ANALYTICAL
BIOCHEMISTRY
195,
63-67
(19%)
Biological
Sulfane Sulfur
A. M. Westley
and John Westley
Department
of Biochemistry,
University
of Chicago, Chicago, Illinois
60637
Received December 7,199O
A voltammetric method for determining cyanidereactive sulfane sulfur in biological materials is described. Samples are incubated with a sulfurtransferase, a thiolic cofactor, and cyanide. Thiocyanate formed and/or residual cyanide may then be determined electrochemically with either a silver rotating disk electrode or a dropping mercury electrode in differential pulse mode to provide estimates of sulfane sulfur content. The thiocyanate-based procedure is preferable, particularly when samples contain either serum albumin or inorganic sulfide. o 1991 Academic PWSS, IIIC.
Sulfane sulfur atoms are divalent sulfur atoms bonded only to other sulfur, except that they may also bear an ionizable hydrogen at some pH values. Identified components of the physiological pool of sulfane sulfur include the outer sulfur atoms of the persulfides (RS-S-) and of inorganic thiosulfate and organic thiosulfonate anions [RS(O),S-1. Also included are the inner chain atoms of polythionates and polysulfides, and all the atoms of elemental sulfur, which is found in the bloodstream in noncovalent association with serum albumin. All of these forms rapidly become labeled when a compound containing [36S]sulfane sulfur is injected into an experimental animal (1). Although depletion of the sulfane pool (e.g., by cyanide intoxication) has been understood to contribute deleterious effects (2,3), no generally valid analytical procedure for total sulfane sulfur in tissues has been available. A recent method for protein-associated sulfur (4) is clearly too restrictive in its specificity to be applicable to this problem. Another recent paper (5) reports measurements of the state of the sulfane pool in blood by use of Koh’s much earlier procedure for polythionates (6), a method based on calorimetric detection of the ferric thiocyanate complex ion. However, the sensitivity and specificity of the method are so limited (4,7,8) as to cast some doubt on the reported analytical values (seeDiscussion). The present method is a further devel0003-269’?/91$3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
opment of the voltammetric procedure for sulfane sulfur that yielded some preliminary tissue sulfane values mentioned in a recent report (9). MATERIALS
AND
METHODS
Materials
Standard solutions of analytes and solutions of reagents were prepared essentially as specified previously for cyanide and thiocyanate determinations (10). Fresh human blood was obtained by fingertip puncture, and bovine tissue samples were obtained from a local meat market. Lyophilized human serum and crystalline human serum albumin were from US. Biochemical Corp. Crystalline bovine serum albumin was purchased from Sigma. Crystalline rhodanese (thiosulfate:cyanide sulfurtransferase EC 2.8.1.1) was preparedby the Horowitz method (11). Sodium methanethiosulfonate, sodium 2-aminoethanethiosulfonate, and sodium benzenethiosulfonate were synthesized (12,13) and characterized (14) as reported previously. A complex of serum albumin with elemental sulfur was prepared by adding a solution of sulfur (99.999%, Aldrich) in HPLC-grade pyridine to a solution of crystalline serum albumin in aqueous buffer, as reported previously (15). Solutions of mixed polythionates (tri- through hexa-) were generated by allowing equimolar sodium thiosulfate and sodium tetrathionate (both from Baker) to react in neutral aqueous solution. The polythionates were characterized by thin-layer chromatography on cellulose in l-butanol-acetone-H,0 (2:2:1) and by differential pulse polarographic analysis in tetrabutylammonium borate buffer, pH 7.9, I = 0.10. A mixture of inorganic polysulfides, consisting mostly of disulfide, was prepared by adding 1 eq of elemental sulfur to a sodium sulfide solution in 0.10 N NaOH. Analysis
A sample of tissue extract or blood plasma is incubated briefly at 25°C after addition of crystalline rho63
Inc. reserved.
64
WESTLEY
AND
danese, glutathione (GSH), and potassium cyanide. After acidification and degassing to remove residual cyanide as HCN, the thiocyanate formed is determined by the voltammetric procedure reported previously (lo), with small modifications and the inclusion of a series of sulfane sulfur additions to serve as internal standards. Alternatively, the residual cyanide can be recovered analytically and the sulfane sulfur content of the sample is then inferred from the cyanide depletion. The thiocyanate-based procedure is the preferable method (see Discussion). Detailed protocols for the procedures follow. Sample preparation. Tissue samples are homogenized in a blade homogenizer with three volumes of cold phosphate-borate buffer, pH 8.6, ionic strength 0.1. To 2.0 ml of iced homogenate are added, successively, 0.20 ml of 50 mM sodium p-toluenesulfinate (an antioxidant) and 0.40 ml of 20% (w/v) Triton X-100. The mixture is centrifuged at 05°C for 15 min at 12,OOOg, and the supernatant is retained as the sample material. When blood, rather than solid tissues, is to be analyzed, the sample material is either titrated fresh plasma or whole blood hemolysate obtained by treatment with Triton in the presence of toluene sulfinate. Thiocyanate-based assay. Five loo-~1 aliquots of the sample material’ and one loo-p1 aliquot of the phosphate-borate buffer are placed in separate l&ml conical plastic centrifuge tubes. Three of the sample tubes are “spiked” with sulfane sulfur by adding 1, 2, and 3 ~1, respectively, of 5.0 mM Na,S,O,. All of the tubes are placed on ice and 0.20 ml of the phosphate-borate buffer is added, followed by 10 ~1 of 10 mM GSH, and 15 pg of crystalline rhodanese. With the exception of one unspiked sample tube, which serves as a control for preformed thiocyanate, the tubes receive 10 ~1 of 5 mM KCN each. All the tubes are then set in a 25°C bath to incubate. After 10 min, the reactions are stopped by the addition of 0.10 ml of 1.0 M H,SO,, and the solutions are degassed to remove HCN by 10 cycles of alternating 10 s N, flow/l0 s vacuum while the tubes are agitated. The degassed tubes are then placed in a simple gassing train that permits trapping of the HCN to be formed from thiocyanate (10). One-half milliliter of 50 RIM KMnO, is added through the gas inlet tube and the HCN produced from SCN- is trapped in 0.50 ml of 0.10 N NaOH in a 5-ml conical glass centrifuge tube during 30 min of gas flow (2 ml of air or N, per second). The cyanide content of the traps is then determined with a rotating disk silver electrode and cyanide internal standards, as reported previously (10). After appropriate subtraction of blanks, data are processed by linear regression of the cyanide signal, ex-
’ When the sample material is a hemolysate of whole blood, aliquots are restricted to 10 to 15 ~1 to minimize the interference caused by large quantities of hemoglobin.
