Estimation of protein sulfhydryl groups with 5,5′-[35S] dithio-bis(2-nitrobenzoate)

ANALYTICAL

BIOCHEMISTRY

Estimation

59,

41&425

of Protein

(1974)

Suifhydryl

Groups

with

BALIGA

AND

5,5’- [ 35S] dithio-bis(2-nitrobenzoate) PETER

114. STEINERT, HAMISH

B. SUREN N. MUNRO

Physiological Chemistry Laboratories, Department of Nutrition and Food Science. Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Received September17, 1973; acceptedNovember 27. 1973 ?%-Labeleddithio-bis(2-nitrobenzoate)has been preparedand its usefulnessfor the measurement of protein sulfhq-dry1groupshasbeeninvestigated. The techniqueis at least 20 times more sensitivethan whf~ndithio-bis(2nitrobenzoate) is usedcalorimetrically.The adduct formed betweenthe reagent and sulfhydrvl groupsis stablebetweenpH 3-9 which shouldenable useof the reagentfor the taggingof sulfhydryl-containingproteinsin n wide variety of biochemicalproredures.Removal of the ndduct from a protein is achieved simply by t.reatment,with 3 thiol reagent. with concomitant restorationof enzymic activitp of the protein. The procedurehashcen successfullytested on alcoholdehgdrogcnase and mammalianIibosomcs. A number of specific calorimetric methods arc available for the analytical determination of protein sulfhydrpl groups, including p-chloromercuribenzoate (pCWIB) (1 j , 5,5’-dithio-bis- (2-nitrobenzoatej (DTNB j (3). (21, and most recently, 4,4’-his-dimcthylaminodiphenylcarbinol Other procedures have involved the use of radioactive reagents, among which are N-ethylmaleimide, iodoacetic acid, iodoacetamide, pCJKB, and tetraethylt,hiuram disulfide (4). In general, t’he radioactive reagents are more sensitive by about two orders of magnitude than the calorimetric reagents. The reaction of DTNB with sulfhydryl groups is unique in that it forms an adduct via a disulfide bond and has the attractive potential of being reversible, but exploitation of this property has not been convenient with the calorimetric procedure. In the present work we have prepared %-labeled DTNB and have examined its usefulness as a reagent for measurement of protein sulfhydryl groups. MATERIALS AND METHODS The following were obtained from the indicated sources. DTNB, Calbiochem; 2-nitro-5-chlorobenzoic acid, Aldrich; /3-nicotinamide adcnine 416 Copyright,@ 1974by Arademic Press,Inc. All rights of reproductionin any form reserved.

[%]DTNB

417

FOR SN GROUPS

dinucleotide (grade III) and lysozyme (three times crystallized) , Sigma Chemical Co. ; yeast alcohol dehydrogenasc (two times crystallized), Schwarz-Mann; sodium [““S]sulfide of specific activity 75.2 Ci/mole, New England Nuclear. E. coli ribosomal subunits were a gift of Dr. S. Acharya. Ribosomes were prepared from the livers of fasting 150-g rat.s (5,6), followed by seclimentat,ion twice through a discontinuous sucrose gradient (0.5-2.0 M sucrose) containi1~~ 0.5 M NH,Cl in a buffer of 50 mu Tris-HCI ($I 7.61, 25 mniLRCl, and 5 rn~ MgCi, to remove all adherent elongation factors (56). To remove cyanate, 10 M solution of urea in water was passed through a mixc(l ion-exchange resin (equal parts Dowex 50 and Dowex 1) immediately prior to use. [““S] DTKB was prepared from 2-nitro-5-chlorobenzoic acid and sodium ]3”S]~~lfid e by adapting the chemical method of Ellman (2). The product was recrystallized from boiling glacial acetic acid as a pale-yellow powder, melting point 236-238°C with decomposition; authentic DTNB (Calbiochem) had a melting point, of 230-232°C; the expected melting point was 238°C (2). The samples were counted with an efficiency of 68% in a niuclcar Chicago Scin~illat,iol~ count,er. Since the product may be col~t:a~~li~late~l with traces of unreacted [:W] suifide, the specific activity of the [““SIDTSB was measuretl by titration against a prot,ein of known SII content, alcohol dehydrogcnase (see belou-). Reaction of DTNB with protein thiol groups occurs in the following manner:

