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Determination of disulfide groups in proteins by a sensitive electroreductive-colorimetric method
ANALYTICAL
BIOCHEMISTRY
76, l70- 176 ( 1976)
Determination of Disulfide Groups Sensitive Electroreductive-Calorimetric
in Proteins by a Method
P. D. J. WEITZMAN Departmetzt
oj’Biochemistry.
Utzi~‘ersity
of Leicester,
Leicester,
EngIrd
Received April 19, 1976: accepted June 8, 1976 A method is described for the determination of the total disulfide content of proteins. It is based on electrolytic cleavage of disulfide bonds at a mercury pool cathode in acidic buffer containing guanidine, followed by calorimetric estimation of the liberated thiol groups with 5,5’-dithiobis@-nitrobenzoate). The general applicability of the method is demonstrated with a range of established proteins and the results obtained are in excellent agreement with known values. The method offers distinct advantages over other available procedures and is applicable to both protein and nonprotein disulfides.
Disulfide groups constitute an important structural feature of proteins. They play a major role in the maintenance of three-dimensional protein structure, and various methods have been devised for their quantitative estimation. Whereas the related thiol groups may be simply and directly determined with a variety of fairly specific reagents, methods for determining disulfide groups are generally indirect and rely on some form of bond cleavage followed by estimation of the reaction products. Three types of cleavage have been used as the basis of disulfide bond analysis. (a) Oxidation with per-formic acid and estimation of cysteic acid after total acid hydrolysis of the protein (1). (b) Reaction with sulfite (sulfitolysis) to produce an equimolar mixture of thiosulfate and thiol, followed by estimation of the latter by amperometric (polarographic) titration with heavy-metal reagents (2,3). (c) Reduction with mercaptoethanol, dithiothreitol, or sodium borohydride, followed by alkylation (generally carboxymethylation) of the cysteine residues, total acid hydrolysis of the protein, and estimation of the derivatized cysteine (4-6). Determination of the thiol groups formed on borohydride reduction has also been done by amperometric titration (7) or calorimetrically (8) after reaction with 5,5’-dithiobis (?-nitrobenzoate) (DTNB). The above procedures are relatively laborious and time consuming and, moreover, require specialized equipment in the form of an amino acid analyzer or polarographic apparatus. My interest in the electrolytic reduction of disulfide bonds prompted the present work aimed at devising an improved procedure for quantitative disulfide analysis. 170 CopyrIght Q 1976 h) Academic Pres Inc. All rights of reproduction in nny form rewired.
DISULFIDE
GROUP
171
DETERMINATION
-.-II
L5V dry battery FIG.
1. Apparatus
for electrolytic
reduction.
See text
for description
Some years ago we showed that protein disulfide bonds could be readily and quantitatively cleaved by electrolytic reduction, each disulfide producing two thiol groups (9). and more recently we have applied this method of reduction to simpler. nonprotein disultides (IO). The apparatus required is inexpensive and simply constructed. The overwhelming advantage of electrolytic reduction is that the reduction is stopped simply by switching off the current and. as no reducing agent is then present, no separating procedures are required. Estimation of thiol groups formed by the reduction may therefore be performed simply by mixing with DTNB and measuring the resulting yellow color at 412 nm. This communication describes the application of electrolytic reduction to the quantitative determination of disulfide groups in a number of proteins. The method is shown to give results in excellent agreement with their known disulfide contents and to have distinct advantages over other established procedures. EXPERIMENTAL