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Purification of xanthine dehydrogenase from rat liver: A rapid procedure with high enzyme yields
OF BIOCHEMISTRY AND BIOPHYSICS Vol. 258, No. 1, October, pp. 219-225, 1987
ARCHIVES
Purification
of Xanthine Dehydrogenase from Rat Liver: A Rapid Procedure with High Enzyme Yields’
SULEIMAN Division
A. SULEIMAN2
AND
JEFFREY
B. STEVENS3
of Entironmental Health, School of Public Health, The University of Minnesota, Minneapolis, Minnesota 55&% Received March 9. 1987, and in revised form June 5,1987
Xanthine dehydrogenase (EC 121.3’7) was purified approximately lOOO-fold from liver homogenates of adult male Sprague-Dawley rats. Enzyme recovery was good (~20% of the starting activity was obtained), and the homogeneously pure enzyme had a molecular mass of approximately 300,000 Da. The purified protein exhibited a specific activity of 2470 units/mg protein and spectral properties identical to those of the best preparations of this enzyme reported by other investigators. Routine preparations of this enzyme also possess higher dehydrogenase:oxidase ratios (typically between 5 and 6) than do other xanthine dehydrogenase preparations so far reported in the literature. Maximum dehydrogenase:oxidase ratios, greater than 10, could be obtained from this procedure if only peak dehydrogenase fractions from the chromatography columns were saved. The present small-scale purification method, which can be completed in 48-60 h, utilizes ammonium sulfate fractionation, Sephadex G-200 column chromatography, Blue Dextran-Sepharose column chromatography, and preparative gel electrophoresis. o 1987 Academic
Prear,
Inc.
Xanthine dehydrogenase (XDH4; EC 1.1.1.204, formerly 1.2.1.37) is one of a group of molybdenum, iron-sulfur flavin hydroxylases that act on a wide variety of purine, pteridine, and pyrimidine substrates (1). In mammalian tissues this enzyme is thought to catalyze the oxidation of hypoxanthine and xanthine to uric acid, with a concomitant reduction of NAD+ (2-6). In vitro, i.e., in crude tissue preparations, XDH is readily converted to its oxidase form, xanr This work was supported by BRSG No. 2-SO7RR 05448, awarded to the University of Minnesota School of Public Health by the Biomedical Research Grant Program, Division of Research and Resources, National Institutes of Health, Bethesda, MD. r Present address: Department of Biological Sciences, Yarmouk University, Irbid, Jordan. 3 To whom correspondence should be addressed. ’ Abbreviations used: XDH, xanthine dehydrogenase; X0, xanthine oxidase; NBT, nitroblue tetrazolium; PMS, phenazine methosulfate; PMSF, phenylmethylsulfonyl fluoride.
thine oxidase (X0; EC 1.1.3.22, formerly 1.2.3.2) by oxidation in air at 37°C or by proteolysis with trypsin (2). When this enzyme was isolated initially from rat liver, it was described as xanthine oxidase (7). The air-oxidized enzyme, but not the proteolytically digested enzyme, can be reconverted to xanthine dehydrogenase upon exposure to thiols such as dithioerythritol (6, 8-10). Under the current IUPAC nomenclature xanthine oxidase is called the type 0 form of xanthine oxidoreductase, whereas xanthine dehydrogenase is designated the type D form. Both types have been purified from various mammalian and nonmammalian tissue sources (9,11,12,13). Most of these purification procedures, however, are either laborious or require harsh procedures, e.g., acetone or heat precipitation. Thus, relatively low yields of enzyme are usually obtained. In the present study we report a simple, quick, and gentle procedure for the 219
0003-9861/87 $3.00 Copyright All rights
0 1987 by Academic Press. Inc. of reproduction in any foorm reserved.
