The Effect of Some Cystine Derivatives on Catalase Activity*

H. HiRAif ANDH. F. DEUTSCH

(Department of Physiological Chemistry, University of Wisconsin, Madison, Wis.)

The decreased liver catalase activity of animals MATERIALS AND METHODS bearing tumors is well known (17, 18). However, Beef liver catalase, crystallized six times and prepared by various explanations of this effect have been of acetone fractionation (8), was utilized as the source of enzyme fered. One of these, as exemplified by the studies in in ritro experiments. It was found to be essentially pure, as evaluated by clectrophoretic and ultracentrifugai assay. Its of Nakahara et al. (Il, 12, 23-26), postulates that hydrogen peroxide-destroying activity, as evaluated by the tumors release a factor designated as toxohor- method of Bonnichsen, Chance, and Theorell (4), varied be mone, which exerts its effect on liver catalase. A tween 2.2 and 2.7 X IO7liters/mole/sec. As in a previous in vestigation (11), the amount of catalase giving a first-order similar view is also taken by other investigators reaction constant of 1 X IO"1 was designated as 1 unit. The (15, 16, 18). dissociation constants of various catalase-inhibitor complexes An alternate suggestion is that the discussed in were calculated from general equilibrium equations. hibition is due to some aberrancy in cystine metab The cystine derivatives used in the various in vitro and in olism by tumor tissues. These show a marked in vivo experiments were cysteine, cystine disulfoxide (18), crease in alkali-labile sulfur (13), and the level of cysteine sulfinic acid1 (21), 2-amino-ethane-sulfinic acid (2, 14, 27, S3),1cysteic acid, taurine, and the decomposition products cystine has been found by Schweigert et al. (29) to resulting from the action of sodium sulfite on cystine di be present in markedly enhanced concentration in sulfoxide. liver hepatomas as compared with adjacent nor The rapid autoxidation of cysteine and sulfinic acids makes mal tissue. The intraperitoneal administration of difficult the preparation of standard solutions for experimental work. Such type compounds were always prepared in 0.01 M cystine also lowers liver catalase activity in vivo sodium sulfite, which prevents oxidation of the above. This (30), but no increased blood level of this amino level of sodium sulfite showed no appreciable in ritro or in viro acid was noted in tumor-bearing rats. effects on catalase in control experiments. Hargreaves and Deutsch (11) have reported Cysteine concentrations were determined by the method of that tumor "Kochsafts" are very active in inhibit Folin and Marenzi (10). Ascending paper chromatography ex periments on Whatman No. 1 paper with the use of a saturated ing catalase in vitro as compared with those of phenol Â¡water(100:40 volumes) solvent was employed to de other tissues. This finding has been criticized by termine the relative purity of compounds such as cysteine, Endo et al. (9), who reported that other tissue cystine, cystine disulfoxide, and the sulfinic acids. The chro- "Kochsafts" likewise inhibited catalase strongly. matograms were developed with ninhydrin. They suggest that this activity is due to sulfhydryl compounds. However, it was found that, whereas RESULTS iodoacetate would reverse the in vitro inhibitory In vitro studies.â€”Various levels of inhibitors effect of cysteine on catalase, it was ineffective in and catalase in 0.1 M,pH 6.8, potassium phosphate reducing the activity of tumor "Kochsafts" (30). buffer were incubated at 0-2Â°C. for 60-90 min The indicated aberrancy in cystine metabolism utes. Sodium suhlte was present at a level of 0.01 by tumors and the known inhibitory action of M, as previously mentioned. The period of incuba cysteine on catalase activity prompted an investi tion employed was found sufficient to give maxi gation on the effect of various known cystine mum inhibition in all cases. These mixtures were metabolites on this enzyme. Such compounds then assayed for catalase activity, which was in might undergo marked elevation in liver tissue as terpreted as free enzyme by reference to a control the result of an increased rate of metabolism of catalase activity-concentration curve of the type this amino acid. presented in Chart 1. * This work was supported in part by a grant from the The results of the experiments with cysteine are shown in Chart 2. The value of the dissociation American Cancer Society (Univ. of Wis. Institutional Grant) constant of cysteine-catalase was nearly 4 X IO"6. and the Wisconsin Alumni Research Foundation. t Receipt of a Fulbright Travel Grant is gratefully ac 1The authors wish to acknowledge with thanks the helpful knowledged. advice of Dr. Kiyoo Satoh during the preparation of these Received for publication October 2, 1957. compounds. 283