WESTLEY
pressed as nanomoles of cyanide from the permanganate oxidation of SCN-, against nanomoles of sulfane sulfur spike: The coefficient of correlation should be greater than 0.99. The fact that different tissue extracts affect the sensitivity of the response differently is compensated for by taking the ratio of intercept f SE to slope + SE of this plot as the best measure of sulfane sulfur in the unspiked sample. This ratio f its SE is the sulfane sulfur content per aliquot of the sample material. Cyanide depletion assay. If desired, an estimate of sulfane sulfur content can also be obtained by an alternative procedure which monitors cyanide utilization rather than thiocyanate production. Six sample tubes with samples, blanks, and sulfane sulfur spikes are set up and incubated exactly as in the thiocyanate-based assay described above. However, termination of the enzyme-catalyzed cyanolysis reactions is carried out after placing the tubes in the trapped gassing train for HCN recovery. The reactions are stopped by the addition of 0.10 ml of 1.0 M H,SOI through the gas inlet tube, and gassing is continued for 30 min to collect the released HCN in 0.50 ml of 0.10 N NaOH. The cyanide content of the traps is then determined electrochemically, as in the thiocyanate-based procedure. Because of the possible occurrence of inorganic sulfide in the samples from the cyanide-depletion assay, however, a differential pulse polarographic method with a dropping mercury electrode may be necessary (10). The data are corrected appropriately for blanks and then processed by linear regression of the nanomoles of recovered cyanide against nanomoles of sulfane sulfur spike. The negative coefficient of correlation should be greater than 0.99. The ratio of intercept to the negative value of the slope of this plot represents the best value of the cyanide recovery from the unspiked sample. The difference between this number f SE and 50 nmol gives the cyanide depletion f SE in nmol per aliquot of the sample material. Where SCN- formation is the only cause of cyanide depletion, this value is equal to the sulfane sulfur content per aliquot of sample. RESULTS
Typical regression plots for three sample materials analyzed by the thiocyanate-based procedure are given in Fig. 1. The signal values are based on mean standard cyanide response values (with the silver electrode) of approximately 12 nA per nanomole. Table 1 details the calculation of sulfane sulfur content for brain tissue, the least precise of the three determinations in Fig. 1. Values obtained by the present analytical procedure for human plasma and whole blood as well as for a number of bovine tissues are presented in Table 2. The analytical values for a given sample were reproducible within the statistical error limits friven.
BIOLOGICAL
SULFANE
65
SULFUR TABLE Total
Sulfane
Sulfur
2
Content
of Various Sulfane
Sample
material
Whole blood Blood plasma Muscle Brain Heart Liver Kidney D The standard error sis, not from replicate
01 0
5
10
Sulfone
Sulfur
“Spike”
15 In mol)
Analytical values from cyanide depletion analysis may be somewhat higher than those from the thiocyanate-based procedure when the sample material contains serum albumin (see Discussion), but the effect is often obscured by the inherently poor precision of the cyanide depletion method. For example, in analyses of a human serum albumin solution (45 mg/ml), the thiocyanate-based value was 21 f 6 nmol/ml from a regression plot with a coefficient of correlation of 0.9962. Although the regression plot from the corresponding cyanide depletion analysis had a better coefficient of correlation (O-9993), the final value of 27 Jo:13 showed a far worse precision, reflecting the fact that the depletion value is necessarily a difference. A cyanide-depletion analysis of a bovine kidney extract displayed the same problem: a
TABLE
1
Sulfane Sulfur
Brain
Regression plot parameters Intercept: 2.579 + 0.302 nmol Slope: 0.825 k 0.032 Coefficient of correlation: 0.9985 Intercept/slope
= 3.127 + 0.386
(Intercept/slope)/(O.lO = 163 f 20 nmol
nmol
ml-aliquot X 0.1923 g of tissue/ml of sulfane sulfur/g of tissue
Species
sulfur (nmol/g)
human human bovine bovine bovine bovine bovine
239 + go+ 115 + 163 -t 300 -+ 416 k 458 f
values are derived determinations.
from
the regression
content”
12 4 16 20 26 51 23 analy-
sulfane sulfur value of 442 +- 178 nmol/g was calculated from a cyanide-depletion regression line with a coefficient of correlation equal to 0.993, which is quite comparable to that (0.998) for a thiocyanate-based line for the same kidney that yielded a value of 446 f 25 nmol/g.