220
SULEIMAN
small-scale purification of xanthine dehydrogenase from rat liver. This procedure offers a high yield of enzyme (>20% of the starting activity) with a relatively high type D/type 0 ratio (typically in the range 5-6). MATERIALS
AND
METHODS
Adult male Sprague-Dawley rats (ZOO-250 g) were purchased from Biolab (Madison, Wisconsin) and were used as liver donors. Bovine serum albumin, horse ferricytochrome c, lactate dehydrogenase, 2-mercaptoethanol, milk xanthine oxidase, NAD+, nitroblue tetrazolium (NBT), phenazine methosulfate (PMS), phenylmethylsulfonyl fluoride (PMSF), ethylenediaminetetraacetic acid, disodium salt (EDTA), reactive blue 2-Sepharose (Blue Dextran-Sepharose), trypsin inhibitor, and xanthine were all obtained from the Sigma Chemical Co. (St. Louis, MO). Acrylamide, bisacrylamide, ammonium persulfate, and N,N,N’,N’tetramethylethylenediamine were purchased from Bio-Rad Laboratories (Richmond, CA). Other chemicals were of reagent quality. Purification proceduv-e. Each animal was initially anesthetized by an ip injection of pentobarbital (60 mg/kg) before the abdomen was opened. The liver was then perfused with lo-15 ml of cold (0-4°C) 0.25 M sucrose solution. The liver was removed from the animal, washed with the 0.25 M sucrose solution, weighed, and then homogenized with a Potter-Elvehjem apparatus in 10 vol of 0.1 M phosphate buffer, pH 7.8, containing 100 mM 2-mercaptoethanol, 3 mM PMSF, 1 mM EDTA, and 80 mg/liter trypsin inhibitor (buffer A). The homogenate was centrifuged at 15,000g for 20 min in a Sorvall RC-5B refrigerated centrifuge. All of the subsequent operations were performed at 0-4°C. Solid ammonium sulfate was added with stirring to the clear, reddish supernatant to obtain a final concentration of 20 g/100 ml. After 30 min of gentle stirring, the suspension was fractionated by centrifugation at 15,OOOgfor 20 min. The pellet was discarded, and an additional 20 g/100 ml ammonium sulfate was added to the supernatant. After a second centrifugation at 15,OOOgfor 20 min the supernatant was decanted, and the pellet was dissolved in 25-30 ml of buffer A. The enzyme was then dialyzed overnight against several volumes of 0.1 M phosphate (pH 7.8,l mM EDTA, 20 mM 2-mercaptoethanol, and 1 mM PMSF (buffer B). The dialyzed enzyme solution was concentrated in an Amicon ultrafiltration cell fitted with a Diaflo PM10 ultrafiltration membrane (Amicon Corporation, Lexington, MA) and then applied to a Sephadex G-200 column (2.5 X 45 cm) pre-equilibrated with buffer B. The enzyme was eluted from the column in the same buffer. Three-milliliter fractions were collected. Fractions containing xanthine dehydrogenase
AND
STEVENS
activity were pooled, concentrated and then reapplied to the G-200 column. After this second elution, the fractions containing active enzyme were again pooled and concentrated. The enzyme solution was then applied to a 1.5 X 14-cm Blue Dextran-Sepharose column (reactive blue 2-Sepharose) and eluted with buffer B. Three-milliliter fractions were collected. The final step in the purification procedure was polyacrylamide gel elution. The fractions from the Blue Dextran-Sepharose column that contained active enzyme were pooled and concentrated in an Amicon filtration cell to a volume of 1 ml. The enzyme preparation was then placed on 7% native polyacrylamide gel discs and electrophoresed for 6 h at 250 V (see below for details). After electrophoresis one of the polyacrylamide gels was stained for xanthine dehydrogenase activity to locate the enzyme. The corresponding sections on the other nine gels were excised and saved. The gel slices were diced with scissors to approximately 1 mm’ and placed in a tube (0.5 X 12 cm) plugged with 1.5 cm of stacking polyacrylamide gel prepared according to the procedure of Laemmli (14). The tube was then filled to near capacity with stacking gel, according to a modification of the procedure by Weber and Kuter (15), with extraordinary care to avoid air bubbles. A dialysis bag was fitted to the bottom end. The tube was filled with 0.1 M phosphate buffer, pH 7.8, and placed back into the electrophoresis unit. The gel containing the enzyme was again electrophoresed at 250 V for 6 h, or until the enzyme was out of the gel and into the dialysis bag. The dialysis bag containing the enzyme (yellow color) was then