THE ENZYMATIC HYDROLYSIS OF GLUTATHIONE BY RAT KIDNEY

THE ENZYMATIC HYDROLYSIS OF GLUTATHIONE BY RAT KIDNEY BY E. F. SCHROEDER AND GLADYS E. WOODWARD (From The Biochemical Research Foundation of the Franklin Institute, Philadelphia) (Received for publication, March 16, 1937) In previous work it was shown (1, 2) that kidney tissue (rat, rabbit, pig, horse) contains an enzyme which exerts a strong hydrolytic action on reduced and oxidized glutathione. Pancreas also contains the enzyme, although in smaller amounts. Titration data and certain color reactions indicated that hydrolysis proceeded only until all the glycine had been removed, leaving the then unknown y-glutamylcysteine (3), or rdiglutamylcystine, in solution. Further work with various rat kidney preparations has led us to revise our original conclusions. We now find that both peptide linkages are completely hydrolyzed, by processes which are without doubt enzymatic. Attempts to isolate y-glutamylcysteine from kidney-glutathione (GSH) incubation mixtures were not successful. However, a crystalline product, identified as cysteine sulfate, was obtained in 53 per cent yield. Since cysteine is the middle amino acid in the tripeptide molecule, this result indicated either that complete enzymatic breakdown into the constituent amino acids had occurred, or that the glutamylcysteine first formed had been hydrolyzed by chemical action during the attempted isolation. The first alternative proved to be the correct one. Using extremely mild conditions for protein removal (heat coagulation, or removal of an insoluble acetone-ether enzyme preparation by filtration) and then oxygenating the protein-free titrates, we were able to recover over 70 per cent of the theoretical cysteine as insoluble cystine. Our original conclusion was based largely on titration data obtained by the method of LinderstrZm-Lang, Weil, and Holter 209

210 Enzymatic Hydrolysis of Glutathione (4). In this method, titration is carried out in 80 per cent acetonealcohol mixture, with tetramethylammonium hydroxide as base and thymol blue as indicator. It has now been found that pure glutathione, when titrated to ph 11 to 12 as called for, gives values which are too high by over 0.75 equivalent of alkali. Cysteine, under like conditions, gives the correct value. When proper correction is made for this abnormal behavior of pure glutathione, results are obtained which agree closely with complete hydrolysis of both peptide linkages. The Harris (5) alcohol-formaldehyde- NaOH method, and the Harris (5) HCl method, both of which were found to give correct titrations for pure glutathione and its constituent amino acids, also indicate that both linkages are hydrolyzed. Oxidized glutathione (GSSG) is likewise readily hydrolyzed into its constituent amino acids by rat kidney, cystine being identified as a product by means of the Sullivan test. Titration data are inconclusive in this case because of interference due to precipitation of cystine during the course of the reaction. The discovery of the complete enzymatic breakdown of glutathione is of interest in view of the high degree of stability towards enzymes usually attributed to this compound (6-8). Grassmann and coworkers (9) were unable to effect an enzymatic hydrolysis of reduced glutathione, although glycine was removed from the oxidized form by means of pancreatic carboxypolypeptidase. Yet the recent work of Dyer and du Vigneaud (lo), who showed that glutathione is able to replace cystine in rats held on a cystinedeficient diet, strongly indicates that hydrolysis to yield cysteine (or cystine) does occur during metabolism. On the basis of rules formulated by Bergmann et ab. (11) the splitting of the glycine-cysteine linkage can be satisfactorily accounted for by the action of carboxypolypeptidase. It is more difficult to explain the hydrolysis of the r-glutamic acid-cysteine linkage. Bergmann and Zervas (12) and Grassmann and Schneider (13) have reported that bdipeptides of aspartic acid are not attacked by any of the known enzymes, although the a! forms are normally hydrolyzed. In view of the close structural similarity between aspartic and glutamic acids, it may be expected that cr-dipeptides of the latter will be normally hydrolyzed, as is in fact the case, while r-dipeptides should be resistant to enzyme attack.

