ANALYTICAL BIOCHEMISTRY 64, 80-84 (1975)

Preparation of Cystathionine Sulfoxide and Sulfone and Some Properties Relating to Their Differentiation ANNE H.

DATKO, JOHN GIOVANELLI,

AND S. H A R V E Y M U D D

National Institute of Mental Health, Laboratory of General and Comparative Biocttemistry, Bethesda, Maryland 20014 Received June 26, 1974; accepted August 30, 1974 Methods are described for the oxidation of cystathionine to yield relatively pure preparations of either cystathionine sulfoxide (a mixture of the diastereoisomers) or of cystathionine sulfone. These products were shown to contain both the three and the four carbon moieties of cystathionine and were identified by their characteristic behavior during treatment with hydriodic acid or with oxidizing agent, and in the case of the sulfone, by elemental analysis. Paper and column chromatographic and electrophoretic mobilities of the sulfoxides and the sulfone were determined.

During investigations in this laboratory on sulfur amino acid metabolism in both animals and plants, we have encountered problems in dealing with cystathionine and its oxidation products. Commonly, cystathionine is subjected to oxidation by H~O2 or performic acid (1-3) to permit its chromatographic separation from compounds such as cyst(e)ine and homocyst(e)ine_ However, the products of these oxidation procedures have not been identified, so that the extent of formation of cystathionine sulfoxide, cystathionine sulfone, or other compounds, is not known (1). We, as well as others (1), have experienced difficulties in the chromatography of these products and of cystathionine. These facts led us to make a systematic investigation of conditions for oxidation of cystathionine, and of methods for separation and identification of the major oxidation products. MATERIALS AND METHODS Cystathionine (Calbiochem) was dissolved in 1 M KOH: distilled H 2 0 was added to yield a solution which was, finally, 0.1 M KOH; distilled H~O and 0.2 M in KOH (pH 8-9). [14C4] Cystathionine (radioactivity located uniformly in the C4 moiety) and [3-I4C] cystathionine (radioactivity located in the fi-carbon of the C3 moiety) were prepared by the method of Mudd et al. (2). Performic acid was prepared by adding 1 ml of 30% H202 to 9 ml of 88% formic acid and allowing the mixture to incubate for 30 min at 27°C before use. Descending paper chroma80 Copyright© 1975 by Academic Press, Inc. Printed in the United States All rights of reproductionin any formreserved.

C Y S T A T H I O N I N E SU.LFOXIDE A N D S U L F O N E

81

tography was performed overnight at room temperature on Whatman No. 1 paper. Electrophoresis was carried out for 135 min at 2500 V at 4°C on Whatman No. 3MM paper. Amino acid analysis by automated column chromatography was performed as described by Spackman et ah (4). Evaporation was carried out in v a c u o at 25°C with a Rotary Evapomix (Buchler Instruments). Peroxide oxidation of small amounts of cystathionine was carried out by incubating 1 /xmole cystathionine with 0.2 ml of the appropriate dilution of 30% H20,,,. Performic acid oxidation of cystathionine was carried out by incubating l /zmole cystathionine with 0.17 ml performic acid. Reduction with H I was performed by incubating 1 tzmole of the material to be reduced with 0.085 ml 1 HI. All incubations were for 30 min at 30°C except where noted, and the reactions were stopped by removing the peroxide, performic acid, or HI by evaporation. RESULTS AND DISCUSSION In preliminary experiments cystathionine was incubated with increasing concentrations of H.,O2. The resulting products were separated by paper chromatography with m e t h a n o l - p y r i d i n e - l . 2 5 N HCI (Solvent 3, Table 1) and visualized with ninhydrin. Incubation with low concentrations of peroxide (0.02-0.13 M) resulted in the disappearance of cystathionine, and, concomitantly, the appearance of material which migrated more slowly as a broad, possibly " t w i n n e d , " spot. Incubation with higher concentrations of peroxide (0.4- 11.6 M), o r with performic acid, resulted in formation of lesser amounts of this "twinned" spot, and, concomitantly, the appearance of yet a different material which remained at the origin of heavily loaded chromatograms The material formed by incubation with low H,,O~ concentrations was identified as a mixture of the two diastereoisomers of cystathionine sulfoxide by the following evidence. The material could be resolved as two closely migrating peaks by automated amino acid column chromatography (see Table 1). Each could be reduced back to cystathionine by incubation with H1 (5,6), and each could be further oxidized to cystathionine sulfone by incubation with performic acid (6-8). Finally, both the three carbon and the four carbon moieties of cystathionine were contained in an unresolved mixture of the two materials, as shown by the incorporation of radioactivity into the mixture when the oxidation was performed on cystathionine labeled with J4C in the three carbon moiety or in the four carbon moiety. The material formed under strong oxidizing conditions was identified as cystathionine sulfone by the following evidence_ Both the three carbon and the four carbon moieties of cystathionine were contained in the material, as shown by the incorporation of radioactivity into the