removed from the tube, and the enzyme was dialyzed for several hours against buffer B. Gel electrophoresis. For the last step in the purification procedure and for determining the purity of the final enzyme preparation, polyacrylamide discontinuous (disc) gel electrophoresis was performed on 7% native gel (under nondenaturing conditions) polymerized with ammonium persulfate according to Laemmli (14). Twenty to fifty microliters of a 1:l mixture of sample buffer (10% glycerol and 0.5 mM bromphenol blue in 100 mM Tris buffer, pH 6.8) and enzyme solution (2.5 mg/ml protein) was added to each tube. The reservoir buffer of the electrophoresis system consisted of 0.05 M Tris and 0.38 M glycine, pH 8.9, according to Jovin et al (16). Electrophoresis was performed at 250 V. For determining enzyme purity the gels were stained after electrophoresis for protein with amido black or for enzyme activity by immersion for 15 min in the following staining mixture (17): 0.1 M phosphate buffer (pH 7.8), 0.05 M xanthine, 0.37 my NBT, 0.75 mM NAD+, and 0.13 mM PMS. Xanthine oxidase (type 0) activity was detected by means of the above staining solution without NAD+ (17,18). Standards for the molecular mass determination included milk xanthine oxidase (-300,000 Da), lactate dehydrogenase (~100,000 Da), bovine serum albumin
PURIFICATION
OF RAT
LIVER
(60,000 Da) and horse ferricytochrome c (14,000 Da). The molecular mass of the purified xanthine dehydrogenase was estimated from a best-fit line obtained with the Rf values of the molecular mass standards above. Enzyme assays. Xanthine oxidoreductase activity (type D + type 0) was monitored in homogenates, supernates, and the chromatography fractions at various steps along the purification procedure by following the amount of uric acid formed at 293 nm, as described by Stirpe and Della Corte (2). All enzyme assays were performed in a Beckman DU-7HS spectrophotometer at 25°C. One unit of enzyme activity represents 1 nmol uric acid produced/min. The assay mixture contained 0.1 M potassium phosphate (pH 7.8), 0.2 mM xanthine, 0.4 mM NAD+, and 20-100 ~1 of the enzyme sample in a final volume of 2.5 ml. Xanthine oxidase activity (type 0 enzyme) was determined in an identical manner, although in the absence of NAD+. Aldehyde oxidase (EC 1.2.3.1) activity was assayed according to the procedure of Rajagopalan and Handler (11). The assay mixture contained 0.05 M KPOI (pH 7.8), 0.005% EDTA, 6 mru N-methylnicotinamide chloride, and enzyme in a final volume of 2.5 ml. After mixing, the reaction was followed at 300 nm in a Beckman DU-7HS spectrophotometer. One unit of enzyme activity represents an absorbancy change of l/cm/min at 25°C. Protein was determined by the method of Lowry et al. (19), with bovine serum albumin as the standard.
Fraction
XANTHINE
DEHYDROGENASE
221
RESULTS
Pur(ficatim
Procedure
Figure 1 shows a typical elution profile obtained when the second 20 g/100 ml ammonium sulfate precipitate is fractionated on Sephadex G-200 (first passage). Both the type D and the type 0 enzyme activity patterns (0 - 0 and 0 - 0, respectively) are shown, along with the total protein pattern (0 - - - 0). It should be noted that the two enzyme activity profiles are not congruent. The type 0 activity peak lags slightly behind the type D activity peak, suggesting that the type 0 activity exhibits a slightly smaller molecular mass. This finding is consistent with the observation that this type 0 contaminating activity cannot be converted to the type D activity with 2mercaptoethanol or dithioerythritol. Thus, this type 0 enzyme activity is most likely associated with the proteolytically digested form of xanthine oxidoreductase. Interestingly, the addition of five times the concentration of trypsin inhibitor and/ or PMSF to buffer A had no effect on the amount of the type 0 contaminating en-
Number
FIG. 1. Total protein and xanthine oxidoreductase activity profiles from a Sephadex G-200 column. This figure represents data obtained during the first passage of the second ammonium sulfate precipitate through this column. Three-milliliter fractions were collected in buffer B. Enzyme activity for type D and type 0 xanthine oxidoreductase was assayed as stated in the text. l - - - 0, Total protein; 0 - 0, xanthine dehydrogenase activity; 0 - 0, xanthine oxidase activity.
