Chromogenic substrates for sulfurtransferases

ANALYTICAL

BIOCHEMISTRY

149,66-7 1 (1985)

Chromogenic

Substrates

for Sulfurtransferases’

MARY R. BURROUSAND Department

of Biochemistry,

University

JOHN WESTLEY

of Chicago,

Chicago,

Illinois

60637

Received January 29, 1985 The azo dye 4-(dimethylamino)-4’-azobenzene (DAB) thiosulfonate anion can serve as a sulfur-donor substrate for rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) and for thiosulfate reductase (EC unassigned) with cyanide anion and GSH, respectively, as acceptor substrates. In either case, the dye product is DAB sulfinate, which differs substantially in light absorption at 500 nm. Moreover, DAB sulfinate can serve as a sulfur-acceptor substrate for rhodanese with either inorganic thiosulfate or a colorless thiosulfonate anion as donor, and this reaction provides a second chromogenic assay procedure. o 19135 Academic mess, IN. KEY WORDS: rhodanese; thiosulfate reductase; sulfurtransferases; thiosulfonates; sulfinates; continuous assay.

Assay methods based on substrate conversions that directly yield spectral changes in the visible region have not previously been available for the sulfurtransferases. However, ultraviolet spectral differences (1) between aromatic thiosulfonate anions (ArS(0)2S-) and the corresponding sulfinates (ArSO;) suggested that such visible chromogenic substrates could be developed. Thiosulfonate anions are excellent sulfur-donor substrates for both rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) (l-4) and thiosulfate reductase (EC unassigned) (5), and sulfinates are sulfur-acceptor substrates for rhodanese (1).

chromatography was Malinckrodt’s CC-7 Silicar, 100-200 mesh. Rhodanese was purified to homogeneity from bovine liver by the procedure of Horowitz (6). Yeast thiosulfate reductase was prepared as previously described (7). Sodium methanethiosulfonate was synthesized as described previously (2). Other reagents and solvents were purchased from commercial sources as reagent grade chemicals and were used without further purification. Thin-layer chromatography of DAB thiosulfonate, sulfinate, and sulfonate (methyl orange) was done as previously described (8). Absorption spectra were measured with a Varian 634 spectrophotometer equipped with a chart recorder. Reaction velocities were MATERIALS AND METHODS determined by continuous recording at 500 Certified methyl orange and 4-(dimethylnm at a sensitivity of 0.100 absorbance unit amino)-4’-azobenzene (DAB)2 sulfonyl chlo- full scale. ride were purchased from Aldrich. HardThe thiosulfonate synthesized was sublayer silica thin-layer chromatography plates jected to analysis for sulfane sulfur by exwere from Analtech. Silica used for column haustive cyanolysis, as reported for previous thiosulfonate syntheses (2,3), and the kinetic ’ This work was supported by National Science Foun- homogeneity of this product was also checked dation Research Grant PCM 8 1- 16006 and United States (3). The only variation in analytical procedure Public Health Service Grant GM-3097 1. that was necessary was the treatment of * Abbreviation used: DAB, 4-(dimethylamino)-4’-azobenzene. completed cyanolysis reactions with sufficient

0003-2697185 $3.00 Copyright 0 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.

66

CHROMOGENIC

SUBSTRATES

activated charcoal to remove the highly colored sulfinate product before aliquots for thiocyanate analysis were taken. RESULTS

DAB sulfinic acid and the sodium salt of DAB thiosulfonate anion were both prepared from DAB sulfonyl chloride by the reactions shown in Fig. I. Syntheses. Formation of DAB thiosulfonate anion was carried out at 65°C in a wellstirred 10% suspension of the solid sulfonyl chloride in an aqueous solution containing one analytical equivalent of Na$. When thin-layer chromatographic analysis showed that the reaction was practically complete (~2 h), the reaction mixture was dried in an atmosphere of N2 under reduced pressure. The solid residue was extracted two times with a volume of hot ethanol equal to half the original reaction volume each time, and the product crystallized from the combined extracts overnight at -20°C. This procedure is a modification of that used previously for other thiosulfonate syntheses (2), based on the method of Traeger and Linde (9). After recrystallization, the yield of sodium DAB thiosulfonate was 60%. Synthesis of DAB sulfinic acid was carried out at 65°C in a well-stirred 5% suspension of the sulfonyl chloride in an aqueous solution containing a 15-fold molar excess of Na2S03. When thin-layer chromatographic analysis showed that the reaction was essentially complete (-2.5 h), the reaction mixture was acidified to pH < 1 with concentrated HCl

