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A comparison of pyridoxal phosphate-cystine and pyridoxal phosphate-homocystine reactions
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
138,
697-699
(1970)
COMMUNICATIONS A Comparison
of Pyridoxal
Phosphate-Cystine Homocystine
and Pyridoxal
Phosphate-
Reactions
In previous researches on the degradation of cystine in noneneymatic model reactions (l-3), it has been demonstrated that pyridoxal phosphate (PALP) catalyzes the cleavage of cystine through a typical ~$3 elimination reaction, originating thiocysteine, ammonia, and pyruvate (4, 5). Thiocysteine then decomposes into cysteine and elementary sulfur. The reaction takes place at room temperature, in slightly alkaline medium, and without added metal ions. It is characterized by the intermediate formation of a thiazolidine derivative of cysteine and PALP, which shows a peculiar absorption spectrum (6, 7). In the same way PALP catalyzes the degradation of S-aminoethylcysteine, with p-elimination of cysteamine (S), and this latter also links PALP in a thiazolidine ring during the reaction (9). It seemed interesting to study if, in the same experimental conditions, homocystine also could be degraded by PALP, possibly through an cu-y elimination reaction. In this case homocysteine should be one of the products of the reaction. To evidentiate it, we have first checked that in our experimental conditions homocysteine readily reacts with PALP originating a tetrahydro-thiazine ring (6, 7) easily detectable spectrophotometrically. Then we have searched for the production of this derivative in incubation mixtures containing PALP and homocystine. Control tests with cystine and PALP have been run in parallel. DL-Homocystine and DL-homocysteine were obtained from Fluka; PALP from Hoffmann-LaRoche; all other compounds and reagents from Merck. Solutions (10V2 M) of cysteine or homocysteine in water were prepared immediately before use. Cystine and homocystine were dissolved in the minimum amount of 1 N HCl, and diluted to 10-a M with 0.5 M sodium phosphate buffer pH 8.5. PALP was dissolved in the same buffer. Optical spectra were recorded, using l-mm or IO-mm silica DB spectrophotometer cells, in a Beckman equipped with a Sargent SRL-G recorder. The cell compartment was thermoregulated at 25” by circulating water from an ultrathermostat.
Figure 1 shows that in 0.5 M sodium phosphate buffer pH 8.5, homocysteine, like cysteine (4), binds to PALP causing the disappearance of the absorption at 390 nm, and the appearance of a peak characteristic for a thiazolidine or thiazine derivative (6,7). There is a small but evident dif ference between the peak of the cysteine-PALP
.6
4 n 6 .2.
FIG. 1. Variations of PALP spectrum after addition of homocysteine. A. Full line: PALP 1O-4 M in 0.5 M sodium phosphate buffer, pH 8.5. Dotted lines: PALP lo+ M and homocysteine lo+ M in the same buffer. Curves 1 to 5 recorded at 1, 5, 15,30, and 60 min after the addition of homocysteine. B. Spectra recorded at the end of the reaction between PALP and cysteine (full line) or homocysteine (dotted line). PALP 1W M, thiols 10V3 M in 0.5 M sodium phosphate buffer, pH 8.5. Light path: 10 mm; temperature: 25”.
thiazolidine, which shows the maximum at 330 nm, and the peak of the thiazine derivative of homocysteine, centered at 325 nm (Fig. 1B). Figure 2 shows that for solutions containing 1OW M PALP and 10 or 20 molar excess of cysteine or homocysteine, the decrease in OD at 390 nm is parallel to the increase at 330 nm. In these conditions, and also for equimolar solutions of PALP 697
COMMUNICATIONS
(i98
min.