FIG. 1. Regression plots of three sample materials. 0, 15-/J aliquots of whole human blood hemolysate (containing 11.3 ~1 of blood); n , loo-p1 aliquots of brain extract (containing 19.23 mg of bovine brain tissue); 0, loo-p1 aliquots of titrated human plasma (containing 97 ~1 of plasma).
Bovine
Tissues
of extract)
DISCUSSION The importance of metabolic sulfane sulfur has been stressed in the contexts of cyanide detoxication (2,9,1518) and the formation (19-24) and control (25) of ironsulfur centers. Clearly, research motivated by the aim of augmenting the physiological sulfane pool, as a means of preventing or minimizing cyanide intoxication, for example, requires an analytical method for total sulfane sulfur that can be applied to biological material. No reliable method for this purpose has been available. An operational definition of sulfane sulfur has been offered in terms of its susceptibility to cyanolysis (26). The definition requires expansion to include a requirement for inorganic thiocyanate to be formed as a product. One shortcoming of cyanide-depletion methods for sulfane sulfur arises from the fact that cyanolysis of disulfide bonds (RSSR) uses up cyanide but does not involve sulfane sulfur. However, inorganic thiocyanate is not formed in the reaction except at pH values approaching 11. Accordingly, monitoring SCN- formation, rather than cyanide depletion, in cyanolysis can provide a measure of true sulfane sulfur. Alternatively, one might seek to establish cyanolysis conditions so mild that RSSR will not react. Unfortunately, some disulfide bonds in proteins, including those in a small percentage of serum albumin molecules, are so reactive with cyanide (27) that this is not a practical option. For this reason as well as the inherently lower precision of difference methods, procedures for biological sulfane sulfur based on SCN- determination are preferable to those based on cyanide depletion, particularly when the sample material contains serum albumin.
66
WESTLEY
AND
Moreover, with procedures such as the present one that utilize volatilization and trapping to isolate HCN for analysis, the thiocyanate-based method has a further significant advantage when the sample material contains inorganic sulfide. In cyanide-depletion measurements, such sulfide contaminates the trapped cyanide, and the silver electrode does not differentiate well between the two. This distinction necessitates the use of a dropping mercury electrode in differential pulse mode (10). In the thiocyanate-based procedure, however, sulfide is removed along with residual cyanide by degassing prior to oxidation of the SCN-, so that the greater convenience of the silver electrode is retained. Several features of the detailed procedure given here require explanation. One is the use of p-toluenesulfinate as an antioxidant in preparing tissue extracts for the present purpose. Since SCN- is subject to peroxidation in extracts, especially those that contain cell-lysing detergents, which often contain low concentrations of organic peroxides, an antioxidant is needed. Specifically, what is required is a material that reduces peroxides and other reactive oxygen species but does not destroy sulfane sulfur by reducing it to sulfide. A sulfinate is ideal for this purpose since its only reaction with sulfane sulfur is the formation of the corresponding thiosulfonate anion [RS(O),S-1, in which the outer sulfur atom retains its cyanide reactivity as sulfane sulfur. Moreover, thiosulfonate anions are excellent substrates for rhodanese. The incubation with cyanide in the presence of rhodanese and GSH is another feature requiring comment. When GSH is present as a cofactor, rhodanese has access to all of the forms of sulfane sulfur known to occur in biological systems, including even elemental sulfur (28). Inclusion of a substantial concentration of rhodanese (-50 fig/ml), along with GSH at 0.3 mM and KCN at 0.15 mM ensures that all of the sulfane sulfur in the aliquot of sample material will be converted irreversibly to SCN- during the lo-min incubation at 25°C. Rhodanese catalyzes the transfer of sulfane sulfur only, and disulfides (RSSR) that react with cyanide produce no inorganic thiocyanate under the conditions of this incubation. The excess cyanide is removed as HCN by degassing after acidification and the SCN- formed from sulfane sulfur is oxidized with acid KMnO,, following Boxer and Rickards (8). The concentration of permanganate used in the present procedure is greater than that specified previously (10) since this practice diminishes the interference caused by hemoglobin when whole blood is used as the sample material. Although only inorganic thiosulfate is used as a standard sulfane sulfur “spike” in the method described here, sulfane sulfur in a variety of other materials has also proved to be analytically recoverable by this procedure when added to human plasma prior to analysis. Included are both aliphatic and aromatic thiosulfonate
WESTLEY
anions, inorganic polysulfides and polythionates, and elemental sulfur complexed to serum albumin-all examples of the principal forms that become labeled in uiuo when [3SS]sulfane sulfur is injected (1). The sensitivity and specificity of the present method appear adequate to provide practical analytical values for total sulfane sulfur in biological materials. As noted previously (lo), the electrochemical procedure on which the method is based is readily capable of handling subnanomolar quantities of cyanide and thiocyanate. The mean standard error in the single determinations reported in Table 2 is less than 9%, and the use of weighted means of replicate determinations (weighted according to reciprocal variance) can reduce this percentage very substantially. The absence of positive interferences other than that caused by inorganic sulfide has been discussed previously (10). The occurrence of negative interferences, seen as a systematic quenching of the signal (diminished slope in regression plots such as those in Fig. l), is corrected for by use of the regression slope in the analytical calculation (Table 1). The sulfane sulfur values obtained in the present work range from -100 nmol/g for plasma and skeletal muscle to over 400 nmol/g for liver and kidney. The values for whole blood (-240 nmol/g) and plasma are of special interest in that they provide a comparison with some values reported by others. Hannestad et al. (4), using a gas chromatographic method for protein-associated sulfane sulfur, found very low concentrations in either human or rat plasma (0.5 nmol/g or less) although some rat tissues contained more than 35 nmol/g. Comparison with the present results indicates that most of the sulfane sulfur in plasma is not protein-associated, or at any rate will not react with the triphenylphosphine reagent that is the basis of the gas chromatographic procedure. This is so despite the fact that serum albumin has discrete binding sites for sulfane sulfur (29-31). A quite different variation on this theme is provided by the data of Buzaleh et al. (5), whose calorimetric analysis of the normal blood levels of cyanide-labile sulfur in mice gave values greater than 2500 nmol/ml. Both the magnitude of this value in comparison with the present results and our experience with the ferric thiocyanatebased methods in this laboratory suggest that the colorimetric procedure has neither the sensitivity nor the specificity to be capable of yielding true values for blood sulfane sulfur. One further comparison might be of interest. The pharmacokinetic data of Sylvester et al. (32) showed that cyanide administered to dogs is largely detoxified in the primary compartment, the bloodstream. According to the analytical value for human blood sulfane sulfur in the present study, a typical human might have approximately 1.3 mmol of cyanide-reactive sulfur available in the bloodstream for rapid reaction with cyanide.