This may be compared with the values for the sideration the rapid loss of cysteine by oxidation analogous complexes of catalase with cyanide, may account for the higher values of the older 2-amino-e thane and cysteine sulfinic acids, and the literature. products of the decomposition of cystine disulf- The products resulting from the decomposition oxide by sodium sulfite (see Table 1). It is appar of cystine disulfoxide by sodium sulfite were dis ent that cysteine was the strongest inhibitor of the tinctly stronger inhibitors than the expected sul cystine derivatives studied, although it was weak- finic acid derivative. In analogy with the postu lated decomposition of cystine disulfoxide and cystine by cyanide (21) and of cystine by sodium THEORETICAL, suhlte (5), the following reactions might be pro iÂ»j posed for cystine disulfoxide with sodium sulfite. ~o 3OAJ O 3.0* ^^ X O OJ Râ€”Sâ€”Sâ€”R+NajSO|-Â»Râ€”Sâ€”Oâ€”Na o 2.0 2.0 Â°i -s il I Sulfenate

j- 1.0 i.oS o -Â»Râ€”Sâ€”Oâ€”Xa+Râ€”Sâ€”Sâ€”Oâ€”Nai n 2.5 5.0 7.5 10 Suifinate CONC. ENZYMECE)xlO 7 The paper chromatograms of the cystine disulf- CMOLES PER LITER) oxide-sodium sulfite reaction mixtures gavejtwo CHART1.â€”Relation of catalase concentration to activity, TABLE 1 ki = -jln^J and K = -j, where E = moles catalase (mol. DISSOCIATIONCONSTANTSOFVARIOUS wt., 225,000)/liter (17). CATALASECOMPLEXES Dissociation Inhibitor constant* Cyanide 8X10-7 Cysteine 4X10-6 Cystine disulfoxide-NajSOa reac 1-2.5X10-Â« 2-Amino-ethanetion products sulfinic acid 4-6 XIO"4 Cysteine sulfinic acid 5-7 X10~4 * Determined in 0.1 M, pH 6.8 potassium phos phate buffer at 25Â°C.(Â±1Â°C).

ninhydrin-reactive spots. One of these had an R, close to that of the sodium salt of cysteine sulfinic acid (RP 0.2), but the second was not identifiable in terms of the other cystine derivatives. The pos sibility or its being cysteine sulfenate is indicated

4 5 by the above postulated equations. This very la -LOG CONC. bile compound should be a strong inhibitor of cata CHART2.â€”Therelation of cysteine and cyanide concentra lase and, if formed, might be maintained in part tion to catalane activity (4.85 X 10~7 moles catalase in test by the presence of sodium sulfite. It will be pointed system). out later that other reactions of the cystine disulfoxide-sodium suhlte reaction products indicate er than cyanide. Our value for the dissociation that the catalase inhibition is not due to the constant of the cyanide-catalase complex was sulfinate. somewhat lower than that of other investigators Mercaptoethanol and mercaptoacetic acid in (6, 7, 20, 22, 32, 34). The value of 4 X 10~6 for the hibit catalase to the same degree as cysteine. Nei dissociation of the cysteine-catalase complex is ther cysteic acid nor taurine shows any activity, in much lower than the 1 X 10~3 for sulfhydryl agreement with previous findings (30). groups reported by Stern (32). However, as pre Spectrophotometric studies.â€”A marked change viously mentioned, the failure to take into con in the absorption spectrum of catalase was seen

