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Products of the reaction of peroxyacetyl nitrate with sulfhydryl compounds
ARCHIVES
OF
BIOCHEMISTRY
Products
AND
BIOPHYSICS
of the Reaction Sulfhydryl J. B. MUDD
Department
of Biochemistry
237-241 (1969)
1%.
of Peroxyacetyl
January
with
Compounds”* T. T. McMANUS
AND
and Statewide Air Pollution Riverside, California
Received
Nitrate
Research Center, 92502
4, 1969; accepted
March
University
of California
25, 1969
Peroxyacetyl nitrate reacts with reduced glutathione producing oxidized glutathione and S-acetyl glutathione. Reaction of peroxyacetyl nitrate with reduced coenzyme A results in the formation of coenzyme A disulfide, accounting for 3545yo of the reacted sulfhydryl. The remaining products can be separated by ion exchange chromatography and are probably higher oxidation states. There is no evidence of formation of S-acetyl coenzyme A. The distribution of products obtained by treatment of coenzyme A with peroxyacetyl nitrate is similar to that obtained by treatment of coenzyme A with hydrogen peroxide. Treatment of proteins with peroxyacetyl nitrate has not provided any evidence of acetylation of sulfhydryl groups detectable by the hydroxamate test. Intramolecular disulfide bonds are formed by treatment of reduced ribonuclease with peroxyacetyl nitrate but no evidence has been obtained in favor of formation of intermolecular disulfide bonds.
Initial studies on the inactivation of enzymes by peroxyacetyl nitrate provided circumstantial evidence that the susceptible site was a sulfhydryl group (1). The reaction of sulfhydryl groups of hemoglobin, papain, and reduced ribonuclease with peroxyacetyl nitrate was subsequently demonstrated (2). In the elucidation of the chemistry of the reaction of peroxyacetyl nitrate with the sulfhydryl groups of glutathione, it was found that as much as 60% of the reacted sulfhydryl could be accounted for as disulfide, and it appeared likely that the remainder was in the form of X-acetyl glutathione (3). The work described in this paper was
designed to assess accurately the proportion of oxidation and acetylation of sulfhydryl groups by peroxyacetyl nitrate. The compounds chosen for study were glutathione, coenzyme A, and reduced pancreatic ribonuclease. It is hoped that these studies will help to elucidate the mechanism of toxicity of the peroxyacyl nitrates found in polluted urban atmospheres.
1 This work was supported in part by Research Grant AP 0071 from the National Air Pollution Control Administration, United States Public Health Service. 2 The authors are grateful to Mr. K. M. Holtzclaw for preparation of the peroxyacetyl nitrate and to Miss Peggy Jones for assistance with some of the experiments. 237
MATERIA’LS
AND
METHODS
Peroxyacetyl nitrate (CHaCO.OON02) was prepared by the method of Stephens et al. (4). The pure preparation was diluted with nitrogen and stored under pressure in a gas cylinder at 5”. Reaction mixtures were exposed to peroxyacetyl nitrate by permitting the gas to bubble through the solution from a capillary tip at controlled flow rates. The amount of peroxyacetyl nitrate dissolved in the reaction mixture was determined by assaying an aliquot of the solution for nitrite by reaction with sulfanilic acid and a-naphthylamine (5). In the absence of dissolved compounds that will react, the peroxyacetyl nitrate breaks down in aqueous media to acetate,
238
MUDD
AND McMANUS