BIOLOGICAL
The minimum lethal dose of cyanide proximately 2 mmol.
SULFANE
for humans
is ap-
was supported GM-30971.
by National
Institutes
of Health
Re-
REFERENCES 1. Schneider, J. F., and Westley, J. (1969) J. Biol. Chem. 244,57355744. 2. Westley, J. (1980) in Enzymatic Basis of Detoxication (Jakoby, W. B., Ed.), Vol. 2, pp. 245-262, Academic Press, New York. 3. Westley, J. (1989) in Sulfur-Containing Drugs and Related Organic Compounds (Damani, L. A., Ed.), Vol. 2B, pp. 87-99, E. Horwood, Chichester. 4. Hannestad, U., Margheri, S., and Sorbo, B. (1989) And. Biochem.
178,394-398. 5. Buzaleh, A. M., Gen. Pharmacol.
Vazquez, E. S., and Batlle, 2 1,27-32.
A. M. de1 C. (1990)
6. Koh, T. (1965) Bull. Chem. Sot. Jpn. 38, 1510-1515. 7. Westley, J. (1987) in Methods in Enzymology (Jakoby, Griffith, 0. W., Eds.), Vol. 143, pp. 22-25, Academic Diego. 8. Boxer, 300.
G. E., and Rickards,
J. C. (1952)
Arch.
Biochem.
W. B., and Press, San 39,292-
9. Westley, J. (1988) in Cyanide Compounds in Biology, Ciba Foundation Symposium 140 (Evered, D., and Harnett, S., Eds.), pp, 201-218, Wiley, Chichester. 10. Westley, A. M., and Westley, J. (1989) And. Biochem. 181,190194. 11. Horowitz, P. M. (1978) Anal. B&hem. 86,751-753. 12. Mintel, 13. Westley,
R., and Westley, J., and Heyse,
14. Westley,
A., and Westley,
J. (1984)
15. Jarabak, R., and Westley, 10,796. 16. Lang, K. (1933) Biochem.
ACKNOWLEDGMENT This work search Grant
67
SULFUR
J. (1966) J. Biol. Chem. D. (1971) J. Biol. Chem.
241,3381-3385. 246,
14681474.
And.
J. (1986)
Biochem.
J. Bid.
Chem.
142,163-166. 261, 10,793-
2. 259,243-256.
17. Siirbo, B. (1975) in Metabolism berg, D. M., Ed.), 3rd ed., Vol. New York.
of Sulfur Compounds, 7, pp. 433-456, Academic
(GreenPress,
18. Westley, J. (1981) in Cyanide in Biology (Vennesland, B., et al., Eds.), pp. 61-76, Academic Press, London. 19. Finazzi-Agro, A., Cannella, C., Graziani, M. T., and Cavallini, D. (1971) FE&S’Lett. 16, 172-174. 20. Taniguchi, T., and Kimura, T. (1974) B&him. Biophys. Ada 364,284-295. 21. Volini, M., Craven, D., and Ogata, Res. Commun. 79,890-896. 22. Bonomi, F., Pagani, S., Cerletti, J. Biochem. 72, 17-24. 23. Volini, M., and Alexander, nesland, B., et al., Eds.),
K. (1977)
B&hem.
P., and Cannella,
Biophys.
C. (1977)
Eur.
K. (1981) in Cyanide in Biology (Venpp. 77-91, Academic Press, London.
24. Cerletti, P. (1986) Trends in Biochem. Sci. 11,369-372. M. (1989) J. Biol. Chem. 264, 25. Ogata, K., Dai, X., and Volini, 2718-2725. in Enzymology (Jakoby, W. B., 26. Wood, J. L. (1987) in Methods and Griffith, 0. W., Eds.), Vol. 143, pp. 25-29, Academic Press, San Diego. 27. Lundquist, P., Rosling, H., and Sorbo, B. (1985) Clin. Chem. 31, 591-595. 28. Westley, J., and Adler, H. (1983) Fed. Proc. 42, 870. [Abstract 33841 29. Jarabak, R., and Westley, 261. 30. Jarabak, R., and Westley, 31. Jarabak, R., and Westley, 32. Sylvester, D. M., Hayton, (1983) Tox. Appl. Pharmacol.
J. (1989)
J. Biochem.
Tozicol.
4,255-
J. (1990) J. Biochem. Toricol. 5, l-8. J. (1991) J. Biochem. Toricol. 6,1-6. W. L., Morgan, R. L., and Way, J. L. 69,265-271.
