitol). Between three and five volumes of water are added and the protein is precipitated with 60% perchloric acid. The precipitate is separated by centrifugation and the supernatant solution is transferred to a volumetric flask. The precipitate is washed at least twice with 5% perchloric acid, recentrifuged, and the supernatant solutions from each wash are bulked in the volumetric flask. The bulked supernatant solutions are made slightly alkaline with 6 N sodium hydroxide,
AND
PORTER
adjusted to volume, and the thionitrophenylate anion is estimated spectrophotometrically as before. The precipitated and washed protein is dissolved in 1 N sodium hydroxide and the protein is estimated by the biuret method of Gornall et al. (7). (In the case of heme proteins, prior decolorization with Hz02 is carried out as in Scheme I, above.) The principal disadvantages of Scheme II are that it is slow and the initial precipitation of protein from the denaturing reagent (for the removal of excess DTNB) is not always complete. It is advocated that this technique be used only for insoluble proteins and for proteins which are rendered insoluble by treatment with denaturants. However, if the solubilized TNB-protein (free of DTNB) is transferred to a clean centrifuge tube all subsequent steps may be carried out in a strictly quantitative manner, and highly reproducible and reliable results are obtained. Under these circumstances, there is a parallel determination of protein and bound thionitrophenylate on a single aliquot of the isolated TNB-protein. Thus quantitative recovery of TNB-protein is not critical to the accuracy of this procedure. RESULTS
AND
DISCUSSION
REDUCED COENZYME A The modified procedure of assay for sulfhydryl groups has been tested through the isolation and assay of the TNB-derivative of coenzyme A. Coenzyme A was fully reduced by incubation of 3.75 pmoles of the trilithium salt with 20 pmoles of dithiothreitol in 1.5 ml of 0.1 M potassium phosphate buffer, pH 7.5, for 1 hour at 38”. The dithiothreitol concentration was then reduced to 0.001 M by passage of the incubation mixture through a column of Bio-Gel P-2 (1.4 X 12 cm), previously equilibrated with 0.1 M potassium phosphate buffer, pH 7.5 and 0.001 M in dithiothreitol. Reduced coenzyme A, 0.215 pmoles eluted from this column was then incubated with a large excess of DTNB (5 pmoles) at room temperature for 30 minutes. (Coenzyme A concentrations were determined by light absorption at 260 rnp, using a molecular extinction coefficient of 15.4 X lo3 K1 cm-l). The incubation mixture was then applied (quantitatively) to a Bio-Gel P-2 column (1.0 X 23 cm) previously equilibrated with 0.1 M potassium phosphate buffer, pH 7.5.
RIODIFIF,D
TITRATION
OF SULFHYl>RYL
One-ml fractions were collected. The fractions contraining the TKB-derivative of coenzyme A were bulked and made up to a known volume with 0.1 M phosphat’e buffer. Excess solid dithiot’hreitol was added and the liberated chromophore was assayed by a determination of light absorption at 412 rnp. Reduced coenzyme A, 0.21.5 pmole, gave rise to 0.220 pmole of thiophenylate anion. This corresponds t’o a molar ratio of SH: Coenzyme A of 1: 1.