E. F. Schroeder and G. E. Woodward 211 Such rdipeptides have not been available for investigation except indirectly in glutathione, in which Grassmann et al. (9) were unable to effect hydrolysis. Further work on the nature of the enzyme (or enzymes) responsible for glutathione hydrolysis should show whether we are dealing with a new rdipeptidase. EXPERIMENTAL Enzyme Preparations--Aqueous kidney extract was made by thoroughly grinding fresh, clean kidneys of albino rats with sand and water (5 to 15 parts, depending on the activity desired), and removing the residual tissue by centrifuging. Acetone-ether preparation was made by mincing the kidneys, extracting four times with 5 volumes of acetone and twice with 5 volumes of ether, drying overnight in air, and sifting the resulting product through a fine sieve. In order to remove as much water-soluble material as possible, 10 gm. of the dried powder were washed four times with 150 cc. of water in a centrifuge tube. The drying with acetone and ether was then repeated. Isolation of Cysteine Suljate from Kidney-Glutathione Digesk- Attempts to isolate the sulfhydryl compound, resulting from the digestion of glutathione by kidney preparations, by means of copper or lead precipitation methods were not successful. The following mercury procedure was found to be suitable. 1 gm. of GSH was dissolved in 50 cc. of water containing 1 equivalent of NaOH (ph 7). 50 cc. of a 1:5 aqueous rat kidney extract were added and the mixture incubated at 25 for 1 hour. At this time GSH was shown to be absent by the glyoxalase method of Woodward (14). The mixture was deproteinized by addition of 11 cc. of 22 per cent sulfosalicylic acid. The proteinfree filtrate was treated with 17 cc. of mercuric sulfate reagent (10 per cent HgSOc in 5 per cent HaSOk) to precipitate the sulfhydryl compound. The mercury precipitate was thoroughly washed at the centrifuge, suspended in 10 cc. of water, and decomposed with H&3. The HgS was centrifuged down and washed with water, the combined washings and supernatant liquid being gassed with NZ to remove H&3. The solution was placed in a vacuum desiccator over H&304 and evaporated to a syrup which on slight scratching readily set to a crystalline mass. For purification the product was taken up in 60 cc. of warm 95 per cent alco-

212 Enzymatic Hydrolysis of Glutathione hol, and the solution filtered and concentrated to 10 cc. in a vacuum desiccator over HzS04. Clumps of crystals in rosette formation appeared, which were filtered off, washed with absolute alcohol, and dried. The following analyses identified the product as cysteine sulfate, (HS-CHZ----CHNHZ-COOH)Z.H&SO~: found, S 27.8, free HzS04 27.0, N(Kjeldah1) 8.2, cysteine (Sullivan) 71.2; calculated, S 28.2, free H&J04 28.8, N 8.23, cysteine 71.2. The yield obtained in the first crop of the recrystallization was 286 mg., or 53 per cent of the theoretical. The melting point was 175-176 (with decomposition). For further identification, the same product was prepared from Z-cystine. 500 mg. of the latter were dissolved in 100 cc. of 2 per cent sulfosalicylic acid, and reduced to cysteine by the addition of zinc dust (total, 0.3 gm.) in small portions during 90 minutes. The reaction was speeded by slight warming, the course of the reduction being followed by iodate titration of the -SH group at 0 (15). After removal of excess zinc by filtration, 16 cc. of HgSOd reagent were added, and the precipitate carried through the procedure described above. On recrystallization of the crude product from alcohol, a first fraction of 50 mg., melting at 222-224 (with decomposition), was obtained. This contained no HzS04, and was identified as free cysteine by means of the Sullivan reaction and by iodate titration. A sample of free cysteinel prepared from the hydrochloride melted at the same point. The second crop of crystals, 220 mg., melted at 176-177 (with decomposition), and has the same microscopic appearance as the cysteine sulfate isolated from the kidney-gsh mixture. Its cysteine content, as shown by Sullivan s test, was identical with that of the cysteine sulfate isolated from kidney-gsh mixtures. Isolation of Cyst&e from Kidney-GSH Digests-500 mg. of GSH were dissolved in 75 cc. of water containing 1 equivalent of NaOH. After addition of 2.1 gm. of acetone-ether kidney, the mixture was incubated at 25 in Nz for 6 hours, at which time the glyoxalase test showed no GSH remaining. The enzyme was removed at the centrifuge and washed with water. The combined washings and supernatant liquid were adjusted to ph 7, oxygenated until the nitroprusside test became negative (90 minutes), and placed in 1 Kindly supplied by Dr. G. E. Toennies and Dr. Mary A. Bennett of the Lankenau Hospital Research Institute, Philadelphia.