BIOCHEMISTRY
195,
63-67
(19%)
Biological
Sulfane Sulfur
A. M. Westley
and John Westley
Department
of Biochemistry,
University
of Chicago, Chicago, Illinois
60637
Received December 7,199O
A voltammetric method for determining cyanidereactive sulfane sulfur in biological materials is described. Samples are incubated with a sulfurtransferase, a thiolic cofactor, and cyanide. Thiocyanate formed and/or residual cyanide may then be determined electrochemically with either a silver rotating disk electrode or a dropping mercury electrode in differential pulse mode to provide estimates of sulfane sulfur content. The thiocyanate-based procedure is preferable, particularly when samples contain either serum albumin or inorganic sulfide. o 1991 Academic PWSS, IIIC.
Sulfane sulfur atoms are divalent sulfur atoms bonded only to other sulfur, except that they may also bear an ionizable hydrogen at some pH values. Identified components of the physiological pool of sulfane sulfur include the outer sulfur atoms of the persulfides (RS-S-) and of inorganic thiosulfate and organic thiosulfonate anions [RS(O),S-1. Also included are the inner chain atoms of polythionates and polysulfides, and all the atoms of elemental sulfur, which is found in the bloodstream in noncovalent association with serum albumin. All of these forms rapidly become labeled when a compound containing [36S]sulfane sulfur is injected into an experimental animal (1). Although depletion of the sulfane pool (e.g., by cyanide intoxication) has been understood to contribute deleterious effects (2,3), no generally valid analytical procedure for total sulfane sulfur in tissues has been available. A recent method for protein-associated sulfur (4) is clearly too restrictive in its specificity to be applicable to this problem. Another recent paper (5) reports measurements of the state of the sulfane pool in blood by use of Koh’s much earlier procedure for polythionates (6), a method based on calorimetric detection of the ferric thiocyanate complex ion. However, the sensitivity and specificity of the method are so limited (4,7,8) as to cast some doubt on the reported analytical values (seeDiscussion). The present method is a further devel0003-269’?/91$3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
opment of the voltammetric procedure for sulfane sulfur that yielded some preliminary tissue sulfane values mentioned in a recent report (9). MATERIALS
AND
METHODS
Materials
Standard solutions of analytes and solutions of reagents were prepared essentially as specified previously for cyanide and thiocyanate determinations (10). Fresh human blood was obtained by fingertip puncture, and bovine tissue samples were obtained from a local meat market. Lyophilized human serum and crystalline human serum albumin were from US. Biochemical Corp. Crystalline bovine serum albumin was purchased from Sigma. Crystalline rhodanese (thiosulfate:cyanide sulfurtransferase EC 2.8.1.1) was preparedby the Horowitz method (11). Sodium methanethiosulfonate, sodium 2-aminoethanethiosulfonate, and sodium benzenethiosulfonate were synthesized (12,13) and characterized (14) as reported previously. A complex of serum albumin with elemental sulfur was prepared by adding a solution of sulfur (99.999%, Aldrich) in HPLC-grade pyridine to a solution of crystalline serum albumin in aqueous buffer, as reported previously (15). Solutions of mixed polythionates (tri- through hexa-) were generated by allowing equimolar sodium thiosulfate and sodium tetrathionate (both from Baker) to react in neutral aqueous solution. The polythionates were characterized by thin-layer chromatography on cellulose in l-butanol-acetone-H,0 (2:2:1) and by differential pulse polarographic analysis in tetrabutylammonium borate buffer, pH 7.9, I = 0.10. A mixture of inorganic polysulfides, consisting mostly of disulfide, was prepared by adding 1 eq of elemental sulfur to a sodium sulfide solution in 0.10 N NaOH. Analysis
A sample of tissue extract or blood plasma is incubated briefly at 25°C after addition of crystalline rho63
Inc. reserved.
64
WESTLEY
AND
danese, glutathione (GSH), and potassium cyanide. After acidification and degassing to remove residual cyanide as HCN, the thiocyanate formed is determined by the voltammetric procedure reported previously (lo), with small modifications and the inclusion of a series of sulfane sulfur additions to serve as internal standards. Alternatively, the residual cyanide can be recovered analytically and the sulfane sulfur content of the sample is then inferred from the cyanide depletion. The thiocyanate-based procedure is the preferable method (see Discussion). Detailed protocols for the procedures follow. Sample preparation. Tissue samples are homogenized in a blade homogenizer with three volumes of cold phosphate-borate buffer, pH 8.6, ionic strength 0.1. To 2.0 ml of iced homogenate are added, successively, 0.20 ml of 50 mM sodium p-toluenesulfinate (an antioxidant) and 0.40 ml of 20% (w/v) Triton X-100. The mixture is centrifuged at 05°C for 15 min at 12,OOOg, and the supernatant is retained as the sample material. When blood, rather than solid tissues, is to be analyzed, the sample material is either titrated fresh plasma or whole blood hemolysate obtained by treatment with Triton in the presence of toluene sulfinate. Thiocyanate-based assay. Five loo-~1 aliquots of the sample material’ and one loo-p1 aliquot of the phosphate-borate buffer are placed in separate l&ml conical plastic centrifuge tubes. Three of the sample tubes are “spiked” with sulfane sulfur by adding 1, 2, and 3 ~1, respectively, of 5.0 mM Na,S,O,. All of the tubes are placed on ice and 0.20 ml of the phosphate-borate buffer is added, followed by 10 ~1 of 10 mM GSH, and 15 pg of crystalline rhodanese. With the exception of one unspiked sample tube, which serves as a control for preformed thiocyanate, the tubes receive 10 ~1 of 5 mM KCN each. All the tubes are then set in a 25°C bath to incubate. After 10 min, the reactions are stopped by the addition of 0.10 ml of 1.0 M H,SO,, and the solutions are degassed to remove HCN by 10 cycles of alternating 10 s N, flow/l0 s vacuum while the tubes are agitated. The degassed tubes are then placed in a simple gassing train that permits trapping of the HCN to be formed from thiocyanate (10). One-half milliliter of 50 RIM KMnO, is added through the gas inlet tube and the HCN produced from SCN- is trapped in 0.50 ml of 0.10 N NaOH in a 5-ml conical glass centrifuge tube during 30 min of gas flow (2 ml of air or N, per second). The cyanide content of the traps is then determined with a rotating disk silver electrode and cyanide internal standards, as reported previously (10). After appropriate subtraction of blanks, data are processed by linear regression of the cyanide signal, ex-
’ When the sample material is a hemolysate of whole blood, aliquots are restricted to 10 to 15 ~1 to minimize the interference caused by large quantities of hemoglobin.