(:HOIW5
i19
1000 g. The supernatant solution was transferred to a lo-ml volumebric flask. The precipitate was washed with 3 ml of 5 % perchloric acid and resedimented at 1000 g. The supernntant solutions were combined and made alkaline with 1.25 ml of 6 K sodium hydroxide. This solution was made up to 10 ml and the chromophore was e&mated spectrophotometrically. The precipitate was dissolved in 1.5 ml of 1 N sodium hydroxide and assayedfor protein by the method of Gornall et al. (7). The resuhs of t)he above duplicate assays PIGEON LIVER FATTP ACID SYKTHETASE are tabulated in Table I. After a lo-minute This enzyme complex is purified in a meincubation with DTNB, 39 moles SH per dium containing 0.001 M dithiothreitol (4). mole fatty arid synthet,ase (450,000 gm) Sulfhydryl analyses have been carried out on the purified complex by (a) Scheme I, in the were Ctrated. Incubation for SO minutes presence and absence of 0.001 M dithio- raised the titer to 58 moles SH per mole of enzyme complex. t’hreitol and (b) Scheme II. In the latter In a second experiment’, the dithiothreitol case, guanidine hydrochloride (4 M) was used present in the medium used to prepare the as the denaturing reagent. The direct Ellman FAS was removed by molecular filtration on titration (1) was used to check the results Sephadex G-50 equilibrated wit’h 0.1 M potasobtained using Scheme I, where thiol was sium phosphate buffer, pH 7.0, and 0.001 v removed by molecular filtration prior to the EDTA. Fatty acid synthetase (8.35 mg in 0.5 sulfhydryl assay. The results of Scheme II ml) prepared in this way was incubated (which should reveal the total sulfhydryl content of the enzyme, i.e., both “fast” and immediately with 1.0 ml Bellmanreagent (1). “slow” reacting sulfhydryl groups) has been The final volume was made up to 2.0 ml with 0.5 ml of 0.1 M phosphate buffer, pH 7..5,and checked by cysteic acid analysis. incubation was carried out at room temperaa. Scheme I. The fatty acid synthetase used in this experiment was prepared in a ture. Aliquot’s, 0.S ml, were removed after 15 medium composed of 0.2 M potassium phos- minut’es and after 70 minut’es, and assayed by Scheme I as above. The results derived phate, pH 6.8, 0.001 M EDTA, and 0.001 M from this assay were checked by assays by dithiothreitol. Two to four mg fatty acid synthetase, in a the direct Ellman method (1) on O.l-ml volume of 0.15 ml, were incubated with 0.5 aliquots t’aken at the same times (15 and 70 ml Ellman reagent (1) at room temperature minutes). Each aliquot was diluted t,o 1.0 ml for 10 minutes and for 80 minutes. The with 0.1 M pot,assium phosphate buffer, pH TKB-protein was separated from excess 7.5, and then assayedspectrophotomet,rically DTNB and other low molecular weight reac- at 412 mp. The results are t’abulated in Table I. It tion products by molecular filtration on a column of Sephadex G-50 (1 X 22 cm) equili- can be seenthat the t,wo techniques [Scheme brated with 0.1 M Tris-acetate buffer, pH I and direct Ellman tit’ration (l)] gave virtu7.5. The peak of TNB-protein eluted from ally identical values, thus confirming t)he each column was collected and then divided validity of the isolation of the TKB-protein as a reliable method of assay for free sulfhyinto two roughly equal fract,ions (approximately 2 ml each). These fractions were dry1 groups in proteins. Removal of the thiol and immediate sulftreated as duplicates. Excess solid dithiohydryl assay did not affect the sulfhydryl threitol was added to the solution of TNBtiter obtained in the assays of this protein protein to release the chromophore. Two (Table I). However, we have found that drops of 60 % perchloric acid were added and the prot’ein precipitate was sedimented at after 6 hours of storage of t)he t,hiol-free fatty
720
BUTTERWORTH,
BAUM, TABLE
SULFH~DRYL Experiment
Buffer medium of protein solutiona
TITRLLTIONS
ON
Assay
technique
THE
AND
PORTER
I
PIGEON LIVER
FATTY
BCID
Additions
1
+DTT
Scheme
I
None
2
-DTT
Scheme
I
None
3
-DTT
Ellman
(1)
None
4
+DTT
Scheme
II
4 M guanidine
hydrochlorided
5
-DTT
Ellman
(1)
4
M guanidine
hydrochlorided
SYNTHET.\SE Incubation SH groups/ time with mole enzyme DTNB” (min) complexC
10 80 15 i0 15 70 G 11 16 3 9 15
39 58 44 55 43 57 66 64 65 59 41 25
a The medium used t,o prepare the fatty acid synthetase contained 0.2 M potassium phosphate, pH 6.8, 0.001 M EDTA, and 0.001 M dithiot’hreitol (+DTT). In experiments where DTT was removed by molecular filtration, the medium was composed of 0.2 M potassium phosphate buffer, pH 6.8, containing 0.001 M EDTA (-DTT). b When a denaturing reagent, was used, it was added immediat,ely after the addition of DTNB. c The molecular weight of the fatty acid synthetase is 450,000. d The final concentration of this compound in the incubation medium.