following its reaction with cysteine. Of particular Solutions of formol and iodoacetate were react interest was the marked decrease in absorption in ed with the various cysteine derivatives in M/15 the region of the Soret band. The general charac potassium phosphate buffer at pH 6.8. Sodium teristics of the curve are similar to those of cata- sulfite was included in the reaction mixture in all lase-tumor "Kochsaft" mixtures (19). If one as cases, since it was a component of the cystine sumes that the minimum value of the 405 mp op disulfoxide decomposition reaction. Appropriate tical density at high cysteine-catalase ratios is due control experiments with the sodium sulfite were entirely to this complex, the concentration of free always performed. Under the conditions used, the enzyme at different cysteine: catalase ratios may reactions of formol and iodoacetate with cysteine be calculated from the following equations: had gone to completion at room temperature in 25 and 75 minutes, respectively. These reaction mix (1) FE. + BE0 = Â£â€žÂ«,. tures were then incubated with catalase for 1-2 hours at 0-2Â°C. following which interval the usual (2) T = F-B activity determination was performed. Data typi From (1) and (2), cal of experiments with formol and iodoacetate are presented in Tables 3 and 4, respectively. It can Eread. ~ TE0 be seen that formol reversed the catalase-inhibiting (3) F = Ea-E/3 action of all the compounds tested, as well as that of the tumor extracts. In control experiments it where T = moles of total enzyme, F = moles of was seen that formol enhanced catalase activity free enzyme, B = moles of bound enzyme, E0 = in some cases. molar extinction of the free enzyme at 405 m/x, In keeping with previous studies, iodoacetate

TABLE 2 DISSOCIATIONCONSTANTOFTHECYSTEINE-CATALASECOMPLEXAS DETERMINEDBYSPECTROPHOTOMETRICANDACTIVITYMEASUREMENTS CONCENTRATIONS* Free Bound ACTIVITT catalase catalase (ItiXlO') METHOD Cysteine X10' X10' KxlO't 0 4.85 0 1.85 0.110 Activity 5X10-6 5X10-' 3.88 1.07 1.53 1.8 Spectro. 3.95 0.9 0.102 2.2 Activity 5X1CT5 5X10-5 1.82 3.03 0.79 3.0 Spectro. 5X10-Â« 1.74 3.11 0.089 2.8 4.85 0.01 0.077 * In moles/liter. t Dissociation constant. These and the extinction coefficients were determined in 0.1 M, pH 6.8, potassium phosphate buffer at 25Â°C.( +1Â°C.).

Eg = molar extinction of the bound enzyme at was found to inhibit catalase slightly, to partially 405 mju, and Eread. = optical density of mixture of reverse the catalase-inhibiting action of cysteine, enzyme and inhibitor. and to show little effect on the activity of the tu Data from experiments on the determination of mor extract (30). A further finding was the ability the dissociation constant of the catalase-cysteine of iodoacetate to reverse the inhibitory action of complex by the spectrophotometric and activity the sodium sulfite decomposition products of methods are shown in Table 2. The results are in cystine disulfoxide. Interestingly, iodoacetate en good agreement. hanced the inhibitory action of cysteine sulfinic Cysteine sulfinic acid, 2-amino-ethane sulfinic acid. acid, and the reaction products of cystine disulf In vivo effects of various cystine derivatives on oxide and sodium suhlte did not affect the absorp liver catalase activity.â€”Various compounds in tion spectrum of catalase in the 350-520-mju range. M/15, pH 7.2, potassium phosphate buffer were The effect of formol and iodoacetate on the inhibi administered by intraperitoneal injection to 150- tory effect of various cystine derivatives.â€”Formol 190-gm. male rats. The low solubility of cystine reacts with cysteine forming a thiazolidine deriva and cystine disulfoxide necessitated their adminis tive and thus effectively neutralizes the activity of tration in the form of fine suspensions. The rats the sulfhydryl group. lodoacetic acid exerts the were killed by decapitation after stated intervals, same effect through its well known alkylation the livers perfused with isotonic NaCl and ho reaction. mogenized in 3 volumes of distilled water. Catalase

activity and dry weight determinations were then period. Cysteine sulfinic acid would appear to be a carried out on the homogenates. The activity stronger in vivoinhibitor than the 2-amino-ethane measurements were made within several hours, al derivative, because it is converted to the latter though no changes were noted in homogenates inhibitory compound on its metabolic pathway to that were allowed to stand for 24 hours at room taurine. temperature. Freezing and thawing of the homoge nates result in enhanced catalase activity, but DISCUSSION these procedures were not employed, since variable The work reported is directed toward the gen results were obtained. A summary of the average eral problem of determining whether the known

TABLE 3 THEEFFECTOFFORMOLONTHECATALASE-INHIBITINGACTION OFVARIOUSCYSTINEDERIVATIVES CATALASEACTIVITY(K,X l"-) m PRESENCEor MOLAR Formol*+ INHIBITOR CONC. Control Formol* Inhibitor inhibitor Cysteine 1.25XKT4 1.07 1.05 0.21 1.05 5.0 X10-* 0.80 0.96 0 1.0 1.0 X10-Â» Cysteine sulfinic acid 0.80 0.96 0.41 0.94 1.25X10-3 0.96 0.97 0.74 0.99 5.0 X10-3 Cystine disulfoxide- 0.96 0.97 0.68 0.99 Na2SOa reaction prod ucts Tumor "Kochsaft" 1.18 1.30 0.15 1.21 *Final concentration of formol was 1 X IO"1M.