The apparatus required is illustrated in Fig. I. It is similar to that previously described ( 10) except that a smaller glass vessel is used as the cathode compartment. A, so that 2 ml of solution can conveniently be electrolyzed. A small pool of Analar grade mercury serves as the cathode, and electrical contact between this and the outside of the vessel is made with a piece of platinum wire fused through the glass side. The surface of the mercury can be stirred with a small magnetic flea. Compartment A is fitted with a rubber stopper carrying two syringe needles, a salt-bridge. and a small capped hole to permit sample removal. The needles allow nitrogen
172
P. D. J. WEITZMAN TABLE PROTEINS Protein
USED.THEIR Source
I
SOURCES AND ULTRAVIOLET Supplier
ABSORBANCES
Absorbance
Reference
Insulin
Bovine
Sigma
e&278
x l(r
(12)
Pepsin
Swine
Worthington
~~~(278 nm) = 5.17 x l(r
(131
cY-Lactalbumin
Bovine
Gift of Mr. Shindler
E;T,,,(280 nm) = 20.1 (MW = 16,200)
(141
Lysozyme
Chicken white
Worthington
E/:,,(280 (MW
nm) = 27.3 = 14.400)
(IS)
Worthington
Ei:,(280 (MW
nm) = 7.3 = 13,700)
(161
Bovine
E:T,,(280 (MW
nm) = 20.4 = 25,000)
(16)
Bovine
E,rTm(280 nm) = 20.4 (MW = 25,000)
(161
E,‘?,,(280 nml = 14.3 (MW = 23,800)
(161
~~~(280 nm) = 4.36
(171
Ribonuclease
A
a-Chymotrypsin
Chymotrypsinogen
A Bovine
Trypsin
Serum
Bovine
egg
albumin
Bovine
Worthington
Mel
nml = 0.61
x l(r
to be flushed through the solution. The salt-bridge (2% agar in saturated KCl) makes electrical contact between the cathode compartment and the anode compartment, B, which consists of a platinum electrode dipping into saturated KC1 solution. The potential is applied directly from a 4.5 V dry battery. Solutions of proteins were made up in 1 mM HCl such that the concentration of disulfide was in the region of 1-2 mM. Precise concentrations of the proteins were determined from their uv absorbances (Table 1). For electroreduction, 0.20 ml of protein solution and 1.80 ml of 6 M guanidine hydrochloride in 50 mM acetate buffer, pH 4, were introduced into the cathode compartment over the mercury pool, stirred continuously, and deoxygenated for 2-3 min by a stream of oxygen-free nitrogen. The syringe needle carrying nitrogen into the solution was then raised until its tip was a little above the surface of the solution so that the continued flow of nitrogen could maintain oxygen-free conditions within the cathode vessel without causing frothing of the protein. Electrolysis was then performed by applying the output potential of the battery across the electrodes. At intervals, O.lO-ml samples were withdrawn and added to a semimicro spectrophotometer cuvette (l-cm path length) containing 0.89 ml of 0.5 M Tris-HCl, pH 8.5, and 0.01 ml of 10 mM DTNB. The contents of the cuvette were mixed, and the absorbance at 412 nm was measured. A Zeiss
DISULFIDE
0 FIG.
2. Electrolytic
GROUP
10 minutes reduction
173
DETERMINATION
30
20
of the disulfide
bonds
of insulin.
PMQ II spectrophotometer was used and readings were made against a blank containing Tris buffer and DTNB but without protein. On the basis of a molar absorption coefficient of 13,600 M-‘cm-‘for the yellow thionitrobenzoate ion (11) the thiol concentration, and hence the number of disulfide bonds cleaved, could be determined. Guanidine hydrochloride was Ultrapure grade from Schwarz/Mann; DTNB (5,5’-dithiobis[2-nitrobenzoic acid]) was from Sigma. Table 1 lists the proteins used, their suppliers, and their molar absorption coefficients (EJ or values of specific absorbance (E,"&,,) and molecular weights (MW) used to calculate precise concentrations of solutions. TABLE
2
COMPARISON OF THE OBSERVED DISULFIDE CONTENTS OF PROTEINS WITH ESTABLISHED VALUES
Protein
Established tSS/mol)
Insulin Pepsin cu-Lactalbumin Lysozyme Ribonuclease A cu-Chymotrypsin Chymotrypsinogen A Trypsin Bovine serum albumin” n Mean of triplicate determinations. ti The fractional SH content of unreduced DTNB and allowed for.