acid synthetase, the total number of titratable sulfhydryl groups was reduced by about 35 %. The rate at which protein sulfhydryl groups autoxidize in the absence of reduced thiol is no doubt variable. Possible errors which may be incurred in this way can be eliminated by adopting the procedure described here (SchemeI), which doesnot depend on the removal of the reducing agent prior to titration. b. SchemeII. In order to titrate both “fast” and “slow” reacting sulfhydryl groups, the fatty acid synthetase was treated with DTNB in the presenceof 4 M guanidine hydrochloride. Dilution of the incubation mixture with water rendered the protein insoluble and facilitated the isolation of the TNB-protein by Scheme II. A sample of fatty acid synthetase (4.5 mg) in a volume of 0.15 ml of 0.2 M potassium phosphate buffer (0.001 M in EDTA and 0.001 M in dithiothreitol) was incubated with 0.4 ml Ellman reagent (1) and 1.2 ml of guanidine hydrochloride (6 M). The final concentration of guanidine hydrochloride was 4 M. Incubation was carried out at room temperature for 6, 11, and 16 minutes. After incubation, the volume of each sample was
made to 8 ml with water. The solution became turbid, and the protein precipitated within a few minutes. The precipitate was sedimented and washed free of excessDTNB and other potential chromophores with 0.2 N acetic acid. The solubilization of the TNBprotein (now free of DTNB) was achieved by addition of 1 ml of 6 M guanidine hydrochloride. The subsequent release of the thionitrophenylate anion, its assay, and the estimation of protein followed the procedure described previously for Scheme II. The results of this experiment are recorded in Table I (experiment 4). It can be seen that about 65 sulfhydryl groups were titrated after a relatively short incubation of the fatty acid synthetase with DTNB in the presence of 4 M guanidine hydrochloride. Varying the incubation time did not affect the sulfhydryl titer in this medium. Parallel assays carried out by the direct titration technique of Ellman (1) using fatty acid synthetase in the presenceof 4 M guanidine hydrochloride (but in the absence of DTT) showed that the sulfhydryl titer steadily declined with time (Table I, experiment 5). The precise causeof lossof chromophore is obscure, but we have consistently
MODIFIED
TITRATION
OF
recorded the same result during sulfhydryl titration of other proteins using the direct Ellman method (1) in the presence of guanidine hydrochloride. By adoption of the modification of the standard technique which we have described, this effect is eliminated. Confirmat,ion of the value for total sulfhydryl content of the fatty acid synthetase derived from Scheme II has been obtained by cysteic acid analysis of the enzyme complex using the technique of Xoore (8). A value of 64 moles of cysteic acid per 450,000 gm of protein was obtained on two separate analyses. COMPLEX
III
OF
TRANSFER
THE ELECTRON CHAIN
Scheme I has been used to invest’igate certain aspects of the structural organization of Complex III of the electron kansfer chain. Complex III (reduced coenzyme Q-cytochrome c reductase) contains two equivalent’s of cytochrome b and one of cytochrome cl per particle weight of 300,000 (9). By virtue of its heme content, the direct determination of sulfhydryl groups by the Ellman procedure (1) is impossible. Moreover, in t’he presenceof mercurials, the complex is rapidly cleaved with the separation of insoluble components and the destruction of the antimycin A-binding site (6). Therefore the determination of the number of titratable sulfhydryl groups in the complex under various conditions cannot satisfactorily be carried out by the standard techniques involving mercurial reagents. On the other hand, titration with DTNB using Scheme I (above) proved very satisfactory. Table II illustrates a number of interesting features revealed by such titrations. It. can be seenfrom the dat,a presented in Table II that,, in the caseof the uninhibit’ed, oxidized enzyme, 8 sulfhydryl groups were readily titratable (1 hour incubation). After a l-hour incubation with DTNB the TNBcomplex was still fully active enzymically. Only after 4 hours of incubat’ion was this titration significantly increased. Under these circumstances there was evidence of cleavage of the complex. Some confirmation of the validity of these titrations was obtained by following t,hc loss of enzymic activity on
SUI,FHYl)RYL
(:R0l*PS
721
TABLE AVAILABILITY PLEX
III
TO
UNDER
GROUPS
TITRSTION
VARIOUS
WITH
(lo?&) (lOyO) (10%)
OF
COM-
DTNB
COKDITIONS.