nitrite, and molecular oxygen (6). In the presence of dissolved compounds that will react with peroxyacetyl nitrate, the nitrite assay shows the maximal amount of peroxyacetyl nitrate that could have reacted. Sulfhydryl groups of glutathione were measured by the method of Ellman (7). GSSG was assayed by measuring the oxidation of TPNH in the presence of glutathione reductase (8). Reaction mixtures contained 209 pmoles Tris-HCI, pH 7.5, 0.5 pmoles TPNH, 1 mg bovine serum albumin, 1 pmole EDTA, GSSG, and enzyme in a final volume of 3.0 ml. Thioesters were assayed by reacting the hydroxamate with ferric chloride (9). Concentrations of the hydroxamate were determined using S-acetyl glutathione as a standard. Chromatography of coenzyme A, its derivatives and oxidation products, was on columns of DEAE cellulose according to the method of Moffat and Khorana (10). Acetyl CoA was prepared by the method of Simon and Shemin (11). Pancreatic ribonuclease was purchased from Nutritional Biochemicals Corporation. Reduced ribonuclease was prepared by the method of Haber and Anfinsen (12). Protein concentration of the ribonuclease solutions was determined by using the extinction coefficients reported by White (13). Sulfhydryl concentration of the protein solutions was determined by titration with p-mercuribenzoate according to the method of Boyer (14). S-acetylation of the protein was assayed for by the hydroxamate method (9). RESULTS
Reaction of peroxyacetyl nitrate with glutathione. The results of an experiment in which peroxyacetyl nitrate reacted with GSH are shown in Table I. The reaction of sulfhydryl groups was 34 moles per mole of nitrite subsequently found in the reaction mixture. Of the reacted sulfhydryl groups, practically all can be accounted for as GSSG and S-acetyl glutathione. No other products have been identified. Approximately twice as much GSH was converted to GSSG as was converted to S-acetyl glutathione in the initial stages of the reaction. However, this ratio increased on extended exposure of GSH to peroxyacetyl nitrate. There was also some variation in the amount of sulfhydry1 reacted per mole of nitrite at different times of reaction-3.3, 3.8, 3.8, and 4.0 at 10, 20, 30, and 40 min treatment, respectively. The details of the reaction sequence have yet to be investigated.
TABLE REACTION
Treatment (min)
10 20 30 40
I
OF PEROXYACETYL GLUTATHIONE’
NOz ~moles)
GSH Oxidized Gunoles)
0.54 1.08 1.62 2.16
1.80 4.10 6.10 8.65
NITRATE
voF; GS-acetyl Olmoles) bmoles)
1.38 2.86 3.92 6.04
0.76 1.37 1.74 2.20
WITH
$5 GSSG/ GS-acetyl
1.82 2.08 2.25 2.74
mReaction mixtures contained 20 rmoles of GSH and 150 rmoles phosphate buffer, pH 7.0, in a final volume of 5.0 ml. At the end of the reaction period duplicate O.l-ml aliquots were taken for analysis of sulfhydryl. Duplicate 0.2~ml aliquots were taken for analysis of nitrite. Duplicate l.O-ml aliquots were taken for the assay of thioester by the hydroxamate method. Duplicate 0.4-ml aliquots were taken for the assay of GSSG by glutathione reductase. The results presented are from one of five time-course experiments and are typical of the data obtained.
Reaction of peroxyacetyl nitrate with coenzyme A. Because of the substantial amount of acetylation of the sulfhydryl groups of glutathione, we decided to examine the possibility of acetylation of other sulfhydryl compounds. Coenzyme A was exposed to varying amounts of peroxyacetyl nitrate and the reaction of sulfhydryl groups followed. The reaction products were separated by ion-exchange chromatography in an attempt to assess the relative amounts of oxidation and acetylation. Figure 1 shows the elution patterns for the coenzyme A before and after 60 min exposure to peroxyacetyl nitrate. The chromatography of the products of oxidation of coenzyme A by hydrogen peroxide is shown for comparison. The sample of coenzyme A used in these experiments contained an impurity which accounted for 12% of the absorbance at 260 W, but this compound, which emerged before CoASH, was unaffected by peroxyacetyl nitrate. Acetyl coenzyme A is eluted in this system in exactly the same position as CoASH. The two can be distinguished in the effluent fractions by the difference in the 260/230 ratio, the absorbance at 230 rnp being much greater for the thioester. The data in Table II summarize the results of an experiment in which CoASH was ex-
SULFHYDRYL COENZYME
4
OS!