Observed ( SSimol)”
3 3 4 4 4 5 5 6 I7
bovine
3.0
2.9 3.9 3.9 4.0 5.0 4.8 5.9 17.1
serum
albumin
was determined
with
174
P. D. J. WEITZMAN
RESULTS
AND
DISCUSSION
Acidic conditions were employed for the electroreduction in order to stabilize the thiol groups during their transfer from the electrolysis vessel to the DTNB solution, and guanidine was included to facilitate rapid and complete reduction of disulfide bonds. Figure 2 shows the progress curve for the electroreduction of insulin; total reduction of the three disulfide bonds was achieved in about 20 min. Essentially similar time courses were obtained with the other proteins examined, though some required an electrolysis time of about 1 hr for complete reduction. The results of these studies are presented in Table 2, where the observed disulfide contents are compared with established values. Very good agreement was obtained in every case. In presenting this method of disulfide analysis. it is appropriate to consider its merits relative to those of other analytical procedures. In the case of sulfitolysis, the reversibility of the reaction requires that a large excess of sulfite be present. This prevents the use of DTNB to determine the extent of disulfide cleavage as DTNB is itself cleaved by sulfite (18), and recourse must be had to amperometric titration of the thiol groups with heavy-metal reagents (2,3). Reductive cleavage of disulfides has the potential advantage over sulfitolysis that two thiol groups, rather than one, are formed from each disulfide. However, the traditional use of thiol compounds themselves to effect disulfide reduction means that the extent of such reduction cannot be determined directly by thiol analysis either with DTNB or by heavymetal titration. Instead, the low molecular weight thiol compound must first be removed from the protein. One method for doing this involves precipitation of the protein with trichloroacetic acid. After further washing the precipitate with acid it is dissolved in the presence of urea and sodium dodecyl sulfate, and then treated with DTNB (19). However, in view of the risk of reoxidation of thiol groups on the reduced protein during its separation from the low molecular weight thiol reagent, the more common procedure is first to block all thiol groups by alkylation. Low molecular weight compounds are then removed, the protein is completely hydrolyzed, and the alkylated cysteine is determined. This is a laborious procedure; it is both time consuming and dependent on an amino acid analyzer. Moreover, this procedure, like that of cysteic acid determination following oxidative cleavage of disulfides by performic acid, may require the application of a correction factor to account for incomplete recovery of the measured species. Reductive cleavage with borohydride offers the possibility of direct estimation of thiol groups by amperometric titration (7), but this again requires access to. and familiarity with, polarographic equipment. Reduction with borohydride has been followed by estimation of thiol groups
DISULFIDE
GROUP
DETERMINATION
175
with DTNB (8). but care must be taken to destroy all excess borohydride which would otherwise itself react with DTNB. When this latter procedure was applied to a number of proteins (8), some gave gelatinous precipitates of insoluble protein and thus required filtration before calorimetric measurement. Furthermore, one of the proteins examined, chymotrypsinogen. failed to give the correct value for disulfide content. Failure to obtain complete reduction has also been encountered when mercaptoethanol was used to cleave disulfides (10) and, in other cases, more vigorous conditions were required for the reduction of particular proteins (2 I ,22). Although most investigators have employed one or another of these indirect methods. several direct methods for disulfide analysis have been proposed. A method based on the disulfide interchange reaction using N.N’-bis(2.4-dinitrophenyl)cystine has been reported (15) but the extremely long reaction periods (weeks) that are required detract from its usefulness. A method based on reduction of disulfides with dithiothreitol and determination of the resulting monothiols with DTNB in the presence of arsenite, which forms a tight complex with the dithiothreitol. has also been presented (23) but was not found to be applicable to disulfides in proteins. More recently, a direct method has been described in which the reduction of protein disulfide bonds with dithiothreitol can be followed by spectrophotometric measurement (at 310 nm) of the oxidized dithiothreitol formed (34). In this method, however. reduction of lysozyme was very sluggish and did not proceed to completion. Finally. it has been proposed that the thionitrobenzoate ion can cleave protein disulfide bonds and provide a means for their color-metric determination (35). but very serious doubt has been cast on this claim (26.27). When dealing with proteins containing both disulfide and thiol groups, the present method. together with other methods of disulfide analysis, relies on measurement of such thiol groups prior to cleavage of the disulfides and measurement of the additional thiol groups so produced. It is conceivable that in proteins containing both disulfide and thiol groups some of the thiols might be unreactive to the specific reagent (DTNB etc.) even in the presence of denaturants but might become reactive in the course of disulfide cleavage as a result of relaxation of structural constraints. Such a situation poses problems for all methods of disulfide analysis and, where suspected, requires careful and extensive examination of diverse conditions and reagents in order that the true thiol group content of the protein may first be established. The analytical procedure described in the present communication offers some distinct advantages over the methods described above. The apparatus required is an inexpensively and simply constructed glass vessel and the electrolysis is powered by a small dry battery. The results obtained indicate that. in all cases examined. reduction proceeds rapidly and to
I76
P. D. J. WEITZMAN
completion, while the determination of the thiol groups with DTNB confers high sensitivity on the method. The absence of any reducing agent in the reaction mixture means that no treatment of the product is required prior to direct estimation of the thiol groups formed. As a result, the complete analysis is simple and quickly performed and no correction factors for incomplete recovery need be applied. These characteristics should commend the method to the examination of new disulfidecontaining proteins and it should also be noted that the method is equally applicable to the analysis of nonprotein disulfides (10). ACKNOWLEDGMENTS 1 thank Professor Nathan Sharon and Dr. Sarah Rogozinsky of the Department of Biophysics, Weizmann Institute of Science. Rehovot, Israel. for their hospitality and laboratory facilities. Financial assistance from the European Molecular Biology Organization and the Wellcome Trust is gratefully acknowledged.