82

DATKO, GIOVANELLI

AND MUDD

TABLE 1 CHROMATOGRAPHIC AND ELECTROPHORETIC MOBILITIES OF CYSTATHIONINE AND ITS OXIDATION PRODUCTS

Procedure

Distance of cystathionine from origin (cm)

Paper c h r o m a t o g r a p h y Sol. 1. 2-Propanol-88% formic acid-H20 14 (70 : 10: 20, v/v/v) Sol. 2. 2-Propanol-88% formic acid 14 (60 : 40, v/v) Sol. 3. M e t h a n o l - p y r i d i n e - l . 2 5 N HC1 28 ~ (37 : 4 : 8, v/v/v) Sol. 4. E t h a n o l - p y r i d i n e - H ~ O - N H 4 O H ( 2 8 - 3 0 % ) 4 (60 : 20 : 12 : 8, v/v/v/v) Paper electrophoresis Sol. 5. 1.76% Formic acid-10 mM 2-mer35 a captoethanol (pH 1.9) A u t o m a t e d amino acid column chromatograph y

Relative mobility" Sulfoxide(s)

Sulfone

0.61

0 and 0.43 b

0.57

0.54

0_77

0.68

0.47

0.91

0.82

0.71

0.64:0.74 e

0.64 ~

" Mobilities are relative to cystathionine for paper chromatography and electrophoresis, and elution v o l u m e s are relative to glycine for column chromatography. The electrophoretic and paper chromatographic mobilities (with solvent 1) of m a n y c o m p o u n d s structurally related to cystathionine have previously been reported (9). A n additional c o m p o u n d of interest, fi-methyllanthionine, h a s n o w been studied_ During electrophoresis, a synthetic preparation of fi-methyllanthionine (10) m o v e d with Rcystathionine = 0.85. A more satisfactory separation was obtained after performic treatment (Rcystathionine = 0.67). b In this solvent, w h e n less than about 10 nmoles sulfone is present, m o s t of it migrates with an Reystathionine of 0.43. As the a m o u n t of sulfone spotted is increased, only a small proportion of it migrates with this mobility, and the r e m a i n d e r stays at the origin. For example, as m e a s u r e d by distribution of radioactivity, all of the material migrated with Rcystathiomne of 0.43 w h e n 5 nmoles tritiated sulfone was chromatographed, but only about 10% of it had this mobility w h e n 100 nmoles was chromatographed. c In this solvent all of the c o m p o u n d s give broad diffuse spots. The diastereoisomers of the sulfoxide separate slightly, giving a twinned appearance. W h e n the sulfone was p r e s e n t in small a m o u n t s (5 nmoles) it migrated very close behind the slower of the two diastereoisomers of the sulfoxide. W h e n the sulfone was present in larger amounts, a variable proproportion m o v e d with Reystathionine 0.68, and the r e m a i n d e r stayed at the origin. The poor ninhydrin color yield observed after c h r o m a t o g r a p h y in this solvent was improved by incorporating 10% pyridine (v/v) into the ninhydrin reagent (1.25% ninhydrin in acetone, w/v). a Migration toward the cathode. The oxidation products, along with glycine, emerge from the column during elution with citrate buffer of p H 3.25. Cystathionine is eluted after the change to pH 4.25 buffer, with an elution v o l u m e relative to glycine of about 1.56. The molar ninhydrin color yields from cystathionine, recrystallized cystathionine sulfone, or the cystathionine sulfoxide diastereoisomers were approximately the same.