E. F. Schroeder and G. E. Woodward 213 the refrigerator. Overnight, 83 mg. of a white precipitate had formed; on concentration of the liquid, more was deposited, a total of 126 mg. being collected. It was identified as cystine by sulfur analysis (found, 26.7; calculated, 26.9), and by iodate titration after zinc reduction. The yield was 72 per cent of the theoretical. Amino Acid Titration Data-The irregular behavior of reduced glutathione in the Linderstrgm-Lang, Weil, and Holter (4) titration method is illustrated in Table I. To 1 cc. portions of 0.02 M glycine, glutamic acid, cysteine hydrochloride, and GSH, and 0.01 M GSSG, were added 10 cc. of a 1:l mixture of acetone and absolute alcohol and several drops of indicator, either thymol blue TABLE Titration of GSH, GSSG, and Amino Acids with Alkali in Acetone-Alcohol Solution titrated Indicator I ThTT?tititration Observed titration (CHah- NOH NaOH cc. cc. cc. GSH,.......... Thymol blue 0.80 1.14 1.16 I<............... Phenolphthalein 0.80 0.82 0.80 GSSG... Thymol blue 0.80 0.79 0.82 Cysteine HCl.. I 0.80 0.83 0.83 Glutamic acid. I 0.80 0.80 Glycine....... 0.40 0.42 or phenolphthalein. Titrations were carried out with 0.05 N aqueous NaOH or alcoholic tetramethylammonium hydroxide, to the blue of thymol blue or the pink of phenolphthalein. Examination of these data shows that pure GSH titrates too high to the extent of over 0.75 equivalent of alkali when thymol blue is the indicator. Since GSSG titrates correctly in the presence of this indicator, it is probable that the high value for GSH is due to partial titration of the -SH group. When phenolphthalein is the indicator, theoretical values are obtained with either base. Cysteine titrates correctly even when thymol blue is the indicator. The same is true for glycine and glutamic acid. In our previous work, with thymol blue as indicator, the -COOH increase during hydrolysis was calculated from the difference

214 Enzymatic Hydrolysis of Glutathione between the fmal titration and that of the original mixture of kidney plus GSH. Since, on the basis of the above evidence, the former value was correct and the latter much too high, the observed -COOH increase was too low, and corresponded more nearly to hydrolysis of one peptide linkage than of two. Titration of Kidney-GSH Digests--Fig. 1 illustrates a typical GSH-kidney hydrolysis experiment in which the course of the reaction was followed by titration. A mixture was prepared 2 4 6 8 IO 24 HOUR5 FIG. 1. Hydrolysis of reduced glutathione by rat kidney preparations. Curve A, acetone-ether kidney; Curve B, aqueous kidney extract. 0 and A, Harris NaOH method; 0, Harris HCl method; 0, Linderstrem-Lang, Weil, and Holter method. containing 307 mg. of GSH, 50 cc. of water, 1 equivalent of NaOH, and 1.5 gm. of acetone-ether kidney. Incubation was carried out at 25, with shaking, in an atmosphere of nitrogen. At intervals, 6 cc. samples were centrifuged, and 1 cc. aliquots of the supernatant fluid titrated by the three methods indicated. Blanks were run on a similar mixture of kidney and water under the same conditions. These blanks were small, increasing from 0 at 2 hours to 0.1 cc. of 0.1 N alkali at 24 hours. The hydrolysis was calculated by deducting from the observed titration the kidney

E. F. Schroeder and G. E. Woodward 215 blank and the titration of the original GSH. Since, as shown in Table I, GSH titrates high in the Linderstrom-Lang, Weil, and Holter method, the blank value used for this calculation was not the actually observed high titration, but rather the theoretical for GSH. All three titration methods used show that complete hydrolysis of both peptide linkages of GSH occurs. The reaction is quite rapid during the 1st hour, the equivalent of one peptide linkage being split; the rate then decreases somewhat, but after 6 hours approximately 90 per cent of all linkages are hydrolyzed. Fig. 1 also shows that fresh kidney extract, like the acetone-ether preparation, hydrolyzes GSH completely. To a solution of GSH neutralized as above was added an equal volume of 1:7.5 fresh, aqueous kidney extract. Incubation was carried out at 25 under anaerobic conditions. 1 cc. samples were titrated at intervals, without further treatment, by the Harris (5) alcohol-formaldehyde method. Blanks were obtained from control kidney extractwater mixtures. Color Reactions-In confirmation of previous results (2) it was found that Sullivan s (16) test, when applied to such completely digested GSH-kidney reaction mixtures, indicates the presence of free cysteine in nearly theoretical amounts (85 to 95 per cent). The slightly low values are probably due to partial oxidation of the -SH group. r-glutamylcysteine,2 to which the red color development was at first ascribed, gives no color in the test. As reported previously (2), the green color which results when pure glycine is treated with Patton s (17) o-phthalaldehyde reagent is not formed in the above reaction mixtures. Instead, one obtains an intense blue color, which we at first also ascribed to y-glutamylcysteine. However, it has been found that the synthetic dipeptide gives no color with the reagent. Further study has shown that the blue color is obtained when glycine and cysteine are present simultaneously. Cysteine, glutamic acid, or GSH, alone, gives practically no color with the reagent. And the latter two, when present with glycine, do not mask the green color development due to the glycine. 2 Acknowledgment is made to Dr. C. R. Harington of the University College Hospital Medical School, London, for supplying a sample of the synthetic product.