REFERENCES 1. Moore. S. (1963) .I. Biol. Chem. 238, 235-237. 2. Cecil, R. (1963) in The Proteins (Neurath, H.. ed.), 2nd ed., Vol. I. pp. 379-476. Academic Press, New York. 3. Carter, J. R. (1959) J. Bid/. Cham. 234, 1705- 1710. 4. Crestfield, A. M.. Moore, S., and Stein. W. H. (1963) J. Biol. Chem. 238, 622-627. 5. Konigsberg. W. (1972) ire Methods in Enzymology (Hirs, C. H. W.. and Timasheff. S. N.. eds.), Vol. 25. pp. 185-188, Academic Press, New York. 6. Weil. L.. and Seibles. T. S. (1961) Arch. Biochrm. Biophys. 95, 470-473. 7. Brown, W. D. (1960) Biochim. Biopphys. Acfa 44. 365-367. 8. Cavallini, D.. Graziani. M. T., and Dupre. S. (1966) Nature (London) 212, 294-295. 9. Cecil, R., and Weitzman. P. D. J. (1964) Biochum. /. 93, I- Il. 10. Weitzman, P. D. J.. Hewson. J. K., and Parker. M. G. (1974)E‘EBS Left. 43, 101-103. 1 I. Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77. 12. Weil, L., Seibles. T. S.. and Herskovits, T. T. (1965) Arch. Biwlwrtr. Biophys. 111. 308-320. 13. Blumenfeld, 0.0.. Leonis. J., and Perlmann, Cl. E. ( I%O)J. Biol. C/ieni. 235, 379-382. 14. Kronman, M. J.. and Andreotti. R. E. (1964) Biwhernisrry 3, 1145- 1151. 15. Glazer, A. N.. and Smith, E. L. (1961)J. Biol. Chrm. 236, 416-421. 16. Worthington Enzyme Manual (1972) Worthington Biochemical Corporation. Freehold, N.J. 17. Wetlaufer. D. B. (1962)Adt~~rz. Protein Chem. 17, 303-390. 18. Weitzman, P. D. J. (1975) Am/. Biochem. 64, 628-630. 19. Habeeb, A. F. S. A. (1972) in Methods in Enzymology (Hirs. C. H. W.. and Timasheff, S. N., eds.). Vol. 25. pp. 457-464. Academic Press. New York. 20. Shapira. E.. and Arnon, R. (1%9) J. Biol. C/ie,,r. 244, 1026- 1032. 21. Ferdinand. W., Moore, S., and Stein. W. H. ( 1965)Riochirn. Biopliy.~. A<,frr 96.524-527. 22. Dopheide, T. A. A., Moore. S.. and Stein, W. H. (1967)5. Viol. Che,u. 242, 1833- 1837. 23. Zahler. W. L., and Cleland, W. W. (1968) J. Biol. Chem. 243, 716-719. 24. Iyer. K. S.. and Klee, W. A. (1973)5. Biol. Chern. 248, 707-710. 25. Robyt, J. F.. Ackerman. R. J.. and Chittenden. C. G. (1971) Arch. Biocherr~. Biophys. 147, 262-269. 26. Brocklehurst, K.. Kierstan, M., and Little. G. (1972) Rioclwm. J. 128, 81 I-816. 27. Weitrman. P. D. J. (1975) Bia(~lie~r. J. 149, 281-283.
BIOCHEMISTRY
76, l70- 176 ( 1976)
Determination of Disulfide Groups Sensitive Electroreductive-Calorimetric
in Proteins by a Method
P. D. J. WEITZMAN Departmetzt
oj’Biochemistry.