TABLE 4 THEEFFECTOFIODOACETATEONTHECATALASE-INHIBITINGACTION OFVARIOUSCYSTINEDERIVATIVES CATALASE ACTIVITY (KiXlO1) IN PRESENCE OF lodoace- MOLAR lodo- tate + INHIBITOR CONC. Control acetate* Inhibitor inhibitorf 5 X10-* Cysteine 0.82 0.71 0.07 1 X10-* 0.51 0.80 0.62 0 0.47 l X10-* Cysteine sulfinic acidf 0.82 0.71 0.68 5 X10-* 0.67 0.80 0.41 5 X10-Â« 0.62 0.05 0.76 0.58 0.41 0 2.5X10-' Cystine disulfoxide- 0.79 0.48 0.74 2.0X10-Â» 0.71 Na2SO3 reaction 0.76 0.58 0.07 0.44 products Tumor "Kochsaft" 1.18 1.03 0.15 0.25 * The concentration of iodoacetate was 1 X IO"2 M. t The concentrations of this inhibitor are approximate, since the preparations contained considerable cysteic acid at the time of these experiments.

effects of cystine and some of its derivatives is effects of cystine in depressing liver catalase ac shown in Table 5. The detailed data are plotted in tivity are related to the analogous in vivoeffects of Chart 3 to show the individual variations en tumors or to the in vitro effects of tumor "Koch- countered. safts." A considerable variation in the liver catalase Sulfhydryl compounds of the type exemplified activity within a given group is indicated. With by cysteine are strong inhibitors of catalase. Our the exception of 2-amino-ethane sulfinic acid, all value of 4 X 10~5for the dissociation constant of the compounds tested except the sulfonic acid the catalase-cysteine complex may be compared derivatives showed greater liver catalase-depress- with a value of 8 X 10~7for catalase cyanide. Rel ing activity than cystine. The former compound is atively strong catalase inhibition was also shown converted to inactive taurine by rat liver (3) and by the sulfinic acid and the sodium sulfite decom shows a definite inhibitory effect only at the 2-hour position products of cystine disulfoxide. The in-

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1958 American Association for Cancer Research. HiRAi ANDDEUTSCHâ€”CystineDerivativesand Catalase Activity 287 hibitory action of the latter does not appear to be with catalase, causes spectral changes similar to due to the sulfinic acid, since Â¡odoacetatewas ef those of cysteine (19). The sulfinic acids and fective in reversing its effect while augmenting the cystine disulfoxide-sodium suhlte reaction prod effect of the cysteine sulfinic acid. The possibility ucts produced no such changes. Spectrophoto- of cysteine sulfenic acid's being responsible was metric studies on various liver catalase prepara previously mentioned. In any event, a series of tions from rats bearing hepatomas reveal that cystine degradation products has been shown to marked changes in absorption spectrum are in inhibit catalase both in vitro and in vivo. Other duced by the presence of the tumor (28). cystine metabolites, such as mercaptopyruvic The ability of "Kochsafts" of other tissues to acid, 2-amino-2-carboxy-ethanethiosulfinic acid, inhibit catalase (9, 30) indicates that tumor tissue and alaninethiosulfinic acid (31), have not been is not unique in this respect, although in our expe- tested but are also probable inhibitors of this en zyme. An aberrancy in sulfur amino acid metabo-

TABLE 5 Â°500 THEEFFECTOFCYSTINEANDSOMEOFITSDERIVA tr LU TIVESONTHEACTIVITYOFRATLIVERCATALASE a. Amount UJ injected t en o â€¢â€¢ (mg/100 No.rats8157556Unitscatalase/gmliver342266167230248220Percentofnormal7849677364 Inhibitor* gm) 3 300 None â€¢ Â°A Cystine 8.5 â€¢"â€¢ Cysteine 8.5 U e 4- Cysteine (2 hr.) 8.5

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H. Hirai and H. F. Deutsch

Cancer Res 1958;18:283-288.

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