222
SULEIMAN I
AND
I
Fraction
Number
FIG. 2. Total protein and xanthine oxidoreductase activity profiles from the Blue Dextran-Sepharose column. Three-milliliter fractions were collected in buffer B. Enzyme activity for type D and type 0 xanthine oxidoreductase was assayed as stated in the text. 0 - - - 0, Total protein; 0 - 0, xanthine dehydrogenase activity; 0 - 0, xanthine oxidase activity.
zyme activity present. Removal of these protease inhibitors from buffer A, however, yielded preparations that contained a much greater amount of oxidase activity. This additional increase in oxidase activity in the absence of the protease inhibitors also was not convertible to the dehydrogenase activity by 2-mercaptoethanol. Figure 2 shows the elution profile of enzyme activity from the Blue Dextran-
STEVENS
Sepharose column. Again, the activity profiles of the type D enzyme and type 0 enzyme are not congruent. It also should be noted in this figure that the type D:type 0 ratio (D/O) of enzyme activity remains high, and correlates well with the data observed in Figure 1. The maximum D/O found in the peak fraction of xanthine dehydrogenase in this figure was 8.0, although the pooled sample applied to the polyacrylamide disc gels exhibited a D/O of 5.5 (see Table I). If the peak dehydrogenase fractions from these columns were collected exclusively, the final enzyme preparation would exhibit a D/O greater than 10. When optimal D/O of the final enzyme preparation was sought, however, much less enzyme was recoverable (6-10% of the starting activity vs. 21% of the starting activity as reported here). Table I describes a complete set of results that is obtained typically with this purification scheme. As can be seen, the final enzyme preparation has a specific activity of 2470 units/mg protein, indicating that an approximately lOOO-fold purification had taken place. Also noted is the fairly high yield of pure enzyme, 21% of the starting activity. The D/O ratio of pooled enzyme of each step is also provided in this table. These latter data indicate that the buffers used throughout this procedure were able to maintain the enzyme in the type D form.
TABLE
I
PURIFICATION OF RAT LIVER XANTHINE OXIDOREDUCTASEa
Purification
step
15,000~ supernatant Ammonium sulfate Sephadex G-200 Blue Dextran-Sepharose Gel elution
Volume (ml) 135 35 24 12 1.6
Total dehydrogenase activity (units&) 8970 5480 4810 3604 1853
Total protein bg) 3654 218 23 4 0.75
Specific activity (units/mg) 2.5 25.1 209 901 2470
Recovery (%I 100 61 54 40 21
D/O’ 6.1 5.8 5.6 5.5 5.5
a Rat liver was perfused initially and then washed and homogenized as described in the text. The data in this table depict a representative experiment of the purification scheme. Liver tissue from a single animal was used. b One unit of enzyme activity is expressed as 1 nmol uric acid produced per minute. ‘Type D:type 0 enzyme activity ratio.
PURIFICATION
OF RAT
LIVER
XANTHINE
DEHYDROGENASE
223
The final enzyme preparation was found to be devoid of detectable aldehyde oxidase activity (data not shown). This latter enzyme exhibits purification characteristics very similar to those of xanthine oxidoreductase and has been reported by other investigators to be present in purified xanthine oxidoreductase preparations.
Enzyme Characteristics The absorption spectra of purified xanthine dehydrogenase are shown in Fig. 3. Both the oxidized and the reduced enzyme spectra are shown. Each spectrum closely resembles that of milk xanthine oxidase, as reported by Massey et al. (20), Nelson and Handler (21), and Kielley (22). A flavin peak at 460 nm is clearly evident in these spectra. The spectra also exhibit absorption in the region, 500-650 nm, characteristic of many iron-containing flavoproteins. Shown in Fig. 4 are the nondenaturing disc gels of purified xanthine dehydrogenase. The gel on the right was stained for protein; the gel on the left for enzyme activity. As can be seen, one major band was present when the gels were stained either for enzyme activity or for protein. No other bands were detected on either gel. In addition, the activity band corresponded identically to the protein band. When compared against molecular mass standards
FIG. 4. Polyacrylamide electrophoresis gels of purified xanthine oxidoreductase (type D). (A) Purified xanthine dehydrogenase stained for enzyme activity; (B) purified xanthine dehydrogenase stained for protein. Staining procedures are described in the text.
run simultaneously on polyacrylamide gels, a molecular mass of approximately 300,000 Da was estimated for this protein (data not shown). DISCUSSION
Wavelength
(nm)
FIG. 3. Absorption spectra of purified xanthine oxidoreductase (type D). The enzyme solution contained 0.25 mg/ml. The solid line represents the oxidized enzyme, while the dotted line represents enzyme reduced with xanthine (1.0 mM).