WESTLEY
pressed as nanomoles of cyanide from the permanganate oxidation of SCN-, against nanomoles of sulfane sulfur spike: The coefficient of correlation should be greater than 0.99. The fact that different tissue extracts affect the sensitivity of the response differently is compensated for by taking the ratio of intercept f SE to slope + SE of this plot as the best measure of sulfane sulfur in the unspiked sample. This ratio f its SE is the sulfane sulfur content per aliquot of the sample material. Cyanide depletion assay. If desired, an estimate of sulfane sulfur content can also be obtained by an alternative procedure which monitors cyanide utilization rather than thiocyanate production. Six sample tubes with samples, blanks, and sulfane sulfur spikes are set up and incubated exactly as in the thiocyanate-based assay described above. However, termination of the enzyme-catalyzed cyanolysis reactions is carried out after placing the tubes in the trapped gassing train for HCN recovery. The reactions are stopped by the addition of 0.10 ml of 1.0 M H,SOI through the gas inlet tube, and gassing is continued for 30 min to collect the released HCN in 0.50 ml of 0.10 N NaOH. The cyanide content of the traps is then determined electrochemically, as in the thiocyanate-based procedure. Because of the possible occurrence of inorganic sulfide in the samples from the cyanide-depletion assay, however, a differential pulse polarographic method with a dropping mercury electrode may be necessary (10). The data are corrected appropriately for blanks and then processed by linear regression of the nanomoles of recovered cyanide against nanomoles of sulfane sulfur spike. The negative coefficient of correlation should be greater than 0.99. The ratio of intercept to the negative value of the slope of this plot represents the best value of the cyanide recovery from the unspiked sample. The difference between this number f SE and 50 nmol gives the cyanide depletion f SE in nmol per aliquot of the sample material. Where SCN- formation is the only cause of cyanide depletion, this value is equal to the sulfane sulfur content per aliquot of sample. RESULTS
Typical regression plots for three sample materials analyzed by the thiocyanate-based procedure are given in Fig. 1. The signal values are based on mean standard cyanide response values (with the silver electrode) of approximately 12 nA per nanomole. Table 1 details the calculation of sulfane sulfur content for brain tissue, the least precise of the three determinations in Fig. 1. Values obtained by the present analytical procedure for human plasma and whole blood as well as for a number of bovine tissues are presented in Table 2. The analytical values for a given sample were reproducible within the statistical error limits friven.
BIOLOGICAL
SULFANE
65
SULFUR TABLE Total
Sulfane
Sulfur
2
Content
of Various Sulfane
Sample
material
Whole blood Blood plasma Muscle Brain Heart Liver Kidney D The standard error sis, not from replicate
01 0
5
10
Sulfone
Sulfur
“Spike”
15 In mol)
Analytical values from cyanide depletion analysis may be somewhat higher than those from the thiocyanate-based procedure when the sample material contains serum albumin (see Discussion), but the effect is often obscured by the inherently poor precision of the cyanide depletion method. For example, in analyses of a human serum albumin solution (45 mg/ml), the thiocyanate-based value was 21 f 6 nmol/ml from a regression plot with a coefficient of correlation of 0.9962. Although the regression plot from the corresponding cyanide depletion analysis had a better coefficient of correlation (O-9993), the final value of 27 Jo:13 showed a far worse precision, reflecting the fact that the depletion value is necessarily a difference. A cyanide-depletion analysis of a bovine kidney extract displayed the same problem: a
TABLE
1
Sulfane Sulfur
Brain
Regression plot parameters Intercept: 2.579 + 0.302 nmol Slope: 0.825 k 0.032 Coefficient of correlation: 0.9985 Intercept/slope
= 3.127 + 0.386
(Intercept/slope)/(O.lO = 163 f 20 nmol
nmol
ml-aliquot X 0.1923 g of tissue/ml of sulfane sulfur/g of tissue
Species
sulfur (nmol/g)
human human bovine bovine bovine bovine bovine
239 + go+ 115 + 163 -t 300 -+ 416 k 458 f
values are derived determinations.
from
the regression
content”
12 4 16 20 26 51 23 analy-
sulfane sulfur value of 442 +- 178 nmol/g was calculated from a cyanide-depletion regression line with a coefficient of correlation equal to 0.993, which is quite comparable to that (0.998) for a thiocyanate-based line for the same kidney that yielded a value of 446 f 25 nmol/g.