(CH,),N+N=N+SO,CI

I\

SzCl-

DAR thmsulfonote

so,zH&J

so,2-

HCI DAB

sulfimc

acid

FIG. 1. Synthetic reactions for DAB thiosulfonate anion and DAB sulfinic acid from DAB sulfonyl chloride.

FOR SULFURTRANSFERASES

67

and then chilled and centrifuged. The supernatant solution was discarded and the solids were extracted with a volume of hot ethanol equal to half the original reaction volume. DAB sulfinic acid crystallized from the extract overnight at -20°C. This procedure is a modification of the general method of Kulka (10). The yield after recrystallization was >90%. Characterization of products. As isolated from the synthesis reaction mixtures, both products contained minor amounts of characteristic contaminants but were suitable for use as routine assay substrates without further purification. For more critical applications, sodium DAB thiosulfonate could be cleared of the small quantity of contaminating sulfinate by isocratic column chromatography on silica with 5:2 ethyl acetate, 65% ethanol (v/ v) as the solvent. Similarly, DAB sulfinic acid could be cleared of its slight contamination with the sulfonic acid in the same liquid chromatography system. The thiosulfonate was the first band eluted from such columns, followed by the sulfonate and the sulfinate, in that order. The same contaminants tend to reappear in solutions of these products because of the general instability of aromatic thiosulfonate anions in aqueous solutions (8) and the sensitivity of sulfinates to air oxidation. Both products are best stored under dry nitrogen. The sulfane sulfur content of chromatographically purified DAB thiosulfonate exceeded 99% of the theoretical value and was kinetically homogeneous in cyanolysis. Visible absorption spectra of DAB thiosulfonate and sulfinate, along with their difference spectrum, are presented in Fig. 2. At its absorption maximum (466 nm), DAB thiosulfonate had an extinction coefficient of 19.8 mK’ cm-‘. DAB sulfinite had an extinction coefficient of 18.8 mM-’ cm-’ at its absorption maximum (460 nm). The AC at 500 nm was 2.94 mM-’ cm-‘. In the aqueous buffer employed, the visible absorption of DAB sulfonate (methyl orange) did not differ from that of the sulfinate.

68

BURROUS

AND WESTLEY

E lmM4cni’l

400

500

600

FIG. 2. Visible absorption spectra. The solvent was 0.10 M acetic acid-sodium acetate buffer containing glytine at 0.10 M, pH 5.0. -, DAB sulfinate; ---, DAB thiosulfonate * . *, difference spectrum.

The chemical identities of DAB thiosulfonate and sulfinate were established by means of both uncatalyzed and enzymecatalyzed reactions, as indicated in Fig. 3. DAB thiosulfonate could be converted quantitatively to the sulfinate by treatment with strong acid (reaction 1) or by treatment with excess cyanide in the presence of rhodanese

(reaction 2). As shown in reaction 3 of Fig. 3, DAB sulfinate was converted quantitatively to the thiosulfonate by treatment with excess inorganic thiosulfate or methane thiosulfonate anion in the presence of rhodanese. Finally (reactions 4 and 5), both the thiosulfonate and the sulfinate were oxidized quantitatively to the sulfonate by treatment with excess H202. All of these reactions occurred rapidly at room temperature. The conversions were followed by silica thin-layer chromatography with the two-solvent systems previously given for the clear resolution of these three DAB derivatives (8). In all of these reactions, the changes in visible light absorption were also in accord with the spectra given in Fig. 2. Assay systems. The difference spectrum reported in Fig. 2 can be used as the basis for sulfurtransferase assays. Fig. 4A displays progress curves of light absorption at 500 nm with a starting concentration of the sulfurdonor substrate DAB thiosulfonate equal to 50 PM in the presence of various concentrations of bovine liver rhodanese and 1.67 mM potassium cyanide as sulfur-acceptor substrate. Similar progress curves were obtained when the catalyst was thiosulfate reductase and the acceptor substrate was GSH. Figure 4B shows the time course of Asp when