3 COENZYME PEROXIDE
OXIDATION
BY PEROXYACETYL
A. --T-------1
A TREATED
WITH
HYDROGEN
4 A
I 061 I 041
02! u 100 zoo ml
300
\,1 -_-_L--l . 400
500
EFFLUENT
FIG. 1. Ion-exchange chromatography of coenzyme A and oxidation products; (1) chromatography of coenzyme A standard; (2) chromatography of coenzyme A after treatment with peroxyacetyl nitrate. Conditions and procedure as for Table II. The chromatogram shown is that for the sample exposed to 2.08~moles peroxyacetyl nitrate; (5) coenzyme A oxidized with excess hydrogen peroxide. Identification of peaks: 1, impurity; 2, reduced coenzyme A and oxidation products; 3, unidentified oxidation products; 4, oxidized coenzyme A (disulfide).
posed to increasing amounts of peroxyacetyl nitrate. The major product of the reaction of CoASH with peroxyacetyl nitrate was the disulfide of coenzyme A (peak 4). In addition, there were other oxidation products which have not yet been identified (peak 3). The products in peaks 3 and 4 did not account for the sulfhydryl which reacted even though the recovery according to absorbance at 260 rnp was 95 %. Assay of sulfhydryl in peak 2 showed that on the basis of 260 rnp absorbance, the compounds in peak 2 contained only 96, 95, 31, and 23 % of the sulfhydryl expected if the peak were pure CoASH after 15, 30, 45, and 60 min exposure, respectively. It appears likely that one of the oxidation products has the
239
NITRATE
same chromatographic properties as unreacted coenzyme A. Attempts to detect acetyl CoA in the reaction mixture by the hydroxamate assay were always negative. Measurement of the 260/230 ratio in both peaks 2 and 4 gave a ratio expected for CoA (either reduced or oxidized) and at no time gave a ratio expected for acetyl coenzyme A. The major reaction product when CoASH was treated with hydrogen peroxide was also the disulfide, and in addition there were compounds comparable to those found after reaction with peroxyacetyl nitrate. It may be concluded that the reaction of peroxyacetyl nitrate with CoASH produces no acetyl coenzyme A: The reaction is analogous to that with peroxide and does not depend on the anhydride character of peroxyacetyl nitrate. Reaction of peroxyacetyl nitrate with suljhydryE yroups of poteins. Attempts to measure the acetylation of human hemoglobin and bovine serum albumin were negative, even after complete reaction of the sulfhydryl groups. Chromatography of the products of the reaction with hemoglobin on Sephadex columns gave no indication of the formation TABLE
II
REACTION OF PEROXYACETYL NITRATE COENZYME Aa
15 30 45 60
0.52 1.04 1.56 2.08
1.05 2.18 2.51 2.81
2.36 1.73 1.60 1.30
0.210 0.273 0.510 0.593
WITH
0.418 0.945 0.915 1.00
0 Reaction mixtures contained 3 mg (approx. 3.5 pmoles) coenzyme A and 50 pmoles phosphate buffer pH 7.0 in a final volume of 5.0 ml. Aliquots of 0.1 ml were taken before and after exposure to peroxyacetyl nitrate for determination of sulfhydryl groups. Aliquots of 0.3 ml were taken at the end of the reaction period for the analysis of nitrite. An aliquot of 3.0 ml was applied to a column of DEAE cellulose (1.2 X 15 cm) and eluted according to Moffat and Khorana. Fractions (volume 7 ml) were measured at 260 rnp and recoveries in micromoles calculated on the basis of the adenine moiety. The results presented are from one of three time course experiments and are typical of the data obtained.