The enzyme xanthine oxidoreductase is believed to exist predominantly, if not exclusively, in the type D form (as xanthine dehydrogenase) in mammalian tissues. However, it gradually changes to an oxidase during most purification procedures or on standing after purification. This phenomenon has been reported by many investigators (2, 3, 8, 9, 23, 24). In our procedure, it was discovered that the addition of protease inhibitor PMSF, trypsin inhibitor, and 2-mercaptoethanol to the homogenization buffer (buffer A) and of PMSF and 2-mercaptoethanol to buffer B were able to minimize the conversion of xanthine dehydrogenase to xanthine oxidase during the purification procedure. Battelli et al (5) have reported that the addition of soybean trypsin inhibitor to the 100,OOOgsuperna-
224
SULEIMAN
tant of various rat tissues was also effective in maintaining xanthine dehydrogenase activity throughout their purification procedure. Ikegami and Nishino recently reported essentially no conversion of the Wistar rat liver enzyme to the proteolytitally digested oxidase form, even though protease inhibitors were absent from their buffers (25). An explanation for this result in view of our findings, as well as those reported by Battelli et cd. (5), is not apparent. In the present study, the rat liver xanthine dehydrogenase was purified at a considerably higher D/O ratio (typically between 5 and 6) for the pure enzyme than the highest ratio (3.6) reported by other investigators (9). The failure of early investigators to obtain such a high D/O ratio in their purification procedures may have been the result of their use of an acetone or heat precipitation step, since either of these steps might have caused partial loss of the dehydrogenase activity (9,13,20,26). Our procedure specifically avoids these steps and, as shown in Table I, is able to maintain a D/O similar to that observed in fresh tissue homogenates. This D/O, however, is a pooled fraction value and not an optimal value. As can be seen in Figs. 1 and 2, a considerably higher D/O value, in the range of 8-12, could be obtained if only the peak xanthine dehydrogenase fractions from the chromatography columns are saved. This practice, however, severely limits the yield of enzyme by this procedure. Only 6-10% of the starting activity was recoverable when maximal D/O was sought. A small amount of type 0 enzyme (the proteolytically digested form of xanthine oxidoreductase) was always observed in the starting tissue homogenates in this investigation. The level of this enzyme form could not be diminished any further than that reported in this article by the addition of up to five times the amount of proteolytic inhibitors to buffer A. Therefore, it is our conclusion that rat liver tissue may actually contain some type 0 enzyme activity in vivo. Other investigators have also suggested that type 0 xanthine oxidoreductase
AND STEVENS
may be present in mammalian tissues, especially in the small intestine (5). The present purification procedure yields a relatively high percentage of the starting xanthine dehydrogenase activity, 21% .This yield is much better than that reported in previous procedures [2.7% (8,9)-10% (25)]. This high yield again may be due to the fact that fewer steps are required in our purification procedure, as well as the fact that only mild purification steps were utilized. The reported specific activity (2470 units/mg protein) obtained with this procedure also compares favorably to the best preparations recorded in the literature. Irie (13) has reported a specific activity of 2500 units/mg protein from chicken liver, and Krenitsky et al. (27) have reported a specific activity of 3000 units/mg protein from human liver. These findings represent the highest specific activities reported to date. By comparison, Murison (28) obtained enzyme exhibiting a specific activity of 1710 units/mg protein from chicken liver. Ikegami and Niskino (25) reported purifying xanthine oxidoreductase from Wistar rat liver, but their final preparation contained enzyme with a relatively low specific activity, 1390 units/mg protein. This low specific activity corresponds with their finding of a significant amount of desulfoxanthine oxidoreductase in their purified sample. We did not find this inactive form of xanthine dehydrogenase in our preparation. Therefore, perhaps the desulfonated enzyme exists naturally in the Wistar rat liver (Ikegami and Niskino preparation) and not in Sprague-Dawley rat liver (our preparation), or perhaps the desulfonated enzyme was produced from the native enzyme during their purification procedure. No other enzyme purification procedure, however, has mentioned this latter possibility. In conclusion, a mild and rapid procedure for the purification of rat liver xanthine dehydrogenase on a small scale has been developed. The enzyme was purified approximately lOOO-fold and exhibits a high state of purity, as evidenced spectrophotometrically by its high specific activity and by disc gel electrophoresis.