:;02H fp”

i;R;/Ne+co21i + J$co*H S-S-Protein

DiNB

S odduct

Colored

anion

In the established procedure of Ellman (2)) the absorbance of the intensely colored anion is measured at 412 nm. In t,he present experiments, the essentially colorless adduct is measured by its radioactivity, and, therefore, it is necessary to remove the radioactive free anion and excess of reagent. Two methods were used routinely; chromatography on Sephadex C-50 equilibrated in 0.1 JI Tris-HCl, pH 8.0 (compare with Ref. 7) and filtration onto nit,rocelluiose membranes (8). In both of these cases,the technique is limited to sulfhydryl compounds of molecular weight greater than about 5000, since smaller molecules are not exeluded by the Sephadex and are not q~la~ltitat,ively adsorbed by the membrane. A third procedure, precipitatioI1 of protein with 10% tri-

418

STEINERT,

BALJGA

AND

MUFiR

chloroacetic acid, was not satisfactory since the adduct is unstable in strongly acid media (see below). Protein was measured by the method of Lowry et al. (9) using bovine serum albumin as standard. Enzymic assays for yeast alcohol dehydrogenase were performed by an established procedure (10). Ribosome concentrations were calculated on the basis that the absorbance of a 1 mg/ml solution is 150 units/cm, and their activity for in vitro protein synthesis was assayed (5). RESULTS

Measurement

of Adduct

and Standardization

of Technique

Pure yeast alcohol dehydrogenase, which has eight sulfhydryl groups per molecule of 150,000 daltons (ll), was used to standardize the technique. The efficiency of the separation of the adduct formed between the [35S]DTNB and the anion released was tested using a Sephadex G-50 column procedure and adsorption onto nitrocellulose membranes (Fig. 1). Quantitative separation between the adduct and excess of reagent was achieved on Sephadex (Fig. 1A). As much as 5 mg of protein could be

5/

Effluent

Volume

(ml)

Amount

of Protein

(pg)

1. Measurement of adduct formation. (A) The reaction mixture (final vol 1.01 ml) contained yeast alcohol dehydrogenase (300 pg. i.e., 16.0 nmoles of -SH), 0.01 ml of [““SIDTNB (10 mM solution in 0.1 M phosphate buffer pH 7.0) and 0.1 M Tris-HCl buffer (pH 8.0) and was held at room temperature for 3 hr. A sample of 0.8 ml was chromatographed on a column of Sephadex G-50 equilibrated in 0.1 M Tris-HCl buffer (pH 8.0) in a 25-ml buret, and 1.0-ml fractions were collected for counting by addition of “Aquasol.” (B) Assays of the same composition as above but with different amounts of alcohol dehydrogenase as indicated were employed. The entire reaction mixture was poured onto a nitrocellulose filter (Millipore filters, pore size 0.45 pm), washed 10 times with about 1 ml of 0.1 M Tris-HCl (pH 8.0) buffer, dried, and counted using a toluene-based fluid. FIQ.

[~$]DTNB

FOR

SH

419

GROUPS

efficiently fractionated by a column of these dimensions. Blank values (no added sulfhydryl compound) were not significantly above background. In the filtration t’echnicpe, up to 150 pg of alcohol dehydrogenase (Fig. 1Bj was rpantitatiyrly bound to the filter. (C’p to 300 pg of ribosomes could be quantitatively adsorbed under similar conditions.) Blank values for the amount of radioactivity bound to the filter in the absence of added sulfhydryl compound were 50-70 counts/min above background, which usually represented less than 576 of the assay activity. More extensive washing of the membrane resulted in partial loss of the bound protein. The kinetics of reactions of [:~rS]DTNB with alcohol dehydrogenase are shown in Fig. 2 which displays both the :!“S adduct bound and the color of the anion released. The reaction achieves a maximum in 2-3 hr at room temperature (,23”C) and 5-10 min in the presence of 8 ix urea. Thus, denaturation of the protein exposes the relatively slowly reacting thiol groups. The reaction is also temlwrature dependent (Fig. 3). From the data shown in Fig. 2, the specific activity of the [3sS]DTNB