240
MUDD
AND McMANUS
of intermolecular disulfides detectable by changes in molecular weight. In order to obtain a protein of low molecular weight with a relatively large number of sulfhydryl groups per molecule, reduced ribonuclease was prepared. A solution containing 20 mg ribonuclease and 5 mg dithiothreitol in 2.0 ml of 8 M urea was flushed with nitrogen and stored overnight at lo. The solution was applied to a column (12 X 15 cm) of Sephadex G25 and eluted with 0.1 M acetic acid. The absorbance of the 3.0-ml fractions was measured at 277 rnE.cand protein concentration determined from this measurement. Aliquots of the protein solution were titrated with lo-3 M PMB at pH 4.5. An aliquot of 2.4 ml protein solution containing 370 rnp moles of protein and 2.76 clmoles sulfhydryl groups (7.45 sulfhydryl groups per molecule of protein) was added to 1.0 ml 0.1 M acetate buffer pH 4.5 and reacted with 3~moles of peroxyacetyl nitrate. At the end of the reaction period an aliquot was taken for titration of the sulfhydryl groups and it was found that 0.36 pmoles of sulfhydryl remained. The protein solution was assayed for thioester by the hydroxamate method and the result was negative. It is apparent in this case that even though the sulfhydryl groups of ribonuclease react with peroxyacetyl nitrate, acetylation is negligible as compared with oxidation. DISCUSSION
The results reported in this paper indicate that the reaction of peroxyacetyl nitrate with glutathione is relatively uncomplicated. The reaction is most simply summarized as the reaction of 3-4 moles of GSH per mole of peroxyacetyl nitrate with the production of equimolar amounts of GSSG and S-acetyl glutathione though deviations from this relationship were apparent in the later stages of reaction. In the case of coenzyme A 2-3 molecules of sulfhydryl groups were oxidized per molecule of peroxyacetyl nitrate. Attempts to detect S-acetyl coenzyme A were completely negative. The absence of S-acetyl coenzyme A cannot be explained on the grounds that it may be formed and then oxidized, because synthetic S-acetyl coenzyme A was resistant to reaction with peroxyacetyl nitrate. In addition to the disulfide of coenzyme A, the
reaction products include a number of compounds, detected after ion-exchange chromatography, which have not yet been identified but probably include compounds of higher oxidation state: sulfoxides, sulfones, sulfinic, and sulfonic acids. As far as the reactions of peroxyacetyl nitrate with proteins are concerned, one might expect from the results with glutathione that both acetylation and disulfrde formation would be measurable reactions and contribute to the toxicity of peroxyacetyl nitrate. On steric grounds one may expect less disulfide formation than with glutathione or coenzyme A. It is quite easy to measure the reaction of protein sulfhydryl groups with peroxyacetyl nitrate (2), but the identification of the reaction products is not so easy. We have not been able to find any evidence for the formation of S-acetylated protein. When reduced ribonuclease is allowed to oxidize slowly, reformation of disulfide bonds results in virtually complete recovery of activity (12). The recovery of some enzymic activity after treatment of reduced ribonuclease with peroxyacetyl nitrate shows that disulfide formation occurs (2). Other products of oxidation include higher oxidation states since cysteic acid is found in the hydrolyzate of papain after treatment with peroxyacetyl nitrate (2). The formation of disulfides from protein sulfhydry1 groups could be intramolecular, as in ribonuclease, or intermolecular. In our experiments with human hemoglobin, we have obtained no evidence for an increase in molecular weight as would be expected for the formation of dimers. The formation of polymeric material has been reported as a result of exposure of protein solution to lipid peroxides (15, 16). It is not clear in these cases, however, that the polymeric material is formed as a result of disulfide formation. REFERENCES 1. MUDD, J. B., Arch. Biochem. Biophys. 102, 59 (1963). 2. MUDD, J. B., LEAVITT, R., AND KERSEY, W. H., J. Biol. Chem. 241,408l (1966). 3. MUDD, J. B., J. Biol. Chem. 24l, 4077 (1966). 4. STEPHENS, E. R., DARLEY, E. F., TAYLOR, 0. C., AND SCOTT, W. E., I&em. J. Air Water Pollution 4, 79 (1961).
SULFHYDRYL
OXIDATION
BY PEROXYACETYL
5. SNELL, F. D., AND SNELL, C. T., “Calorimetric Methods of Analysis,” p. 644. Van Nostrand, Princeton, New Jersey, (1936). 6. STEPHENS, E. R., Atmos. Envir. 1, 19 (1967). 7. ELLMAN, G. L., Arch. Biochem. Biophys. 82, 70 (1959). 8. RACKER, E., Methods Enzymol., II, 722 (1955). 9. STADTMAN, E. R., Methods Enzymol., III, 228 (1957). 10. MOFFAT, J. G., AND KHORANA, M. G., J. Am. Chem. Sot. 63, 663 (1961).