Time of incubation at 25’ (hours)
Additiona
None None None None Taurocholate Taurocholate Taurocholate
II
OF SULFHYDRYL
0.17 1.0 2.0 4.0 0.75 2.0 18.0
Sulfhydryl equivalents
mole cytochrome
per
OxidizedC
AntimycirP
6 8 8 13 19 23 23
6 8
c,*
9 9 11 12
a All incubation mixtures contained, in 0.6 ml, 4 mg of Complex III protein, 2 Nmoles of DTNB, and 0.02 M Tris-acetate, pII 8.0. The other components added to the incubation media were present in the final concentrations as indicated. * After incubation at room temperature for the time indicated, samples were introduced onto a column of Sephadex G-25 (medium grade) equilibrated with 0.1 M Tris-acetate, pH 7.Ti. Protein was eluted with the same buffer (i.e., Scheme I was applied). The fraction containing the TNB-protein was collected and the amount of bound thiophenylate was estimated according t,o the procedure described for colored proteins in the section on Experimental Procedure. c Preparations of the complex were oxidized with a trace of 0.01 M potassium ferricyanide. d Antimycin A (10 equiv per mole of cyt,ochrome ~1) was added as a solution in ethanol (10 DIM) to these samples (following oxidation with a trace of 0.01 M potassium ferricyanide).
t#itration with mersalyl (10). The addition of S equivalents of mersalyl did not affect the activit)y of the complex even upon prolonged incubation; but 12 equivalents of mersalyl caused complete inactivation, which could be reversed by treat’ment with mercaptans (10). Table II also shows that in the presence of high concentrations of detergent, a total of 23 sulfhydryl groups per complex could be titrated within 2 hours, the titration remaining constant on further prolonged incubation. It can also be seenthat, treatment of the complex with the specific inhibitor antimycin A did not affect the number of readily titratable sulfhydryl groups, but very significantly inhibited t’he titration of further
72’1
BUTTERWORTH,
BAUM,
groups in the presenceof concentrated detergent. The significance of this effect of antimycin A in apparently increasing the conformational stability of the complex is discussed elsewhere (10). Nonetheless, it is relevant to point out that only by the use of the modified titration procedure described in this communication was it possible to obtain t,hesedata. “CORE
PROTEIN”
A new major protein component of Complex III of the electron transfer chain has recently been isolated and characterized (6). It is a colorless, insoluble protein with a molecular weight of about 47,000. In view of the finding that separation of this component from the complex was accomplished by the use of any of a number of sulfhydryl group reagents, it was of interest to determine the sulfhydryl content of the protein. This posed experiment)al problems for two reasons. The first was that the protein as isolated contained bound mersalyl (there was also some evidence of disulfide bond formation during isolation). The second was the insolubility of ‘(core protein.” These difficulties were overcome by using a suitable adaptation of Scheme II as outlined below. A suspension of “core protein” (about 5 mg) in 3 ml of 0.01 M dithiothreitol was incubated overnight with stirring. The reduced protein was washed three times with 12-ml portions of water and was then dissolved in 0.4 ml of 6 M guanidine hydrochloride. To this solution was added 0.4 ml of a 0.01 M solution of DTNB in 0.1 M potassium phosphate, pH 7.5. The mixture was incubated for 1 hour at 25” and t’hen diluted to 10 ml with water, at which stage the mixture be-came turbid. The suspensionwas then adjusted to pH 5.0 with dilute acetic acid and the TNB-protein was separated by centrifugation. The subsequent procedure : washand ing, release of thionitrophenylate, precipitation and estimation of protein, was as described in the section on Experimental Procedure. By this procedure, a value of 150 rnl.cequiv of sulfhydryl groups per milligram of protein was obtained, corresponding to about 7 sulfhydryl groups per molecule of protein (molecular weight 47,000).