FIG. 1. Regression plots of three sample materials. 0, 15-/J aliquots of whole human blood hemolysate (containing 11.3 ~1 of blood); n , loo-p1 aliquots of brain extract (containing 19.23 mg of bovine brain tissue); 0, loo-p1 aliquots of titrated human plasma (containing 97 ~1 of plasma).
Bovine
Tissues
of extract)
DISCUSSION The importance of metabolic sulfane sulfur has been stressed in the contexts of cyanide detoxication (2,9,1518) and the formation (19-24) and control (25) of ironsulfur centers. Clearly, research motivated by the aim of augmenting the physiological sulfane pool, as a means of preventing or minimizing cyanide intoxication, for example, requires an analytical method for total sulfane sulfur that can be applied to biological material. No reliable method for this purpose has been available. An operational definition of sulfane sulfur has been offered in terms of its susceptibility to cyanolysis (26). The definition requires expansion to include a requirement for inorganic thiocyanate to be formed as a product. One shortcoming of cyanide-depletion methods for sulfane sulfur arises from the fact that cyanolysis of disulfide bonds (RSSR) uses up cyanide but does not involve sulfane sulfur. However, inorganic thiocyanate is not formed in the reaction except at pH values approaching 11. Accordingly, monitoring SCN- formation, rather than cyanide depletion, in cyanolysis can provide a measure of true sulfane sulfur. Alternatively, one might seek to establish cyanolysis conditions so mild that RSSR will not react. Unfortunately, some disulfide bonds in proteins, including those in a small percentage of serum albumin molecules, are so reactive with cyanide (27) that this is not a practical option. For this reason as well as the inherently lower precision of difference methods, procedures for biological sulfane sulfur based on SCN- determination are preferable to those based on cyanide depletion, particularly when the sample material contains serum albumin.
66
WESTLEY
AND
Moreover, with procedures such as the present one that utilize volatilization and trapping to isolate HCN for analysis, the thiocyanate-based method has a further significant advantage when the sample material contains inorganic sulfide. In cyanide-depletion measurements, such sulfide contaminates the trapped cyanide, and the silver electrode does not differentiate well between the two. This distinction necessitates the use of a dropping mercury electrode in differential pulse mode (10). In the thiocyanate-based procedure, however, sulfide is removed along with residual cyanide by degassing prior to oxidation of the SCN-, so that the greater convenience of the silver electrode is retained. Several features of the detailed procedure given here require explanation. One is the use of p-toluenesulfinate as an antioxidant in preparing tissue extracts for the present purpose. Since SCN- is subject to peroxidation in extracts, especially those that contain cell-lysing detergents, which often contain low concentrations of organic peroxides, an antioxidant is needed. Specifically, what is required is a material that reduces peroxides and other reactive oxygen species but does not destroy sulfane sulfur by reducing it to sulfide. A sulfinate is ideal for this purpose since its only reaction with sulfane sulfur is the formation of the corresponding thiosulfonate anion [RS(O),S-1, in which the outer sulfur atom retains its cyanide reactivity as sulfane sulfur. Moreover, thiosulfonate anions are excellent substrates for rhodanese. The incubation with cyanide in the presence of rhodanese and GSH is another feature requiring comment. When GSH is present as a cofactor, rhodanese has access to all of the forms of sulfane sulfur known to occur in biological systems, including even elemental sulfur (28). Inclusion of a substantial concentration of rhodanese (-50 fig/ml), along with GSH at 0.3 mM and KCN at 0.15 mM ensures that all of the sulfane sulfur in the aliquot of sample material will be converted irreversibly to SCN- during the lo-min incubation at 25°C. Rhodanese catalyzes the transfer of sulfane sulfur only, and disulfides (RSSR) that react with cyanide produce no inorganic thiocyanate under the conditions of this incubation. The excess cyanide is removed as HCN by degassing after acidification and the SCN- formed from sulfane sulfur is oxidized with acid KMnO,, following Boxer and Rickards (8). The concentration of permanganate used in the present procedure is greater than that specified previously (10) since this practice diminishes the interference caused by hemoglobin when whole blood is used as the sample material. Although only inorganic thiosulfate is used as a standard sulfane sulfur “spike” in the method described here, sulfane sulfur in a variety of other materials has also proved to be analytically recoverable by this procedure when added to human plasma prior to analysis. Included are both aliphatic and aromatic thiosulfonate
WESTLEY
anions, inorganic polysulfides and polythionates, and elemental sulfur complexed to serum albumin-all examples of the principal forms that become labeled in uiuo when [3SS]sulfane sulfur is injected (1). The sensitivity and specificity of the present method appear adequate to provide practical analytical values for total sulfane sulfur in biological materials. As noted previously (lo), the electrochemical procedure on which the method is based is readily capable of handling subnanomolar quantities of cyanide and thiocyanate. The mean standard error in the single determinations reported in Table 2 is less than 9%, and the use of weighted means of replicate determinations (weighted according to reciprocal variance) can reduce this percentage very substantially. The absence of positive interferences other than that caused by inorganic sulfide has been discussed previously (10). The occurrence of negative interferences, seen as a systematic quenching of the signal (diminished slope in regression plots such as those in Fig. l), is corrected for by use of the regression slope in the analytical calculation (Table 1). The sulfane sulfur values obtained in the present work range from -100 nmol/g for plasma and skeletal muscle to over 400 nmol/g for liver and kidney. The values for whole blood (-240 nmol/g) and plasma are of special interest in that they provide a comparison with some values reported by others. Hannestad et al. (4), using a gas chromatographic method for protein-associated sulfane sulfur, found very low concentrations in either human or rat plasma (0.5 nmol/g or less) although some rat tissues contained more than 35 nmol/g. Comparison with the present results indicates that most of the sulfane sulfur in plasma is not protein-associated, or at any rate will not react with the triphenylphosphine reagent that is the basis of the gas chromatographic procedure. This is so despite the fact that serum albumin has discrete binding sites for sulfane sulfur (29-31). A quite different variation on this theme is provided by the data of Buzaleh et al. (5), whose calorimetric analysis of the normal blood levels of cyanide-labile sulfur in mice gave values greater than 2500 nmol/ml. Both the magnitude of this value in comparison with the present results and our experience with the ferric thiocyanatebased methods in this laboratory suggest that the colorimetric procedure has neither the sensitivity nor the specificity to be capable of yielding true values for blood sulfane sulfur. One further comparison might be of interest. The pharmacokinetic data of Sylvester et al. (32) showed that cyanide administered to dogs is largely detoxified in the primary compartment, the bloodstream. According to the analytical value for human blood sulfane sulfur in the present study, a typical human might have approximately 1.3 mmol of cyanide-reactive sulfur available in the bloodstream for rapid reaction with cyanide.