Utzi~‘ersity
of Leicester,
Leicester,
EngIrd
Received April 19, 1976: accepted June 8, 1976 A method is described for the determination of the total disulfide content of proteins. It is based on electrolytic cleavage of disulfide bonds at a mercury pool cathode in acidic buffer containing guanidine, followed by calorimetric estimation of the liberated thiol groups with 5,5’-dithiobis@-nitrobenzoate). The general applicability of the method is demonstrated with a range of established proteins and the results obtained are in excellent agreement with known values. The method offers distinct advantages over other available procedures and is applicable to both protein and nonprotein disulfides.
Disulfide groups constitute an important structural feature of proteins. They play a major role in the maintenance of three-dimensional protein structure, and various methods have been devised for their quantitative estimation. Whereas the related thiol groups may be simply and directly determined with a variety of fairly specific reagents, methods for determining disulfide groups are generally indirect and rely on some form of bond cleavage followed by estimation of the reaction products. Three types of cleavage have been used as the basis of disulfide bond analysis. (a) Oxidation with per-formic acid and estimation of cysteic acid after total acid hydrolysis of the protein (1). (b) Reaction with sulfite (sulfitolysis) to produce an equimolar mixture of thiosulfate and thiol, followed by estimation of the latter by amperometric (polarographic) titration with heavy-metal reagents (2,3). (c) Reduction with mercaptoethanol, dithiothreitol, or sodium borohydride, followed by alkylation (generally carboxymethylation) of the cysteine residues, total acid hydrolysis of the protein, and estimation of the derivatized cysteine (4-6). Determination of the thiol groups formed on borohydride reduction has also been done by amperometric titration (7) or calorimetrically (8) after reaction with 5,5’-dithiobis (?-nitrobenzoate) (DTNB). The above procedures are relatively laborious and time consuming and, moreover, require specialized equipment in the form of an amino acid analyzer or polarographic apparatus. My interest in the electrolytic reduction of disulfide bonds prompted the present work aimed at devising an improved procedure for quantitative disulfide analysis. 170 CopyrIght Q 1976 h) Academic Pres Inc. All rights of reproduction in nny form rewired.
DISULFIDE
GROUP
171
DETERMINATION
-.-II
L5V dry battery FIG.
1. Apparatus
for electrolytic
reduction.
See text
for description
Some years ago we showed that protein disulfide bonds could be readily and quantitatively cleaved by electrolytic reduction, each disulfide producing two thiol groups (9). and more recently we have applied this method of reduction to simpler. nonprotein disultides (IO). The apparatus required is inexpensive and simply constructed. The overwhelming advantage of electrolytic reduction is that the reduction is stopped simply by switching off the current and. as no reducing agent is then present, no separating procedures are required. Estimation of thiol groups formed by the reduction may therefore be performed simply by mixing with DTNB and measuring the resulting yellow color at 412 nm. This communication describes the application of electrolytic reduction to the quantitative determination of disulfide groups in a number of proteins. The method is shown to give results in excellent agreement with their known disulfide contents and to have distinct advantages over other established procedures. EXPERIMENTAL The apparatus required is illustrated in Fig. I. It is similar to that previously described ( 10) except that a smaller glass vessel is used as the cathode compartment. A, so that 2 ml of solution can conveniently be electrolyzed. A small pool of Analar grade mercury serves as the cathode, and electrical contact between this and the outside of the vessel is made with a piece of platinum wire fused through the glass side. The surface of the mercury can be stirred with a small magnetic flea. Compartment A is fitted with a rubber stopper carrying two syringe needles, a salt-bridge. and a small capped hole to permit sample removal. The needles allow nitrogen
172
P. D. J. WEITZMAN TABLE PROTEINS Protein
USED.THEIR Source
I
SOURCES AND ULTRAVIOLET Supplier
ABSORBANCES
Absorbance
Reference
Insulin
Bovine
Sigma
e&278
x l(r
(12)
Pepsin
Swine
Worthington
~~~(278 nm) = 5.17 x l(r
(131
cY-Lactalbumin
Bovine
Gift of Mr. Shindler
E;T,,,(280 nm) = 20.1 (MW = 16,200)
(141
Lysozyme
Chicken white
Worthington
E/:,,(280 (MW
nm) = 27.3 = 14.400)
(IS)
Worthington
Ei:,(280 (MW
nm) = 7.3 = 13,700)
(161
Bovine
E:T,,(280 (MW
nm) = 20.4 = 25,000)
(16)
Bovine
E,rTm(280 nm) = 20.4 (MW = 25,000)
(161
E,‘?,,(280 nml = 14.3 (MW = 23,800)
(161
~~~(280 nm) = 4.36
(171
Ribonuclease
A
a-Chymotrypsin
Chymotrypsinogen