NITRATE
241
11. SIMON, E. J., AND SHEMIN, D., J. Am. Chem. Sot. 76, 2520 (1953). 12. HABER, E., AND ANFINSEN, C. B., J. Biol. Chem. 237, 1839 (1962). 13. WHITE, F. H., JR., J. Biol. Chem. 236, 1353 (1961). 14. BOYER, P. D., J. Am. Chem. Sot. 76, 4331 (1954). 15. ROUBAL, W. T., AND TAPPEL, A. L., Arch. Biochem. Biophys. 113,150 (1966). 16. O’BRIEN, P. J., Biochem. J. 102, 28P (1967).
OF
BIOCHEMISTRY
Products
AND
BIOPHYSICS
of the Reaction Sulfhydryl J. B. MUDD
Department
of Biochemistry
237-241 (1969)
1%.
of Peroxyacetyl
January
with
Compounds”* T. T. McMANUS
AND
and Statewide Air Pollution Riverside, California
Received
Nitrate
Research Center, 92502
4, 1969; accepted
March
University
of California
25, 1969
Peroxyacetyl nitrate reacts with reduced glutathione producing oxidized glutathione and S-acetyl glutathione. Reaction of peroxyacetyl nitrate with reduced coenzyme A results in the formation of coenzyme A disulfide, accounting for 3545yo of the reacted sulfhydryl. The remaining products can be separated by ion exchange chromatography and are probably higher oxidation states. There is no evidence of formation of S-acetyl coenzyme A. The distribution of products obtained by treatment of coenzyme A with peroxyacetyl nitrate is similar to that obtained by treatment of coenzyme A with hydrogen peroxide. Treatment of proteins with peroxyacetyl nitrate has not provided any evidence of acetylation of sulfhydryl groups detectable by the hydroxamate test. Intramolecular disulfide bonds are formed by treatment of reduced ribonuclease with peroxyacetyl nitrate but no evidence has been obtained in favor of formation of intermolecular disulfide bonds.
Initial studies on the inactivation of enzymes by peroxyacetyl nitrate provided circumstantial evidence that the susceptible site was a sulfhydryl group (1). The reaction of sulfhydryl groups of hemoglobin, papain, and reduced ribonuclease with peroxyacetyl nitrate was subsequently demonstrated (2). In the elucidation of the chemistry of the reaction of peroxyacetyl nitrate with the sulfhydryl groups of glutathione, it was found that as much as 60% of the reacted sulfhydryl could be accounted for as disulfide, and it appeared likely that the remainder was in the form of X-acetyl glutathione (3). The work described in this paper was
designed to assess accurately the proportion of oxidation and acetylation of sulfhydryl groups by peroxyacetyl nitrate. The compounds chosen for study were glutathione, coenzyme A, and reduced pancreatic ribonuclease. It is hoped that these studies will help to elucidate the mechanism of toxicity of the peroxyacyl nitrates found in polluted urban atmospheres.
1 This work was supported in part by Research Grant AP 0071 from the National Air Pollution Control Administration, United States Public Health Service. 2 The authors are grateful to Mr. K. M. Holtzclaw for preparation of the peroxyacetyl nitrate and to Miss Peggy Jones for assistance with some of the experiments. 237
MATERIA’LS
AND
METHODS
Peroxyacetyl nitrate (CHaCO.OON02) was prepared by the method of Stephens et al. (4). The pure preparation was diluted with nitrogen and stored under pressure in a gas cylinder at 5”. Reaction mixtures were exposed to peroxyacetyl nitrate by permitting the gas to bubble through the solution from a capillary tip at controlled flow rates. The amount of peroxyacetyl nitrate dissolved in the reaction mixture was determined by assaying an aliquot of the solution for nitrite by reaction with sulfanilic acid and a-naphthylamine (5). In the absence of dissolved compounds that will react, the peroxyacetyl nitrate breaks down in aqueous media to acetate,
238
MUDD
AND McMANUS