BIOLOGICAL
The minimum lethal dose of cyanide proximately 2 mmol.
SULFANE
for humans
is ap-
was supported GM-30971.
by National
Institutes
of Health
Re-
REFERENCES 1. Schneider, J. F., and Westley, J. (1969) J. Biol. Chem. 244,57355744. 2. Westley, J. (1980) in Enzymatic Basis of Detoxication (Jakoby, W. B., Ed.), Vol. 2, pp. 245-262, Academic Press, New York. 3. Westley, J. (1989) in Sulfur-Containing Drugs and Related Organic Compounds (Damani, L. A., Ed.), Vol. 2B, pp. 87-99, E. Horwood, Chichester. 4. Hannestad, U., Margheri, S., and Sorbo, B. (1989) And. Biochem.
178,394-398. 5. Buzaleh, A. M., Gen. Pharmacol.
Vazquez, E. S., and Batlle, 2 1,27-32.
A. M. de1 C. (1990)
6. Koh, T. (1965) Bull. Chem. Sot. Jpn. 38, 1510-1515. 7. Westley, J. (1987) in Methods in Enzymology (Jakoby, Griffith, 0. W., Eds.), Vol. 143, pp. 22-25, Academic Diego. 8. Boxer, 300.
G. E., and Rickards,
J. C. (1952)
Arch.
Biochem.
W. B., and Press, San 39,292-
9. Westley, J. (1988) in Cyanide Compounds in Biology, Ciba Foundation Symposium 140 (Evered, D., and Harnett, S., Eds.), pp, 201-218, Wiley, Chichester. 10. Westley, A. M., and Westley, J. (1989) And. Biochem. 181,190194. 11. Horowitz, P. M. (1978) Anal. B&hem. 86,751-753. 12. Mintel, 13. Westley,
R., and Westley, J., and Heyse,
14. Westley,
A., and Westley,
J. (1984)
15. Jarabak, R., and Westley, 10,796. 16. Lang, K. (1933) Biochem.
ACKNOWLEDGMENT This work search Grant
67
SULFUR
J. (1966) J. Biol. Chem. D. (1971) J. Biol. Chem.
241,3381-3385. 246,
14681474.
And.
J. (1986)
Biochem.
J. Bid.
Chem.
142,163-166. 261, 10,793-
2. 259,243-256.
17. Siirbo, B. (1975) in Metabolism berg, D. M., Ed.), 3rd ed., Vol. New York.
of Sulfur Compounds, 7, pp. 433-456, Academic
(GreenPress,
18. Westley, J. (1981) in Cyanide in Biology (Vennesland, B., et al., Eds.), pp. 61-76, Academic Press, London. 19. Finazzi-Agro, A., Cannella, C., Graziani, M. T., and Cavallini, D. (1971) FE&S’Lett. 16, 172-174. 20. Taniguchi, T., and Kimura, T. (1974) B&him. Biophys. Ada 364,284-295. 21. Volini, M., Craven, D., and Ogata, Res. Commun. 79,890-896. 22. Bonomi, F., Pagani, S., Cerletti, J. Biochem. 72, 17-24. 23. Volini, M., and Alexander, nesland, B., et al., Eds.),
K. (1977)
B&hem.
P., and Cannella,
Biophys.
C. (1977)
Eur.
K. (1981) in Cyanide in Biology (Venpp. 77-91, Academic Press, London.
24. Cerletti, P. (1986) Trends in Biochem. Sci. 11,369-372. M. (1989) J. Biol. Chem. 264, 25. Ogata, K., Dai, X., and Volini, 2718-2725. in Enzymology (Jakoby, W. B., 26. Wood, J. L. (1987) in Methods and Griffith, 0. W., Eds.), Vol. 143, pp. 25-29, Academic Press, San Diego. 27. Lundquist, P., Rosling, H., and Sorbo, B. (1985) Clin. Chem. 31, 591-595. 28. Westley, J., and Adler, H. (1983) Fed. Proc. 42, 870. [Abstract 33841 29. Jarabak, R., and Westley, 261. 30. Jarabak, R., and Westley, 31. Jarabak, R., and Westley, 32. Sylvester, D. M., Hayton, (1983) Tox. Appl. Pharmacol.
J. (1989)
J. Biochem.
Tozicol.
4,255-
J. (1990) J. Biochem. Toricol. 5, l-8. J. (1991) J. Biochem. Toricol. 6,1-6. W. L., Morgan, R. L., and Way, J. L. 69,265-271.