DAB sulfinate

DAB sulfonote FIG. 3. Reactions establishing the chemical identities of DAB thiosulfonate anion and DAB sulfinate. The symbol Cl under reaction arrows 2 and 3 indicates rhodanese catalysis. Products were identified by thin-layer chromatography (8).

CHROMOGENIC

SUBSTRATES

[Ffhodanesd

Cpg/ml)

69

FOR SULFURTRANSFERASES

060

I 6

tr 5-pg adulttons thvxulfote reductase

065

2

0 Reactlon

4 Time

0

(mm)

5

of

10 Time

15

Imm)

FIG. 4. Progress curves for enzyme-catalyzed sulfur transfer from DAB thiosulfonate. (A) Rhodanese; (B) thiosulfate reductase. Arrows mark times of enzyme addition. Mixing discontinuities (5-10 s) not shown.

aliquots of a yeast thiosulfate reductase solution were added to 40 pM DAB thiosulfonate in the presence of 1.2 mM GSH. The data show that DAB thiosulfonate is active as a sulfur-donor substrate for these two sulfurtransferases. Similar progress curves, except that As00 increased rather than decreased with time, were obtained when DAB sulfinate was used as a sulfur-acceptor substrate in the presence of rhodanese, with (colorless) methane thiosulfonate as the donor. Figure 5 shows that initial velocities in this system were proportional to enzyme concentration over at least a 20-fold range. Since methane thiosulfonate proved to be somewhat inhibitory at donor/ acceptor ratios > 5, inorganic thiosulfate was also tested as a donor to DAB sulfinate in rhodanese-catalyzed reactions. The data presented in Fig. 6 as a pattern of double reciprocal plots represent steady-state initial velocity measurements in this system.

analysis on which the formal and chemical mechanisms of the rhodanese-catalyzed reaction are based (2-4,11-14) was conducted by discontinuous analysis of thiocyanate production ( 15). Continuous procedures based on the ultraviolet absorbance of lipoate (16), inorganic thiosulfate (17,18), or aromatic thiosulfonate anions (l), or on the visible absorbance of multiply coupled redox indi-

AA,,o/mm x 10’

DISCUSSION

The sulfurtransferases rhodanese and thiosulfate reductase have been studied primarily by methods that involve discontinuous assay procedures. Even much of the detailed kinetic

[Rhodonese]

(nM)

FIG. 5. Initial velocities of rhodanese-catalyzed sulfur transfer from methane thiosulfonate (300 PM) to DAB sulfinate (60 FM).

70

BURROUS

AND WESTLEY

FIG. 6. Initial velocity pattern of rhodanese-catalyzed sulfur transfer from inorganic thiosulfate to DAB s&mate. The concentrations of DAB sulfinate were 0, 38 pM; A, 7 1.O PM; 0, 144 PM. The rhodanese concentration was 150 nM. UO,initial velocity in arbitrary units.

cator dye systems (19,20) have been described but much less used than the discontinuous procedures. Similarly, the first kinetic mechanism studies of thiosulfate reductase (21) used an assay procedure based on discontinuous analysis for product sulfite (22), but more recent studies have been conducted with continuous procedures that involve either coupling to NADPH oxidation by glutathione reductase or use of an aromatic thiosulfonate anion as the sulfur-donor substrate (5). All of the continuous procedures for both enzyme activities display marked lag periods or have other deficiencies of sensitivity, reproducibility, or convenience (20,23,24), and it has therefore seemed worthwhile to develop assay methods based directly on visible chromogenic substrates. The present materials provide two assay systems for rhodanese, one with DAB thiosulfonate as sulfur donor and cyanide as acceptor, the other with inorganic thiosulfate or a colorless thiosulfonate anion as donor and DAB sulfinate as acceptor. In addition, thiosulfate reductase can be assayed with DAB thiosulfonate as donor and GSH as acceptor. All of these assay systems are direct, requiring only buffered solutions of the substrates.