9-rL, L.-.8-

+ UREA

4 Time

000

-0

(hrsi

2. Standardization of [““SIDTNB specific activity. Two pairs of incubations were set up, each of 1.01 ml and 8.08 ml final volume. To one pair, urea was added to a final concentration of 8 M. In addition, each reaction mixture contained (per 1.01 ml) ; buffer (0.1 M Tris-HCl, pH S.O), 255 pg (1.7 nmoles of protein, i.e., 13.6 nmoles of -SH) of yeast alcohol dehydrogenase. and 0.01 ml of [““SIDTNB solution (see Fig. lb). and were incubated at room temperature. At the times indicated, a sample (0.8 ml) from each large reaction mixture was removrd and passed through the Sephadex G-50 column as in Fig. 1A. The effluent between fractions %15 inc!usive (6 ml), which contained the adduct (eluted at the void volume), were collected, poolrd, and a 3-ml aliquot was used for counting. Appropriate corrections were made for sampling and quenching. At the same time. the absorbance at 412 nm of the small reaction volume of each pair was measured, i.e., the anion formed on reaction between the DTKB and protein was measured. FIG.

420

STEINERT,

BALIGA

AR’D

.1KSRO

, Time

(hrs)

FIG. 3. Three reaction mixtures were incubated separately at 37”, 23” (room temperature), and about 0°C. Each reaction contained per 1.01 ml; buffer (0.1 M TrisHCI, pH 8.0), 248 ,~g (1.65 nmoles of protein or 13.2 nmoles of -SH) of yeast alcohol dehydrogenase, and 0.01 ml of DTNB. Samples (0.5 ml) were removed at the indicated times and poured onto nitrocellulose membrane filters for counting as in Fig. 1B.

can be obtained by comparing the amount of the anion released (molar extinction coefficient is 13,600 at 412 nm (Ref. 2)) and the radioactivity in counts/minute of the adduct formed on reaction with t,he alcohol dehydrogenase. From these two, a value of 660 counts/min/nmole of adduct was okained. This value gave the functional specific activity of the [“;S] DTNB sample and was used as a base for the estimation of the sulfhydryl content of other proteins after appropriate correction for decay of the 35S (half-life is 87.3 days). Reaction

with

Sulfhydrgl

Groups

in Various

Compounds

In Table 1 are summarized data obtained from reaction of a variety of proteins with [%]DTNB in the presence and absence of denaturing agents (8 M urea or 1% sodium dodecyl sulfate) using both the column and filtration procedures. Lysozyme and bovine serum albumin show only slight reaction, which is consistent with the fact that these proteins do not possess a free sulfhydryl group, although other workers (8,12) have shown that albumin gives a slight rea’ction with sulfhydryl reagents. Thus, the DTKB reacts specifically with protein sulfhydryl groups. Ribosomes contain at least two different classes of sulfhydryl groups ; one group which is superficial and fast-reacting and a second group of buried sulfhydryls which can bc exposed for reaction only on denaturation with urea or sodium dodecyl sulfate. The value of 44 sulfhydryls for a native 80 S liver ribosome is very similar to the value of 4&42 obtained by McAllister (13). The values obtained for E. coli ribosomal subunits are identical to those of Acharya and Moore (14).