nitrite, and molecular oxygen (6). In the presence of dissolved compounds that will react with peroxyacetyl nitrate, the nitrite assay shows the maximal amount of peroxyacetyl nitrate that could have reacted. Sulfhydryl groups of glutathione were measured by the method of Ellman (7). GSSG was assayed by measuring the oxidation of TPNH in the presence of glutathione reductase (8). Reaction mixtures contained 209 pmoles Tris-HCI, pH 7.5, 0.5 pmoles TPNH, 1 mg bovine serum albumin, 1 pmole EDTA, GSSG, and enzyme in a final volume of 3.0 ml. Thioesters were assayed by reacting the hydroxamate with ferric chloride (9). Concentrations of the hydroxamate were determined using S-acetyl glutathione as a standard. Chromatography of coenzyme A, its derivatives and oxidation products, was on columns of DEAE cellulose according to the method of Moffat and Khorana (10). Acetyl CoA was prepared by the method of Simon and Shemin (11). Pancreatic ribonuclease was purchased from Nutritional Biochemicals Corporation. Reduced ribonuclease was prepared by the method of Haber and Anfinsen (12). Protein concentration of the ribonuclease solutions was determined by using the extinction coefficients reported by White (13). Sulfhydryl concentration of the protein solutions was determined by titration with p-mercuribenzoate according to the method of Boyer (14). S-acetylation of the protein was assayed for by the hydroxamate method (9). RESULTS
Reaction of peroxyacetyl nitrate with glutathione. The results of an experiment in which peroxyacetyl nitrate reacted with GSH are shown in Table I. The reaction of sulfhydryl groups was 34 moles per mole of nitrite subsequently found in the reaction mixture. Of the reacted sulfhydryl groups, practically all can be accounted for as GSSG and S-acetyl glutathione. No other products have been identified. Approximately twice as much GSH was converted to GSSG as was converted to S-acetyl glutathione in the initial stages of the reaction. However, this ratio increased on extended exposure of GSH to peroxyacetyl nitrate. There was also some variation in the amount of sulfhydry1 reacted per mole of nitrite at different times of reaction-3.3, 3.8, 3.8, and 4.0 at 10, 20, 30, and 40 min treatment, respectively. The details of the reaction sequence have yet to be investigated.
TABLE REACTION
Treatment (min)
10 20 30 40
I
OF PEROXYACETYL GLUTATHIONE’
NOz ~moles)
GSH Oxidized Gunoles)
0.54 1.08 1.62 2.16
1.80 4.10 6.10 8.65
NITRATE
voF; GS-acetyl Olmoles) bmoles)
1.38 2.86 3.92 6.04
0.76 1.37 1.74 2.20
WITH
$5 GSSG/ GS-acetyl
1.82 2.08 2.25 2.74
mReaction mixtures contained 20 rmoles of GSH and 150 rmoles phosphate buffer, pH 7.0, in a final volume of 5.0 ml. At the end of the reaction period duplicate O.l-ml aliquots were taken for analysis of sulfhydryl. Duplicate 0.2~ml aliquots were taken for analysis of nitrite. Duplicate l.O-ml aliquots were taken for the assay of thioester by the hydroxamate method. Duplicate 0.4-ml aliquots were taken for the assay of GSSG by glutathione reductase. The results presented are from one of five time-course experiments and are typical of the data obtained.
Reaction of peroxyacetyl nitrate with coenzyme A. Because of the substantial amount of acetylation of the sulfhydryl groups of glutathione, we decided to examine the possibility of acetylation of other sulfhydryl compounds. Coenzyme A was exposed to varying amounts of peroxyacetyl nitrate and the reaction of sulfhydryl groups followed. The reaction products were separated by ion-exchange chromatography in an attempt to assess the relative amounts of oxidation and acetylation. Figure 1 shows the elution patterns for the coenzyme A before and after 60 min exposure to peroxyacetyl nitrate. The chromatography of the products of oxidation of coenzyme A by hydrogen peroxide is shown for comparison. The sample of coenzyme A used in these experiments contained an impurity which accounted for 12% of the absorbance at 260 W, but this compound, which emerged before CoASH, was unaffected by peroxyacetyl nitrate. Acetyl coenzyme A is eluted in this system in exactly the same position as CoASH. The two can be distinguished in the effluent fractions by the difference in the 260/230 ratio, the absorbance at 230 rnp being much greater for the thioester. The data in Table II summarize the results of an experiment in which CoASH was ex-
SULFHYDRYL COENZYME
4
OS!