FIG. 2. Changes of PALP absorption at 390 and 330 nm after the addition of cysteine or homocysteine. Full lines, cysteine; dotted lines, homocysteine. A and B, decrease in OD at 390 nm; A’ and B’, increase at 330 nm for solutions containing PALP 1OW M, and aminothiols 1OW M and 2.10v3 M, respectively, in sodium phosphate buffer 0.5 M, pH 8.5. Light path, 10 mm; temperature, 25”. and cysteine or homocysteine at 10v3 M final concentration, a second-order rate constant of 90 f 5 M-I mini has been calculated for both thiols. This indicates that in the experimental conditions followed, homocysteine links PALP in a cyclic derivative as well as cysteine does, without appreciable differences in the reaction rate. Therefore, if homocysteine is formed from homocystine in the presence of PALP, it should be easily detectable from the appearance of the thiazine peak at 325 nm. Homocystine and PALP were then incubated in equimolar amounts (10m3M) at 25“, in 0.5 M sodium phosphate buffer, pH 8.5, and the spectral changes were recorded. For comparison cystine and PALP were incubated in the same conditions. Figure 3 reports the results obtained. It is evident that cystine is degraded, as already known (4,5), giving rise to a thiazolidine derivative with the typical absorption peak at 330 nm. Homocystine instead gives rise to the only formation of a Schiff base, as indicated by the shift of the PALP absorption to longer wavelengths. Also, when the
FIG. 3. Variations of PALP spectrum after addition of homocystine (A) or cystine (B). A. Full line. PALP 1OW M in 0.5 M sodium phosphate buffer, pH 8.5. Dotted lines. PALP 1OW M and homocystine lo+ M in the same buffer. Spectra recorded 1 min (Curve 1) and 10 min (Curve 2) after the addition of homocystine. The spectrum remained unchanged after 3 hr. B. Full line. PALP 1OW M in 0.5 M sodium phosphate buffer, pH 8.5. Dotted lines. PALP 10s3 M and cystine 1OW M in the same buffer. Curves 1 to 4 recorded 1, 30, 60, and 150 min after the addition of cystine. Light path, 1 mm; temperature, 25”. incubation was performed at 38” for several hours, in a pH range from 7.5 to 9; or when the homocystine-to-PALP ratio was varied from 0.5 to 5, no appreciable variations of the Schiff-base spectrum were observed. On the other hand, it was ascertained that homocysteine can bind PALP even if it is present as Schiff base with homocystine: in fact, after the addition of homocysteine the Schiff -base spectrum readily turns into that of the thiazine derivative. It may be concluded, therefore, that no homocysteine is released from homocystine incubated with PALP; that is, contrary to what happens with cystine, in the experimental conditions followed, PALP cannot catalyze the cleavage of homocystine. This may reflect a greater stability of the Schiff base formed between PALP and homocystine, in respect to that with cystine. The above conclusion has been confirmed by the negative results obtained in searching the possible products of the homocystine cleavage: ammonia, sulfur, and ketoacids. The results of the present note, even if limited only to homocystine, may be an indication that PALP, while catalyze without added metal ions, at room temperature, and in slightly alkaline
COMMUNICATIONS medium, a-0 elimination reactions of sulfur-containing aminoacids, like cystine and S-aminoethylcysteine, cannot give rise, in the same conditions, to a-y elimination reactions on analogous compounds. This hypothesis obviously deserves further confirmation, and it will be interesting to examine, in the experimental conditions above reported, the behavior of cystathionine or of the mixed disulfide between cysteine and homocysteine. REFERENCES 1. CAVALLINI, B., Arch.. 2. DE MARCO, D., Boll. 3. Ds MARCO, D., Arch.
D., DE MARCO, C., AND MONDOV?, Biochem. Biophys. 87, 281 (1960). C., COLETTA, M., AND CAVALLINI, Sot. It. Biol. Sper. 38, 1904 (1962). C., COLETTA, M., AND CAVALLINI, Biochem. Biophys. 109, 51 (1963).