422

STEINERT,

BALIGA

AND

MUXRO

Sodium dodecyl sulfate cannot be used as a denaturant when the filtration technique is employed since the detergent interferes with binding of the protein to the membrane (Table 1). With the exception of this problem, no significant differences were observed between tllc chromatographic and filtration techniques. However, in our hands, some protein complexes are not retained by filtration; for example, EF I-GTP and EF 1-GTP-aminoacyl tRNA complexes do not bind quantitatively (unpublished observations). Thus, it is necessary to check each protein for complete retention on the filter before this technique may be used. This problem does not arise when the chromatographic procedure is employed. When ribosomes are reacted with DTNB, no adduct is formed with the 18 S or 28 S ribosomal RKA species (Fig. 4).

Stability

of Adduct and Activity

of Enzymes

In order to assess the usefulness of this reagent for the measurement of sulfhydryl groups and their function under a variety of different applications, the stability of the adduct formed was investigated (Table 2’~. The adduct formed with alcohol dehydrogenase is stable for at least 1

15

JO

FIG. 4. Does [““SJDTNB react with RNA? A reaction mixture (0.5 ml) in 0.1 M Tris-HCI (pH 8.0) containing approximaiely 3 mg rat liver rihosomes, 1% sodium dodecyl sulfate, and 0.05 ml 10 mM [“BIDTNB solution was incubated at room temperature for 1 hr. At this time the solution was made to 0.1 M LiCl (Ref. 15) and incubated for a further 10 min. A 0.15-ml sample (containing about 320 pg of RNA) was layered over a 520% sucrose density gradient in 10 RIM Tris-HCl, pH 7.5, 0.1% sodium dodecyl sulfate and centrifuged at 45000 rpm for 3 hr at 2°C in a Spinco SW 50 rotor. The gradient was fractionated and the effluent was monitored at 260 nm and collected into 5-drop fractions for counting. The direction of sedimentation is toward the right.

[z5S]~~x~

St,ability

FOR

SH

GROUPS

429

TABLE 3 of the Adductfl 71 Activity 2-‘-kyc

BuH’ct Glycine-acetir

Sodium

arid

acet,ate

Tris-HCl

Potassium

Tris-HCI

phosphate

p1-l S

pH 2.2 pH 3.0 pH 4.0 pH 5.0 pH 6.O pH 7.0 pH x 0 pH 9.0 pH 10.0 pH 11 .o pH 12.0 + 10 no 2-mercaptoet.hanol + 1 mnl dithiot,hreit,ol i-0. 1’;; sodium dodecyl +S ~1 urea +0.5 M KC1 +I .oM KC1

sulfate

recovered __~2:3 “C 11 95 100 101 100 98 9s 95 61 30 10 3 3 96 9x

il The reaction mixt,ure contained, in a final volume of 2.02 ml, 0.1 M Tris-WC1 (pH 8.0) buffer, 8 M urea, 1.5 mg of yeast alcohol dehydrogenase, and 0.1 ml of [“%]L)TNB, and incubated at room temperatllre for 30 min. The adduct was collect,ed by chromatography on Hephadex G-50, and the specific activity of the protein assayed. Samples of the labeled protein were t,hen dialyzed against buffers of ionic strength about 0.1 at the indicated pH and in t,he presence of added reagents listed in the table. 1Iialysis was carried out with two changes of 100 vol at either room temperature or 2m4°C for approximately 24 hr. Samples were then dialyzed briefly against 0.1 M Tris-HCl buffer (pH 7.0) to neutralize and remove added reageut,s. -4 sample was used for mei*surement of protein concentration by the method of Lowry CL al. (9) and the remainder for counting. The remaining activity is expressed as the percentage of the initial specific activit,y of the protein.

day in the cold, or at 23”C, between the pH ranges of 3-9, but is rapidly discharged at extremes of pH. Further, the adduct is retained within this pH range under a variety of other conditions such as high ionic strength, sodium dodecyl sulfate and urea, but exposure to reducing agents results in virtually complete loss of the adduct, as anticipated. Accordingly, this adduct should remain stable under most potential protein chemical applications. In an additional experiment, the reversibility of the adduct in relation to enzymic function was measured (Table 3). In the case of both alcohol dehydrogenase and rat liver ribosomes, the adduct inhibited function to a large extent, but on treatment with reducing agent, almost all activity was recovered.