3 COENZYME PEROXIDE
OXIDATION
BY PEROXYACETYL
A. --T-------1
A TREATED
WITH
HYDROGEN
4 A
I 061 I 041
02! u 100 zoo ml
300
\,1 -_-_L--l . 400
500
EFFLUENT
FIG. 1. Ion-exchange chromatography of coenzyme A and oxidation products; (1) chromatography of coenzyme A standard; (2) chromatography of coenzyme A after treatment with peroxyacetyl nitrate. Conditions and procedure as for Table II. The chromatogram shown is that for the sample exposed to 2.08~moles peroxyacetyl nitrate; (5) coenzyme A oxidized with excess hydrogen peroxide. Identification of peaks: 1, impurity; 2, reduced coenzyme A and oxidation products; 3, unidentified oxidation products; 4, oxidized coenzyme A (disulfide).
posed to increasing amounts of peroxyacetyl nitrate. The major product of the reaction of CoASH with peroxyacetyl nitrate was the disulfide of coenzyme A (peak 4). In addition, there were other oxidation products which have not yet been identified (peak 3). The products in peaks 3 and 4 did not account for the sulfhydryl which reacted even though the recovery according to absorbance at 260 rnp was 95 %. Assay of sulfhydryl in peak 2 showed that on the basis of 260 rnp absorbance, the compounds in peak 2 contained only 96, 95, 31, and 23 % of the sulfhydryl expected if the peak were pure CoASH after 15, 30, 45, and 60 min exposure, respectively. It appears likely that one of the oxidation products has the
239
NITRATE
same chromatographic properties as unreacted coenzyme A. Attempts to detect acetyl CoA in the reaction mixture by the hydroxamate assay were always negative. Measurement of the 260/230 ratio in both peaks 2 and 4 gave a ratio expected for CoA (either reduced or oxidized) and at no time gave a ratio expected for acetyl coenzyme A. The major reaction product when CoASH was treated with hydrogen peroxide was also the disulfide, and in addition there were compounds comparable to those found after reaction with peroxyacetyl nitrate. It may be concluded that the reaction of peroxyacetyl nitrate with CoASH produces no acetyl coenzyme A: The reaction is analogous to that with peroxide and does not depend on the anhydride character of peroxyacetyl nitrate. Reaction of peroxyacetyl nitrate with suljhydryE yroups of poteins. Attempts to measure the acetylation of human hemoglobin and bovine serum albumin were negative, even after complete reaction of the sulfhydryl groups. Chromatography of the products of the reaction with hemoglobin on Sephadex columns gave no indication of the formation TABLE
II
REACTION OF PEROXYACETYL NITRATE COENZYME Aa
15 30 45 60
0.52 1.04 1.56 2.08
1.05 2.18 2.51 2.81
2.36 1.73 1.60 1.30
0.210 0.273 0.510 0.593
WITH
0.418 0.945 0.915 1.00
0 Reaction mixtures contained 3 mg (approx. 3.5 pmoles) coenzyme A and 50 pmoles phosphate buffer pH 7.0 in a final volume of 5.0 ml. Aliquots of 0.1 ml were taken before and after exposure to peroxyacetyl nitrate for determination of sulfhydryl groups. Aliquots of 0.3 ml were taken at the end of the reaction period for the analysis of nitrite. An aliquot of 3.0 ml was applied to a column of DEAE cellulose (1.2 X 15 cm) and eluted according to Moffat and Khorana. Fractions (volume 7 ml) were measured at 260 rnp and recoveries in micromoles calculated on the basis of the adenine moiety. The results presented are from one of three time course experiments and are typical of the data obtained.
