Agr. Biol. Chem., 39 (7), 1417-1422,1975

Purification of Thiol- Interchange

from Candida claussenii•õ

Ryuichiro KURANE and Yasuji MINODA

Department of Agricultural Chemistry , Faculty of Agriculture, University of Tokyo, Bunkyo-ku, Tokyo Received January 22, 1975

Thiol-disulfide interchange enzyme which catalyzes the thiol-disulfide interchange was purified from -free extracts of Candida claussenii by acid treatment, ammonium sulfate fractionation, aqueous polymer two phase method (Dextran-PEG system), CM-Sephadex column chromatography, Sephadex G-100 and Sephadex G-200 gel filtrations. More than four active fractions were obtained on CM-Sephadex column. Further purification steps from one of these fractions resulted in two purified enzyme preparations D-1-1 and D-2 of which the increase in specific activities was 8150- and 8450-folds respectively, over the crude extract. Both purified were homogeneous in ultracentrifugal analysis.

Although the interchange between the sul by the present authors." The paper here fhydryl groups and disulfide bond plays very reports on the purification of the enzyme from important roles in the biological system,'-" Candida claussenii. little work on the purification of the enzyme catalyzing thiol-disulfide interchange is report- MATERIALS AND METHODS ed, except for reductase. Materials. Crystalline reduced glutathione (GSH) Several enzymes have been known which was obtained from Boehringer Mannheim Co., Ltd. catalyze thiol-disulfide interchange (glutathione Dextran T500, CM-Sephadex C-50, Sephadex G-100 reductase, , disulfide reductase and G-200 were obtained from Pharmacia Fine Chemi and thiol-disulfide transhydrogenase). They cals. Other chemicals used in this study were of may be divided generally into two types. One reagent grade.

is disulfide reductase which reduces disulfide Preparation of crude extracts. Candida claussenii bond with reduced co-enzymes and the other having the highest enzyme activities was used in the is thiol-disulfide transhydrogenase, catalyzing investigation. The was cultured in a jar-fer transhydrogenation between the thiol group mentor, washed with phosphate buffer (7.06•~10-2M, and disulfide bond. Except for glutathione pH 6.5) containing 1 mm EDTA and r.-ascorbic acid (250 mg/100 ml), resulting in a 100 % suspension (100 g reductase, purification of these enzymes was wet cells per 100 g of the buffer). The suspension was reported on only thiol-disulfide transhydro pressed through a manton gaulin (400 kg/cm2) or french genase from Baker's yeast by S. Black.' Parti- press and centrifuged at 11,000 x g for 10 min. The al purifications were reported on thiol-disul supernatant was used as the crude extract. A con fide transhydrogenase from Baker's yeast by siderable amount of enzyme activity was detected in the precipitate fraction. Attempts were made to solubilize Y. Minoda and R. Kurane5) and on disulfide the enzyme from the precipitate fraction by autolysis, reductase from spore of Bacillus cereus T by maceration, acetone treatment and the addition of L. C. Blankenship.6) thioglycorate or detergent, but favorable results were Thiol-disulfide interchange activity was de not obtained. tected previously in about 20 kinds of Enzyme assay. Enzymatic reduction of disulfide bonds was carried out at pH 8.0 in Thumberg tubes •õ Disulfide Reduction and Sulfhydryl Oxidation by exchanged with N2 gas. The reaction mixture (total Microbial Enzyme. Part III. This paper was present- volume 4.0 ml) contained 2.0 ml of enzyme solution, ed at the Annual Meeting of Agricultural Chemical Society of Japan, Sendai, April 4, 1972. 10 mg diphenyl disulfide, 2 mg GSH and 2.0 ml of 1418 R. KURANE and Y. MINODA

Tris-buffer (0.37 M, is=0.1, pH 8.4). After incubating the mixture at 30°C for 60 min, the enzyme activity was RESULTS estimated spectrophotometrically by NPDS method, as previously described.51 One unit was defined as Purification of thiol-disulde interchange en the amount of enzyme catalyzing the cleavage of 1 It zyme from Candida claussenii mole diphenyl disulfide per min. The enzyme was purified according to the

Determination of protein concentration. Protein following steps at 5°C, except where noted concentration was routinely determined spectrophoto otherwise. An outline of the purification metrically at O.D.280. procedures is shown in Diagram 1.