The buffer solution used in these assay systems (0.1 rvt acetate containing glycine at 0.1 M, pH 5.0) was chosen to favor the stability of the enzymes. Both enzyme-catalyzed reactions are much faster at higher pH values, but the enzymes are also much less stable, particularly at the extreme dilutions required by the high sensitivity of the methods under these conditions. Glycine was previously shown to have a stabilizing effect on rhodanese (25). Criteria for establishing a valid enzyme assay procedure are much the same as those for establishing true steady-state initial velocity conditions. The progress curve must be practically linear for a sufficient period to establish tangent velocities at zero time, and the velocities so measured must be proportional to total enzyme concentration. The present systems meet these conditions well. Such assay velocities were used to generate the steady-state initial velocity pattern of Fig. 6, which indicates that the established doubledisplacement mechanism of rhodanese catalysis via a sulfur-substituted enzyme intermediate (14) is as applicable to reactions involving the DAB substrates as it is to those of the other thiosulfonates (3). REFERENCES 1. Sorbo, B. (1962) Acta Chem. Seand. 16,243-245. 2. Mintel, R., and Westley, J. (1966) J. Biol. Chem. 241, 3381-3385. 3. Westley, J., and Heyse, D. (1971) J. Biol. Chem. 246, 1468-1474. 4. Jarabak, R., and Westley, J. (1974) Biochemistry 13, 3237-3239. 5. Chauncey, T. R., and Westley, J. (1983) J. Biol. Chem. 258, 15037-l 5045. 6. Horowitz, P. (1978) Anal. Biochem. 86, 75 l-753. 7. Chauncey, T. R., and Westley, J. (1983) Biochim. Biophys. Acta 744, 304-3 11. 8. Westley, A., and Westley, J. (1984) Anal. Biochem. 142, 163-166. 9. Traeger, J., and Linde, 0. (1901) Arch. Pharmacol. 239, 121-145. 10. Kulka, M. (1950) J. Amer. Chem. Sot. 72, 12151218.

CHROMOGENIC

SUBSTRATES

11. Leininger, K. R., and Westley, J. (1968) J. Biol. Chem. 243, 1892- 1899. 12. Schlesinger, P., and Westley, J. (1974) J. Biol. Chem. 249, 780-788. 13. Wang, S.-F., and Volini, M. (1973) .I. Biol. Chem. 248, 7376-7385. 14. Westley, J. (1977) in Bioorganic Chemistry (Van Tamelen, E. E., ed.), Vol. 1, pp. 371-390, Academic Press, New York. 15. Siirbo, B. H. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 2, pp. 334-337, Academic Press, New York. 16. Volini, M., and Westley, J. ( 1966) J. Biol. Chem. 241, 5 168-5 176. 17. Davidson, B., and Westley, J. (1965) .I. Biol. Chem. 240, 4463-4469.

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18. Chou, S. F., Horowitz, P. M., Westley, J., and Jarabak, R. (1985) J. Biol. Chem. 260, 27632770. 19. Smith, A. J., and Lascelles, J. (1966) J. Gen. Microbiol. 42, 357-370. 20. Cannella, C., Berni, R., and Ricci, G. (1984) Anal. Biochem. 142, 159- 162. 21. Uhteg, L., and Westley, J. (1979) Arch. Biochem. Biophys. 195, 2 1 l-222. 22. Koj, A. (1968) Acfa Biochim. Polon. 15, 161-169. 23. Westley, J. (1973) Adv. Enzymol. 39, 327-368. 24. SGrbo, B. (1975) in Metabolic Pathways, 3rd Ed. (Greenberg, D. M., ed.), Vol. 7, pp. 433-456, Academic Press, New York. 25. Westley, J. (1959) J. Biol. Chem. 234, 1857-1860.