A Bovine
Trypsin
Serum
Bovine
egg
albumin
Bovine
Worthington
Mel
nml = 0.61
x l(r
to be flushed through the solution. The salt-bridge (2% agar in saturated KCl) makes electrical contact between the cathode compartment and the anode compartment, B, which consists of a platinum electrode dipping into saturated KC1 solution. The potential is applied directly from a 4.5 V dry battery. Solutions of proteins were made up in 1 mM HCl such that the concentration of disulfide was in the region of 1-2 mM. Precise concentrations of the proteins were determined from their uv absorbances (Table 1). For electroreduction, 0.20 ml of protein solution and 1.80 ml of 6 M guanidine hydrochloride in 50 mM acetate buffer, pH 4, were introduced into the cathode compartment over the mercury pool, stirred continuously, and deoxygenated for 2-3 min by a stream of oxygen-free nitrogen. The syringe needle carrying nitrogen into the solution was then raised until its tip was a little above the surface of the solution so that the continued flow of nitrogen could maintain oxygen-free conditions within the cathode vessel without causing frothing of the protein. Electrolysis was then performed by applying the output potential of the battery across the electrodes. At intervals, O.lO-ml samples were withdrawn and added to a semimicro spectrophotometer cuvette (l-cm path length) containing 0.89 ml of 0.5 M Tris-HCl, pH 8.5, and 0.01 ml of 10 mM DTNB. The contents of the cuvette were mixed, and the absorbance at 412 nm was measured. A Zeiss
DISULFIDE
0 FIG.
2. Electrolytic
GROUP
10 minutes reduction
173
DETERMINATION
30
20
of the disulfide
bonds
of insulin.
PMQ II spectrophotometer was used and readings were made against a blank containing Tris buffer and DTNB but without protein. On the basis of a molar absorption coefficient of 13,600 M-‘cm-‘for the yellow thionitrobenzoate ion (11) the thiol concentration, and hence the number of disulfide bonds cleaved, could be determined. Guanidine hydrochloride was Ultrapure grade from Schwarz/Mann; DTNB (5,5’-dithiobis[2-nitrobenzoic acid]) was from Sigma. Table 1 lists the proteins used, their suppliers, and their molar absorption coefficients (EJ or values of specific absorbance (E,"&,,) and molecular weights (MW) used to calculate precise concentrations of solutions. TABLE
2
COMPARISON OF THE OBSERVED DISULFIDE CONTENTS OF PROTEINS WITH ESTABLISHED VALUES
Protein
Established tSS/mol)
Insulin Pepsin cu-Lactalbumin Lysozyme Ribonuclease A cu-Chymotrypsin Chymotrypsinogen A Trypsin Bovine serum albumin” n Mean of triplicate determinations. ti The fractional SH content of unreduced DTNB and allowed for.
Observed ( SSimol)”
3 3 4 4 4 5 5 6 I7
bovine
3.0
2.9 3.9 3.9 4.0 5.0 4.8 5.9 17.1
serum
albumin
was determined
with
174
P. D. J. WEITZMAN
RESULTS
AND
DISCUSSION
Acidic conditions were employed for the electroreduction in order to stabilize the thiol groups during their transfer from the electrolysis vessel to the DTNB solution, and guanidine was included to facilitate rapid and complete reduction of disulfide bonds. Figure 2 shows the progress curve for the electroreduction of insulin; total reduction of the three disulfide bonds was achieved in about 20 min. Essentially similar time courses were obtained with the other proteins examined, though some required an electrolysis time of about 1 hr for complete reduction. The results of these studies are presented in Table 2, where the observed disulfide contents are compared with established values. Very good agreement was obtained in every case. In presenting this method of disulfide analysis. it is appropriate to consider its merits relative to those of other analytical procedures. In the case of sulfitolysis, the reversibility of the reaction requires that a large excess of sulfite be present. This prevents the use of DTNB to determine the extent of disulfide cleavage as DTNB is itself cleaved by sulfite (18), and recourse must be had to amperometric titration of the thiol groups with heavy-metal reagents (2,3). Reductive cleavage of disulfides has the potential advantage over sulfitolysis that two thiol groups, rather than one, are formed from each disulfide. However, the traditional use of thiol compounds themselves to effect disulfide reduction means that the extent of such reduction cannot be determined directly by thiol analysis either with DTNB or by heavymetal titration. Instead, the low molecular weight thiol compound must first be removed from the protein. One method for doing this involves precipitation of the protein with trichloroacetic acid. After further washing the precipitate with acid it is dissolved in the presence of urea and sodium dodecyl sulfate, and then treated with DTNB (19). However, in view of the risk of reoxidation of thiol groups on the reduced protein during its separation from the low molecular weight thiol reagent, the more