DIAGRAM 1. PURIFICATION PROCEDURE OF THIOL-DISULFIDE INTERCHANGE ENZYME FROM Candida claussenii Purification of Thiol-disulfide Interchange Enzyme 1419

Step 1. (Acid treatment) When the acetate was located in the lower phase (Dextran phase: buffer (u = 0.1, pH 5.1) of the same volume Dex I) and the upper phase (PEG phase: PEG as the crude extract (Sl) was added to the crude I) was discarded. extract, the pH of the mixture became 5.3. After standing overnight at 5°C, the precipitate B) Phase system II. In phase system II, was removed by centrifugation and the 4.0 g (w/w) of 30 % PEG6000,9.0 g of phosphate supernatant was used as S2. buffer (7.06 x 10-` M, ,o = 0.1, pH 6.4) con Step 2. (Ammonium sulfate fractionation) taining 1 mm EDTA and NaCl at a final con

A saturated ammonium sulfate solution, centration of 0.6 M were added to Dex I. After which was adjusted at pH 6.5 with 1 N NaOH, stirring at 4°C for 30 min, centrifugation was carried out at 10,000 x g for 10 min. The was added to the supernatant fraction (S2) to bring the saturation to 0.5. After the solu enzyme activity was distributed entirely in the tion was stirred for 30 min and allowed to stand upper phase (PEG phase: PEG 2), and the lower phase (Dex 2) was discarded. for 4 hr, the precipitate was removed by centrifugation. The supernatant fraction was C) The removal of PEG and the recovery brought to 0.75 saturation by addition of of the enzyme from polymer. The saturated saturated ammonium sulfate solution and al ammonium sulfate solution was added to the lowed to stand for 4 hr at 15°C after stirring PEG 2 to bring the saturation to 1/3. After the for 30 min. The precipitate was collected by PEG 2 fraction was stirred for 10 min and centrifugation, dissolved in K, Na-phosphate allowed to stand for 30 min at 4°C, the enzyme buffer (7.06•~10-2M, ,u = 0.1, pH 6.4) con and PEG were recovered in the lower phase taining 1 mm EDTA and L-ascorbic acid (salt solution) and in the upper phase, respecti (1 mg/ml) and dialyzed thoroughly against the vely, by centrifugation at 10,000 x g for 10 min. same buffer at 5°C. The dialyzed solution The upper phase (PEG phase) was discarded. was used as S3. The lower phase (salts solution) fraction was Step 3. (APTP method: Dextran-PEG brought to 0.75 saturation of ammonium system) Aqeous Polymer Two Phase method sulfate, stirred for 30 min and allowed to stand (APTP method) by P. A. Albertson8) was ap- for 4 hr at 15°C. The precipitated protein plyed to the dialyzed solution (S3) in Dextran was collected by centrifugation at 15,000x g T500-PEG6000(Polyethylene glycol) system (See for 10 min and dissolved in the phosphate Diagram 1). buffer (,u = 0.1, pH 6.4) containing 1 mm

A) Phase system I. In the phase system EDTA and L-ascorbic acid (1 mg/ml). The I, a mixture of 1.7 g (w/w) of 30 % PEG.... and solution was dialyzed against the same buffer 2.3 g (w/w) of 30% Dextran T500 was added to overnight at 5°C. The dialyzed solution was

6.0 g of the dialyzate (S3). After stirring at used as S4.

4°C for 30 min, the mixture was centrifuged for Step 4. (CM-Sephadex C-50 column chro 10 min at 10,000•~g. The enzyme activity matography) The dialyzed solution (S4) 1420 R. KURANE and Y. MINODA obtained above was dialyzed thoroughly against disulfide interchange activities were separately acetate buffer (u = 0.05, pH 5.05) at 4°C. eluted from the column in about 4 fractions, This dialyzate was loaded on a column of tentatively called fraction A, B, C and D CM-Sephadex previously equilibrated with according to the order of elution. the same acetate buffer. After the column Step 5. (Gel filtration on Sephadex G-100) was washed with the same acetate buffer, it Fraction D was dialyzed against deionized was subjected to a linear gradient elution water, concentrated with PEG6000and then was with the same buffer containing ,u = 0.05 to dialyzed against K. Na-phosphate buffer ,u = 1.0 NaCl at a flow rate of 5 g/hr. The (,u = 0.1, pH 6.4) containing 1 mm EDTA. elution pattern is shown in Fig. 1. Thiol- The dialyzed solution obtained was applied to a column of Sephadex G-100 previously equilibrated with the same phosphate buffer. The enzyme was eluted with the same buffer at a flow rate of 10 g/hr. The elution patterns by gel filtration are shown in Fig. 2. Two peaks of thiol-disulfide interchange activity were separately eluted from the column. The two active fractions were called D-1 and D-2. The chromatogram of D-2 was symmetric. Step 6. (Gel filtration on Sephadex G-200) After fraction D-1 was dialyzed under reduced pressure, the concentrated solution was loaded on a column of Sephadex G-200 previously equilibrated with K, Na-phosphate buffer (y= 0.1; pH 6.4) containing 1 mm EDTA. The enzyme was eluted with the same buffer. FIG. 1. Chromatography on CM-Sephadex C-50 The flow rate was 10 g per hour. The chro Column. matogram is shown in Fig. 3. Two protein Each fraction was 5g. •›•\•› enzyme activity (O.D.450); ----- O.D.280; peaks appeared and the enzyme activity was ---, ionic strength of acetate buffer (pH 5.05). recovered in the main protein peak. This