424 TABLE

Inactivation

3

and Reactivation C,E Activity

untreated

Protein source Yeast alcohol dehydrogenase

relative

Thiol

13.5 4 1.5

a The yeast alcohol dehydrogenase DTNB as before, and the adduct

and rat liver

t,o

protein

Prot’ein-addueL tomplex after DTNU -0.3 4 0.3

Rat liver ribosomes

of Proleinsa

Regenerated protein activity

concentrationb for regeneration

97.0 f 0.7 90.0 & 5.2 94.3 + 5.7

1 ma DTT 1 rn% Z-ME

5 ma

DTT

78.2

5

2”ME

ribosomes

i

2.9 were reactsed

rnM

with

unlabeled

formed was recovered by the column method. A sample of the adduct in each case was then incubated with the thiol reagent, indicated for 30 min at 23°C.

The

activities

of the proteins

were

assayed

before

and

after

-SH

regeneration

for dehydrogenase activity (10) or in vitro protein synthesis (5). The values of activity after IJTNB treatment and -8H regeneration are expressed as a percentage of the activity of the original proteins. Note that the regenerated samples were assayed in the presence of the thiol reagent,, and in each case the values are corrected for t,he increase in activity that was apparent, when control samples were assa.yed in the presence of the thioi reagent. b Abbreviations

are: DTT, dithiothrei~~l;

%XE,

~-rne~~aptoe~ha~~ol.

DISCUSSION

The advantages of the use of [3”S]DTNB for the determination of protein sulfhydryl groups are as follows. The sensitivity of this radioactive procedure is at least 20 times greater than the calorimetric method, and furthermore, can be increased substantially by increasing the specific activity of the compound. It should be pointed out, however, that, when the nitrocellulose membrane filtration procedure is employed, the background level of radioactivity bound to the membrane in the absence of added sulf~lydryl will increase with increases in specific activity of the compound. On the other hand, this problem s~lou~dnot arise when the column chronlatograp~lic technique is utilized. Accordingly, it should be possible to achieve the same degree of sensitivity with this reagent as is currently obtained with some other reagents (e.g., l’C-labeled N-ethylmaleimide). A second principal advantage of the use of DTNB is that, unlike other existing techniques, the adduct may be subsequently removed by an excess of reducing agent such as 2-mercaptoethanol or dithiothreitol. With radioactive DTNB, full utilization of this property is possible in a wide range of prot’ein chemical, structural, and kinetic studies. For example, in the isolation of an enzyme or protein known to

[3”s]

DTRTB

FOR

SH

425

GROUPS

possess free sulfhydryl groups, it should be possible to tag these groups with the radioactive DTNB. This would enable monitoring of the enzyme or protein during purification steps and furthermore protect losses of activity. Full activity c,zn be restored simply by addition of reducing agent. The principal handicap of the use of radioactive DTNB, in which 35S is used as the labeling isotope, is the limitation imposed by the comparatively short half-life of the isotope. In this conhxt, either “H- or 14Clabeled DTNB may be prepared with appropriately lab&d precursors. A second theoretical limitat’ion is that the DTNB bound to a sulfhydryl group on a protein may transfer to a neighboring sulfhydryl exposed during later manipulation of the DTSB-labeled protein, as for example in the study of subunits obtained by dissociation of a prelabeled multisubunit protein. Accordingly, appropriate cont’rol experiments are necessary to either prevent or circumvent this possibility. ACKNOWLEDGMEXT This

work

was supported

by

grant

AM

15346 from

the US Public

Health

Service.

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G.

(1954) I,.

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B.

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AND

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GR~SET’~ D. R., .47x1 MUBRAY. J. F. (1967) AI& 13. CHENG, T-C., AND MCALLISTER, H. C. (1973) J. 14. ACHARY.&, S., AND MOORE, P. B. (1973) J. Mol. 15. MOORE. P. B. (1971) J. Mol. Biol. 60, 169. 12.

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