240
MUDD
AND McMANUS
of intermolecular disulfides detectable by changes in molecular weight. In order to obtain a protein of low molecular weight with a relatively large number of sulfhydryl groups per molecule, reduced ribonuclease was prepared. A solution containing 20 mg ribonuclease and 5 mg dithiothreitol in 2.0 ml of 8 M urea was flushed with nitrogen and stored overnight at lo. The solution was applied to a column (12 X 15 cm) of Sephadex G25 and eluted with 0.1 M acetic acid. The absorbance of the 3.0-ml fractions was measured at 277 rnE.cand protein concentration determined from this measurement. Aliquots of the protein solution were titrated with lo-3 M PMB at pH 4.5. An aliquot of 2.4 ml protein solution containing 370 rnp moles of protein and 2.76 clmoles sulfhydryl groups (7.45 sulfhydryl groups per molecule of protein) was added to 1.0 ml 0.1 M acetate buffer pH 4.5 and reacted with 3~moles of peroxyacetyl nitrate. At the end of the reaction period an aliquot was taken for titration of the sulfhydryl groups and it was found that 0.36 pmoles of sulfhydryl remained. The protein solution was assayed for thioester by the hydroxamate method and the result was negative. It is apparent in this case that even though the sulfhydryl groups of ribonuclease react with peroxyacetyl nitrate, acetylation is negligible as compared with oxidation. DISCUSSION
The results reported in this paper indicate that the reaction of peroxyacetyl nitrate with glutathione is relatively uncomplicated. The reaction is most simply summarized as the reaction of 3-4 moles of GSH per mole of peroxyacetyl nitrate with the production of equimolar amounts of GSSG and S-acetyl glutathione though deviations from this relationship were apparent in the later stages of reaction. In the case of coenzyme A 2-3 molecules of sulfhydryl groups were oxidized per molecule of peroxyacetyl nitrate. Attempts to detect S-acetyl coenzyme A were completely negative. The absence of S-acetyl coenzyme A cannot be explained on the grounds that it may be formed and then oxidized, because synthetic S-acetyl coenzyme A was resistant to reaction with peroxyacetyl nitrate. In addition to the disulfide of coenzyme A, the
reaction products include a number of compounds, detected after ion-exchange chromatography, which have not yet been identified but probably include compounds of higher oxidation state: sulfoxides, sulfones, sulfinic, and sulfonic acids. As far as the reactions of peroxyacetyl nitrate with proteins are concerned, one might expect from the results with glutathione that both acetylation and disulfrde formation would be measurable reactions and contribute to the toxicity of peroxyacetyl nitrate. On steric grounds one may expect less disulfide formation than with glutathione or coenzyme A. It is quite easy to measure the reaction of protein sulfhydryl groups with peroxyacetyl nitrate (2), but the identification of the reaction products is not so easy. We have not been able to find any evidence for the formation of S-acetylated protein. When reduced ribonuclease is allowed to oxidize slowly, reformation of disulfide bonds results in virtually complete recovery of activity (12). The recovery of some enzymic activity after treatment of reduced ribonuclease with peroxyacetyl nitrate shows that disulfide formation occurs (2). Other products of oxidation include higher oxidation states since cysteic acid is found in the hydrolyzate of papain after treatment with peroxyacetyl nitrate (2). The formation of disulfides from protein sulfhydry1 groups could be intramolecular, as in ribonuclease, or intermolecular. In our experiments with human hemoglobin, we have obtained no evidence for an increase in molecular weight as would be expected for the formation of dimers. The formation of polymeric material has been reported as a result of exposure of protein solution to lipid peroxides (15, 16). It is not clear in these cases, however, that the polymeric material is formed as a result of disulfide formation. REFERENCES 1. MUDD, J. B., Arch. Biochem. Biophys. 102, 59 (1963). 2. MUDD, J. B., LEAVITT, R., AND KERSEY, W. H., J. Biol. Chem. 241,408l (1966). 3. MUDD, J. B., J. Biol. Chem. 24l, 4077 (1966). 4. STEPHENS, E. R., DARLEY, E. F., TAYLOR, 0. C., AND SCOTT, W. E., I&em. J. Air Water Pollution 4, 79 (1961).
SULFHYDRYL
OXIDATION
BY PEROXYACETYL
5. SNELL, F. D., AND SNELL, C. T., “Calorimetric Methods of Analysis,” p. 644. Van Nostrand, Princeton, New Jersey, (1936). 6. STEPHENS, E. R., Atmos. Envir. 1, 19 (1967). 7. ELLMAN, G. L., Arch. Biochem. Biophys. 82, 70 (1959). 8. RACKER, E., Methods Enzymol., II, 722 (1955). 9. STADTMAN, E. R., Methods Enzymol., III, 228 (1957). 10. MOFFAT, J. G., AND KHORANA, M. G., J. Am. Chem. Sot. 63, 663 (1961).
NITRATE
241
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