FIG. 2. Chromatography on Sephadex G-100 Column.

Fractions of 7 g each were collected at a flow rate of 10 g/hr.

•›•\•›, enzyme activity; •œ---•œ O.D.280. Purification of Thiol-disulfide Interchange Enzyme 1421

active fraction was called D-1-1.

Summary of purification. The purification steps and the results are summarized in Table I. D-1-1 was purified approximately 8150-fold over the original crude extract with a 2.3 recovery of the activity and D-2 by approxi mately 8450-fold with a 2.7 % recovery.

Homogeneity of thiol-disulfide interchange enzyme FIG. 3. Chromatography on Sephadex G-200 Homogeneity of D-1-1 and D-2 was tested Column. with a Hitachi model UCA-lA analytical Fraction of 7 g each were collected at a flow rate of 10 g/hr. ultracentrifuge at 20°C by synthetic interface. 0-0, enzyme activity (O.D.450); •œ---•œO.D.280. In the ultracentrifuge, thiol-disulfide inter-

TABLE I. PURIFICATION OF THIOL-DISULFIDE INTERCHANGE ENZYME FROM Candida claussenii Specific activity: unit/ml/O.D.280.

FIG. 4. Ultracentrifugal Patterns of Purified Thiol-disulfide Interchange Enzyme (D-1-1) and (D-2). (a) purified enzyme (D-1-1), each frame was recorded at 7 min intervals. (b) purified enzyme (D-2); each frame was recorded at 4 min intervals. 1422 R. KURANE and Y. MINODA change enzyme (D-1-1) showed a single EDTA and L-ascorbic acid was used. symmetrical peak at K, Na-phosphate buffer Further purification will be conducted on

(u = 0.1, 7.06•~10-2M, pH 6.4), as shown in fraction A, B and C obtained separately in Fig. 4-a. The enzyme D-2 seemed to be CM-Sephadex column. The properties of almost symmetrical. (Fig. 4-b) D-1-1 and D-2 have been studied and will be reported in the next paper. It is uncertain at

DISCUSSION present whether these fractions and particle bound enzymes are the same. Thiol-disulfide interchange enzyme were purified from cells of Candida claussenii. REFERENCES These might be the first enzymes having thiol- disulfide interchange activity isolated in an 1) N. Mugibayashi, Kagaku to Seibutsu, 8,278 (1970). almost homogeneous form. 2) H. Moor, Arch, Microbiol., 57,135 (1967). 3) M. Hayashibe, Seikagaku, 41, 532 (1969). Only 5 - 6 mg of the lyophilized weight of 4) S. Black, J. Biol. Chem., 243, 1942 (1968). purified enzyme (D-1-1) and 2 - 3 mg of 5) Y. Minoda, R. Kurane and K. Yamada, Agr. purified D-2 were obtained from 18 kg of wet Biol. Chem., 37, 2511 (1973). intact cells which were cultured in about 300 6) L. C. Blankenship and J. R. Mencker, Can. J. liter broth. The enzymes are likely to be Microbiol., 17,1273 (1971). unstable particularly in the presence of transi 7) R. Kurane and Y. Minoda, Agr. Biol. Chem., 39,1411 (1975). tion metals and . Hence, in prepara 8) P. A. Albertson, Advances in Protein Chem., 24, tion and purification, a buffer contained 309(1970).