CYSTATH1ONINE

SULFOXIDE

AND

SULFONE

83

material when the oxidation was performed on cystathionine labeled with 14C in the three carbon moiety or in the four carbon moiety. It was the major product obtained even when the performic acid oxidation was carried out under mild conditions such as to minimize any degradative side reactions (incubation at 15°C for 1 rain). U p o n treatment with a large excess of H I (7.6 M, 27°C, 3 hr) it remained unchanged (8)_ Finally, when [all] cystathionine sulfone, prepared by performic acid oxidation and recrystallized to constant specific radioactivity (3), was subjected to elemental analysis, the results were consistent with the calculated values for the sulfone (Found: C, 32.75; H, 5.75; N, 10.90%. CrH~aN20~S requires C, 33.06; H, 5.54; N, 11.01%). A preparation of relatively pure cystathionine sulfoxide was obtained by incubating 10 tzmoles cystathionine with 2_0 ml of 0.13 M H20,,- T h e reaction was carried out at the somewhat alkaline pH obtained when cystathionine solutions were prepared as described in Methods, since the oxidation proceeded more rapidly under these conditions than at acid or neutral pH. The concentration of peroxide was chosen so that the bulk of the starting material was converted to the sulfoxide, while little sulfone was produced. A small amount of cystathionine remained, but this was relatively easily separated from the sulfoxide (see Table 1). The reaction was stopped by the addition of 0_25 ml 5.7 N formic acid, and the solution was evaporated to dryness. (Larger volumes of HeOe should be removed by lyophilization to prevent further oxidation.) One milliliter of 10 mM 2-mercaptoethanol was added and the resulting solution was evaporated to dryness. The last step was repeated, and the material was subjected to preparative paper electrophoresis (Solvent 5, Table 1) to remove residual oxidant and unreacted cystathionine. A preparation of relatively pure cystathionine sulfone was obtained by incubating 2_7 mmoles cystathionine with 50 ml performic acid for 60 rain. The performic acid was removed by lyophilization. Cystathionine sulfone, prepared by this method and recrystallized four times (3), contained two minor ninhydrin positive impurities with R,.,.~t~,m~o,l~m,in Solvent 1 of about 1.6 and 3.6_ The relative mobilities of cystathionine sulfoxlde and sulfone in several systems are listed in Table 1. Cystathionine sulfone tended to overload relatively easily during paper chromatography. F o r example, with Solvent 1, when the amount of sulfone spotted was increased from 5 nmoles, or less, to 100 nmoles, most of the c o m p o u n d failed to move with its characteristic mobility, but rather remained at the origin of the developed c h r o m a t o g r a m (Table 1). This p h e n o m e n o n showed some specificity, since small amounts of sulfone m o v e d normally when spotted together with 100 nmoles of either cystathionine sulfoxide or cystathionine. Homolanthionine sulfone, 100 nmoles, caused a portion of cys-

84

DATKO, GIOVANELLI AND MUDD

tathionine sulfone to remain at the origin. A similar tendency to overload was noted with Solvent 3, and has apparently previously been encountered with other solvents (1). The information presented here makes it possible to analyze a preparation for the presence of cystathionine, the sulfoxides and the sulfone. Cystathionine is easily distinguished from its oxidation products by its mobility in several solvents (Table 1). Cystathionine sulfoxides and sulfone are not clearly separated from one another by column chromatography nor by most paper chromatographic solvents with the exception of Solvent 4 (Table 1). Therefore, to distinguish these compounds, one may take advantage of the fact that the sulfoxides, but not the sulfone, may be reduced by HI. A further distinguishing characteristic of the sulfone is its tendency to remain at the origin when the chromatograms are overloaded. These methods for producing relatively pure cystathionine sulfoxides and sulfone and for identifying them, should prove useful in dealing with these materials.

ACKNOWLEDGMENTS We wish to thank Brinson Conerly for performing the automated amino acid analyses, and Paula M. Parisius for performing the elemental analysis. Dr. Erhard Gross generously made available a sample of synthetic/3-methyllanthionine.

REFERENCES 1. DELAVIER-KLUTCrtKO,C., AND FLAVIN, M. (1965) J. Biol. Chem. 240, 2537-2549. 2. MUDD, S_ H., FINKEESTEIN, J. D., IRREVERRE, F., AND LASTER, L. (1965) J. Biol. Chem. 240, 4382-4392. 3. GIOVANELLI,J., OWENS, L. D., AND MUDD, S. H. (1973) Plant Physiol. 51, 492-503. 4. SPAEKMAN,D. H,, STEIN, W. H., AND MOORE, S. (1958)Anal. Chem. 30, 1190-1206. 5. TOENNIES, G., AND KOLB, J_ J. (1939) J. Biol. Chem. 128, 399-405. 6. SZMANT, H. H_ (1961) in Organic Sulfur Compounds (Kharasch, N. ed.), Vol. 1, pp. 154-169, Pergamon Press, New York. 7. BARNARD, D., BATEMAN, L., AND CUNNEEN, J. I. (1961) in Organic Sulfur Compounds (Kharasch, N., ed.), Vol. 1, pp. 229-247, Pergamon Press, New York. 8. SUTER, C_ M. (1944) Organic Chemistry of Sulfur, J. Wiley and Sons, Inc., New York. 9. DATKO, A_ H., MUDD, S. H., AND GIOVANELLI, J. (1974) Anal. Biochem. 62, 531545. 10. SCHOBERL, A., GROSS, E., MORELL, J. L., AND WlTKOP, B. (1966) Biochim. Biophys. Acta 121, 406-409.