699
4. DE MARCO, C., BOGNOLO, D., AND CAVALLINI, D., G. Biochim. 12, 187 (1963). 5. CAVALLINI, D., MONDOV~, B., AND DE MARCO, C., Proc. Symp. Chem. Biol. Aspects Pyridoxal Catalysis, p. 361. Pergamon (1963). 6. BUELL, M. V., AND HANSEN, R. E., J. Amer. Chem. Sot. 82, 6042 (1960). 7. MATSUO, Y., J. Amer. Chem. Sot. 79, 2911 (1957). 8. DE MARCO, C!., Biochim. Biophys. Acta 85, 162 (1964). 9. DE MARCO, C., AND BOGNOLO, D., Arch. Biothem. Biophys. 98,526 (1962). A. RINALDI C. DE MARCO Department of Biochemistry University of Cagliari Cagliari, Italy Received January 6, 197’0; accepted March 6, 1970
OF
BIOCHEMISTRY
AND
BIOPHYSICS
138,
697-699
(1970)
COMMUNICATIONS A Comparison
of Pyridoxal
Phosphate-Cystine Homocystine
and Pyridoxal
Phosphate-
Reactions
In previous researches on the degradation of cystine in noneneymatic model reactions (l-3), it has been demonstrated that pyridoxal phosphate (PALP) catalyzes the cleavage of cystine through a typical ~$3 elimination reaction, originating thiocysteine, ammonia, and pyruvate (4, 5). Thiocysteine then decomposes into cysteine and elementary sulfur. The reaction takes place at room temperature, in slightly alkaline medium, and without added metal ions. It is characterized by the intermediate formation of a thiazolidine derivative of cysteine and PALP, which shows a peculiar absorption spectrum (6, 7). In the same way PALP catalyzes the degradation of S-aminoethylcysteine, with p-elimination of cysteamine (S), and this latter also links PALP in a thiazolidine ring during the reaction (9). It seemed interesting to study if, in the same experimental conditions, homocystine also could be degraded by PALP, possibly through an cu-y elimination reaction. In this case homocysteine should be one of the products of the reaction. To evidentiate it, we have first checked that in our experimental conditions homocysteine readily reacts with PALP originating a tetrahydro-thiazine ring (6, 7) easily detectable spectrophotometrically. Then we have searched for the production of this derivative in incubation mixtures containing PALP and homocystine. Control tests with cystine and PALP have been run in parallel. DL-Homocystine and DL-homocysteine were obtained from Fluka; PALP from Hoffmann-LaRoche; all other compounds and reagents from Merck. Solutions (10V2 M) of cysteine or homocysteine in water were prepared immediately before use. Cystine and homocystine were dissolved in the minimum amount of 1 N HCl, and diluted to 10-a M with 0.5 M sodium phosphate buffer pH 8.5. PALP was dissolved in the same buffer. Optical spectra were recorded, using l-mm or IO-mm silica DB spectrophotometer cells, in a Beckman equipped with a Sargent SRL-G recorder. The cell compartment was thermoregulated at 25” by circulating water from an ultrathermostat.
Figure 1 shows that in 0.5 M sodium phosphate buffer pH 8.5, homocysteine, like cysteine (4), binds to PALP causing the disappearance of the absorption at 390 nm, and the appearance of a peak characteristic for a thiazolidine or thiazine derivative (6,7). There is a small but evident dif ference between the peak of the cysteine-PALP
.6
4 n 6 .2.
FIG. 1. Variations of PALP spectrum after addition of homocysteine. A. Full line: PALP 1O-4 M in 0.5 M sodium phosphate buffer, pH 8.5. Dotted lines: PALP lo+ M and homocysteine lo+ M in the same buffer. Curves 1 to 5 recorded at 1, 5, 15,30, and 60 min after the addition of homocysteine. B. Spectra recorded at the end of the reaction between PALP and cysteine (full line) or homocysteine (dotted line). PALP 1W M, thiols 10V3 M in 0.5 M sodium phosphate buffer, pH 8.5. Light path: 10 mm; temperature: 25”.
thiazolidine, which shows the maximum at 330 nm, and the peak of the thiazine derivative of homocysteine, centered at 325 nm (Fig. 1B). Figure 2 shows that for solutions containing 1OW M PALP and 10 or 20 molar excess of cysteine or homocysteine, the decrease in OD at 390 nm is parallel to the increase at 330 nm. In these conditions, and also for equimolar solutions of PALP 697
COMMUNICATIONS
(i98
min.