AND
PORTER
Confirmation of t’his value was obtained by preparing “core protein” using 203Hgchloromerodrin instead of mersalyl as the cleaving agent4. In this case, a somewhat lower tit’ration was obt,ained: 130 rnp equiv of mercury bound per milligram of protein. However, since there was some evidence of disulfide bond formation during t#heisolation of ‘(core protein” and of the reduction of such bonds when “core protein” was treated with dithiothreito14, his relatively small discrepancy could be accounted for. PROTEIN
DISULFIDE
BONDS
The rationale of the procedures discussed in this communication depends upon the presumption that, under the conditions of incubation described, there is no disulfide exchange between DTNB and protein disulfide bonds. That is to say, it is assumed that the method is specific for free sulfhydryl groups. This presumption was tested by applying Scheme I to insulin and to chymotrypsin, species which contain no free sulfhydryl groups but which do possessintramolecular disulfide bonds. In neither case, even upon prolonged incubation with an excess of DTNB, was any chromophore bound to the protein. One example which we have encountered does, however, indicate that there are circumstanceswhen disulfide exchange can give rise to anomalous results. A number of samples of bovine serum albumin were examined both by the direct Ellman procedure (1) and by SchemesI and II. As is common experience, there was somevariability of the direct sulfhydryl titer from sample to sample. In all cases, however, the modified procedures gave consistently higher titers, as many as two extra sulfhydryl groups per molecule of albumin being titrated. The most reasonable interpretation of this finding is that preparations of BSA contain anomalous disulfide bonds which do undergo disulfide exchange with DTNB. This interpretation is in keeping with the observation (11) that BSA samplesare heterogeneousand contain components which are mixed disulfides between 4 Silman, H. I., Rieske, J. S., Lipton, and Baum, H. (unpublished data).
S. H.,
MOI)IFIED
TITRATION
OF
mercaptalbumin (or a modified form of mercapt,albumin) and cyst&e or glutatjhione. Alt,hough such an explanation of t’he anomalous titration obtained with BSA is atkactive, it has not been rigorously tested. With the reservation, therefore, that occasional examples of disulfide exchange with at,ypical protein disulfide bonds might be encountered, it is believed that the general principle of the isolat,ion and estimation of TiYB-protein derivatives allows for an extremely versatile and useful approach to the problem of the determination of protein sulfhydryl groups
8~LFHYI)RYL
1. ELLM~N,
G. L.,
bch.
Biochem.
Biophys.
82,
70 (1959). 2. NoRnwN, A., Llrkzv. Kwzi 8, 331 (1955). 3. Hsu, 1~. Y., W.USON, G., .IND PORTER, ,J. W., J. Biol. Chem. 240, 3637 (1965). 4. BUT.PERWORTH, I'. H. W., C+U~HH.UT, R. B.,
5.
6.
7. The authors are grateful for the advice and interest of Dr. 1)avid E. Green in the development of this work. We also wish to express ollr appreciation to Dr. A. G. Haavik for his assistance in this work and for his help in the preparat,ion of the manuscript. We thank I)r. H. I. Silman for permission to cite his nnpuhlished work, and for a generous gift of “core protein.” One of 17s (H. B.) is grateful to the Wellcome Trustees for the award of a Wellcome Research Travel Grant.
2x3
CROUPS
8. 9.
10.
Il.
B.IUM, H., OLSOX, E. B., &bRGOLIS, S. A., .\NL) I'ORTER, J. W., Arch. Biochem. Hiophys. 116, 453 (19G6). KIESKE, J. S., in “Methods in Enzymology” (R. W. Estabrook and &I. E. Pullman eds.), \-01. X. Academic Press, New York (1967). SILErl.\N, ti. I.. k.\UM, I%., .kND I,IPTOE, 8. H., Federution Proc. (1967). (In press). GORN.U,L, A. G., B2\~uz~w~~t, C. J., MD ~)avr~,~~.M.,J.Riol. Chem. 1’77,751 (1949). MOORE, S., J. Hiol. Chem. 238, 235 (1963). TBIGOLOFF, -4., Y.~NG, P. C., WHARTON,D. C., .~XD RIESKE, J. S., Riochim. Biophys. Acta 96, 1 (1965). B.\uM, II., RIESKE, J. S., HILM.IN, H. I., .IND LIPTON, s. II., PrOC.1Z'all. ACUd. &i. (1967). (In press). AKDERSSON, L., Uiochim. Biophys. Acta 117, 115 (1966).