common procedure is first to block all thiol groups by alkylation. Low molecular weight compounds are then removed, the protein is completely hydrolyzed, and the alkylated cysteine is determined. This is a laborious procedure; it is both time consuming and dependent on an amino acid analyzer. Moreover, this procedure, like that of cysteic acid determination following oxidative cleavage of disulfides by performic acid, may require the application of a correction factor to account for incomplete recovery of the measured species. Reductive cleavage with borohydride offers the possibility of direct estimation of thiol groups by amperometric titration (7), but this again requires access to. and familiarity with, polarographic equipment. Reduction with borohydride has been followed by estimation of thiol groups
DISULFIDE
GROUP
DETERMINATION
175
with DTNB (8). but care must be taken to destroy all excess borohydride which would otherwise itself react with DTNB. When this latter procedure was applied to a number of proteins (8), some gave gelatinous precipitates of insoluble protein and thus required filtration before calorimetric measurement. Furthermore, one of the proteins examined, chymotrypsinogen. failed to give the correct value for disulfide content. Failure to obtain complete reduction has also been encountered when mercaptoethanol was used to cleave disulfides (10) and, in other cases, more vigorous conditions were required for the reduction of particular proteins (2 I ,22). Although most investigators have employed one or another of these indirect methods. several direct methods for disulfide analysis have been proposed. A method based on the disulfide interchange reaction using N.N’-bis(2.4-dinitrophenyl)cystine has been reported (15) but the extremely long reaction periods (weeks) that are required detract from its usefulness. A method based on reduction of disulfides with dithiothreitol and determination of the resulting monothiols with DTNB in the presence of arsenite, which forms a tight complex with the dithiothreitol. has also been presented (23) but was not found to be applicable to disulfides in proteins. More recently, a direct method has been described in which the reduction of protein disulfide bonds with dithiothreitol can be followed by spectrophotometric measurement (at 310 nm) of the oxidized dithiothreitol formed (34). In this method, however. reduction of lysozyme was very sluggish and did not proceed to completion. Finally. it has been proposed that the thionitrobenzoate ion can cleave protein disulfide bonds and provide a means for their color-metric determination (35). but very serious doubt has been cast on this claim (26.27). When dealing with proteins containing both disulfide and thiol groups, the present method. together with other methods of disulfide analysis, relies on measurement of such thiol groups prior to cleavage of the disulfides and measurement of the additional thiol groups so produced. It is conceivable that in proteins containing both disulfide and thiol groups some of the thiols might be unreactive to the specific reagent (DTNB etc.) even in the presence of denaturants but might become reactive in the course of disulfide cleavage as a result of relaxation of structural constraints. Such a situation poses problems for all methods of disulfide analysis and, where suspected, requires careful and extensive examination of diverse conditions and reagents in order that the true thiol group content of the protein may first be established. The analytical procedure described in the present communication offers some distinct advantages over the methods described above. The apparatus required is an inexpensively and simply constructed glass vessel and the electrolysis is powered by a small dry battery. The results obtained indicate that. in all cases examined. reduction proceeds rapidly and to
I76
P. D. J. WEITZMAN
completion, while the determination of the thiol groups with DTNB confers high sensitivity on the method. The absence of any reducing agent in the reaction mixture means that no treatment of the product is required prior to direct estimation of the thiol groups formed. As a result, the complete analysis is simple and quickly performed and no correction factors for incomplete recovery need be applied. These characteristics should commend the method to the examination of new disulfidecontaining proteins and it should also be noted that the method is equally applicable to the analysis of nonprotein disulfides (10). ACKNOWLEDGMENTS 1 thank Professor Nathan Sharon and Dr. Sarah Rogozinsky of the Department of Biophysics, Weizmann Institute of Science. Rehovot, Israel. for their hospitality and laboratory facilities. Financial assistance from the European Molecular Biology Organization and the Wellcome Trust is gratefully acknowledged.
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