216 Enzymatic Hydrolysis of Glutathione Hydrolysis of GSSG by Kidney-307 mg. of GSH were dissolved in 25 cc. of water, neutralized with 1 equivalent of NaOH, and oxygenated until iodate titration showed that practically all of the -SH had disappeared. Two reaction mixtures were prepared, one containing 10 cc. of the GSSG solution and 10 cc. of 1: 15 aqueous kidney extract, and the other, 10 cc. of GSSG, 10 cc. of water, and 0.6 gm. of acetone-ether kidney. Both were incubated at 25, samples being titrated at intervals for -COOH increase by the alcohol-formaldehyde method of Harris. The titrations increased steadily up to a value, at 4 hours, equivalent to the hydrolysis of approximately half of the total peptide linkages. This value then remained constant up to 24 hours, when the experiment was interrupted. It was at first thought that this indicated a difference from GSH in the behavior of GSSG towards the action of kidney. However, it was observed that at 24 hours the mixture made with fresh extract contained a fairly heavy precipitate. Due to interference of the acetoneether powder, it was impossible to detect any precipitation in that reaction mixture. It was surmised that the precipitate was cystine, and that its removal from the reaction mixture was the cause of low titrations. This view was shown to be correct by means of the Sullivan cystine (16) test. The reaction mixtures were thoroughly shaken and uniform samples removed. The fresh extract mixture was found to contain 81 per cent of the theoretical amount of free cystine. The acetone-ether mixture was centrifuged and cystine determinations were made separately on the residue and the supernatant liquid. The former contained 40 per cent, and the latter 26 per cent of the theoretically possible free cystine, or a total of 66 per cent. These results, although not as quantitative as those obtained with GSH because of the experimental diiculties involved, nevertheless indicate that all of the peptide linkages of GSSG are attacked by rat kidney preparations. SUMMARY Reduced and oxidized glutathione are completely hydrolyzed into their constituent amino acids by an enzyme (or enzymes) present in rat kidney, as indicated by titration data, the Sullivan cysteine test, and the isolation of cysteine (as sulfate) and cystine in high yields from the reaction mixtures. The conclusion drawn

E. F. Schroeder and G. E. Woodward 217 in earlier work, that only glycine is removed enzymatically, must be revised in view of the observation that reduced glutathione titrates abnormally high in the Linderstrgm-Lang, Weil, and Holter tetramethylammonium hydroxide method employed at that time. BIBLIOGRAPHY 1. Woodward, G. E., Munro, M. P., and Schroeder, E. F., J. Bid. Chem., 109, 11 (1935). 2. Schroeder, E. F., Munro, M. P., and Weil, L., J. Biol. Chem., 110, 181 (1935). 3. Harington, C. R., andmead, T. H., Biochem. J., 29,1602 (1935). 4. Linderstrflm-Lang, K., Weil, L., and Holter, H., 2. physiol. Chem., 233, 174 (1935). 5. Harris, L. J.,.I. Biol. Chem., 84,296 (1929). 6. Hopkins, F. G., Biochem. J., 16,286 (1921). 7. Lewis, G. T., and Lewis, H. B., J. Biol. Chem., 73,536 (1927). 8. Kendall, E. C., Mason, H. L., and McKenzie, B. F., J. BioZ. Chem., 87, 55 (1930). Mason, H. L., J. BioZ. Chem., 99,25 (1931). 9. Grassmann, W., Dyckerhof, H., and Eibeler, H., 2. physiol. Chem., 189, 112 (1930). 10. Dyer, H. M., and du Vigneaud, V., J. BioZ. Chem., 116,543 (1936). 11. Bergmann, M., Zervas, L., and Schleich, H., 2. physiol. Chem., 224, 45 (1934). 12. Bergmann, M., and Zervas, L., 2. physiol. Chem., 224,17 (1934). 13. Grassmann, W., and Schneider, F., B&hem. Z., 273,452 (1934). 14. Woodward, G. E., J. BioZ. Chem., 169,l (1935). 15. Woodward, G. E., and Fry, E. G., J. BioZ. Chem., 97,465 (1932). 16. Sullivan, M. X., Pub. Health Rep., U. S. P. H. S., 44,142l (1929). 17. Patton, A. R., J. BioZ. Chem., 198,267 (1935).

THE ENZYMATIC HYDROLYSIS OF GLUTATHIONE BY RAT KIDNEY E. F. Schroeder and Gladys E. Woodward J. Biol. Chem. 1937, 120:209-217. Access the most updated version of this article at http://www.jbc.org/content/120/1/209.citation Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites 0 references, 0 of which can be accessed free at http://www.jbc.org/content/120/1/209.citation.full.h tml#ref-list-1