FIG. 2. Changes of PALP absorption at 390 and 330 nm after the addition of cysteine or homocysteine. Full lines, cysteine; dotted lines, homocysteine. A and B, decrease in OD at 390 nm; A’ and B’, increase at 330 nm for solutions containing PALP 1OW M, and aminothiols 1OW M and 2.10v3 M, respectively, in sodium phosphate buffer 0.5 M, pH 8.5. Light path, 10 mm; temperature, 25”. and cysteine or homocysteine at 10v3 M final concentration, a second-order rate constant of 90 f 5 M-I mini has been calculated for both thiols. This indicates that in the experimental conditions followed, homocysteine links PALP in a cyclic derivative as well as cysteine does, without appreciable differences in the reaction rate. Therefore, if homocysteine is formed from homocystine in the presence of PALP, it should be easily detectable from the appearance of the thiazine peak at 325 nm. Homocystine and PALP were then incubated in equimolar amounts (10m3M) at 25“, in 0.5 M sodium phosphate buffer, pH 8.5, and the spectral changes were recorded. For comparison cystine and PALP were incubated in the same conditions. Figure 3 reports the results obtained. It is evident that cystine is degraded, as already known (4,5), giving rise to a thiazolidine derivative with the typical absorption peak at 330 nm. Homocystine instead gives rise to the only formation of a Schiff base, as indicated by the shift of the PALP absorption to longer wavelengths. Also, when the
FIG. 3. Variations of PALP spectrum after addition of homocystine (A) or cystine (B). A. Full line. PALP 1OW M in 0.5 M sodium phosphate buffer, pH 8.5. Dotted lines. PALP 1OW M and homocystine lo+ M in the same buffer. Spectra recorded 1 min (Curve 1) and 10 min (Curve 2) after the addition of homocystine. The spectrum remained unchanged after 3 hr. B. Full line. PALP 1OW M in 0.5 M sodium phosphate buffer, pH 8.5. Dotted lines. PALP 10s3 M and cystine 1OW M in the same buffer. Curves 1 to 4 recorded 1, 30, 60, and 150 min after the addition of cystine. Light path, 1 mm; temperature, 25”. incubation was performed at 38” for several hours, in a pH range from 7.5 to 9; or when the homocystine-to-PALP ratio was varied from 0.5 to 5, no appreciable variations of the Schiff-base spectrum were observed. On the other hand, it was ascertained that homocysteine can bind PALP even if it is present as Schiff base with homocystine: in fact, after the addition of homocysteine the Schiff -base spectrum readily turns into that of the thiazine derivative. It may be concluded, therefore, that no homocysteine is released from homocystine incubated with PALP; that is, contrary to what happens with cystine, in the experimental conditions followed, PALP cannot catalyze the cleavage of homocystine. This may reflect a greater stability of the Schiff base formed between PALP and homocystine, in respect to that with cystine. The above conclusion has been confirmed by the negative results obtained in searching the possible products of the homocystine cleavage: ammonia, sulfur, and ketoacids. The results of the present note, even if limited only to homocystine, may be an indication that PALP, while catalyze without added metal ions, at room temperature, and in slightly alkaline
COMMUNICATIONS medium, a-0 elimination reactions of sulfur-containing aminoacids, like cystine and S-aminoethylcysteine, cannot give rise, in the same conditions, to a-y elimination reactions on analogous compounds. This hypothesis obviously deserves further confirmation, and it will be interesting to examine, in the experimental conditions above reported, the behavior of cystathionine or of the mixed disulfide between cysteine and homocysteine. REFERENCES 1. CAVALLINI, B., Arch.. 2. DE MARCO, D., Boll. 3. Ds MARCO, D., Arch.
D., DE MARCO, C., AND MONDOV?, Biochem. Biophys. 87, 281 (1960). C., COLETTA, M., AND CAVALLINI, Sot. It. Biol. Sper. 38, 1904 (1962). C., COLETTA, M., AND CAVALLINI, Biochem. Biophys. 109, 51 (1963).
699
4. DE MARCO, C., BOGNOLO, D., AND CAVALLINI, D., G. Biochim. 12, 187 (1963). 5. CAVALLINI, D., MONDOV~, B., AND DE MARCO, C., Proc. Symp. Chem. Biol. Aspects Pyridoxal Catalysis, p. 361. Pergamon (1963). 6. BUELL, M. V., AND HANSEN, R. E., J. Amer. Chem. Sot. 82, 6042 (1960). 7. MATSUO, Y., J. Amer. Chem. Sot. 79, 2911 (1957). 8. DE MARCO, C!., Biochim. Biophys. Acta 85, 162 (1964). 9. DE MARCO, C., AND BOGNOLO, D., Arch. Biothem. Biophys. 98,526 (1962). A. RINALDI C. DE MARCO Department of Biochemistry University of Cagliari Cagliari, Italy Received January 6, 197’0; accepted March 6, 1970