PURIFICATION
OF RAT
LIVER
REFERENCES 1. COUGHLAN, M. P. (1980) in Molybdenum and Molybdenum-Containing Enzymes (Coughlan, M. P., Ed.), pp. 119-185, Pergamon, Oxford. 2. STIRPE, F., AND DELLA CORTE, E. (1969) J. Bill Chem. 244,3855-3863. 3. DELLA CORTE, E., GOZZETTI, G., NOVELLO, F., AND STIRPE, F. (1969) Biochim Biquhvs. Acta 191, 164166. 4. JOYCE, P., AND DUKE, E. J. (1971) Biochem. J. 125, 111P. 5. BATTELLI, M. G., DELLA CORTE, E., AND STIRPE, F. (1972) B&hem. J. 126, ‘747-749. 6. SCHOUTSEN, B., DEJONG, J. W., HARMSEN, E., DE TOMLO, P., AND ACHTERBERG, P. W. (1983) Biochim Biophys. Acta 762,519-524. 7. ROWE, P. B., AND WYNGAARDEN, J. B. (1966) J. Biol. Chem 241,5571-5576. 8. DELLA CORTE, E., AND STIRPE, F. (1972) Biochem J. 126,739-745. 9. WAUD, W. R., AND RAJAGOPALAN, K. V. (1976) Arch Biochem Biophys. 172,354-364. 10. BA~ELLI, M. G., LORENZONI, E., AND STIRPE, F. (1973) BiocAem J. 131,191-198. 11. RAJAGOPALAN, K. V., AND HANDLER, P. (1967) J. Biol. Chem 242,4097-4107. 12. VALENTINE, R. C., JACKSON, R. L., AND WOLFE, R. S. (1962) Biochewz. Biophys. Res. Commun 7,453-456. 13. IRIE, S. (1984) J. B&hem. 95,405-412.
XANTHINE
DEHYDROGENASE
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14. LAEMMLI, U. K. (1970) Nature @ondon) 227,680685. 15. WEBER, K., AND KUTER, D. J. (1971) J. Biol. Chem. 246,4504-4509. 16. JOVIN, T., CHRAMBACH, A., AND NAUGHTON, M. A. (1964) And Biochem 9,350-369. 17. LYERLA, T. A., AND FOURNIER, P. C. (1983) Comp. Biochem Physiol. 76B, 497-502. 18. SMYTH, M. J., AND DUKE, E. J. (1976) in Flavins and Flavoproteins (Singer, T. P., Ed.), pp. 572575, Elsevier, Amsterdam. 19. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 20. MASSEY, V., BRUMBY, P. E., KOMAI, H., AND PALMER, G. (1969) J. BioL Chew. 244,1682-1691. 21. NELSON, C. A., AND HANDLER, P. (1968) J. Biol. Chem 243,5360-5373. 22. KIELLEY, R. K. (1955) J. BioZ. Chem. 216,405-412. 23. WAUD, W. R., AND RAJAGOPALAN, K. V. (1976) Arch. Biochem. Biophys. 172,35&379. 24. BRAY, R. C. (1975) in The Enzymes (Boyer, B. D., Ed.), Vol. 12, pp. 300-419, Academic Press, New York. 25. IKEGAMI, T., AND NISHINO, T. (1986) Arch. Biochem, Biophys. 247,254-260. 26. CLEERE, W. F., AND CAUGHLAN, M. P. (1975) Cmp. B&hem. Physiol. 50B, 311-322. 27. KRENITSKY, T. A., SPECTOR, T., AND HALL, W. W. (1986) Arch. Biochem Biophys. 247,108-119. 28. MURISON, G. (1969) Dev. BioL 20,518-543.
ARCHIVES
Purification
of Xanthine Dehydrogenase from Rat Liver: A Rapid Procedure with High Enzyme Yields’
SULEIMAN Division
A. SULEIMAN2
AND
JEFFREY
B. STEVENS3
of Entironmental Health, School of Public Health, The University of Minnesota, Minneapolis, Minnesota 55&% Received March 9. 1987, and in revised form June 5,1987
Xanthine dehydrogenase (EC 121.3’7) was purified approximately lOOO-fold from liver homogenates of adult male Sprague-Dawley rats. Enzyme recovery was good (~20% of the starting activity was obtained), and the homogeneously pure enzyme had a molecular mass of approximately 300,000 Da. The purified protein exhibited a specific activity of 2470 units/mg protein and spectral properties identical to those of the best preparations of this enzyme reported by other investigators. Routine preparations of this enzyme also possess higher dehydrogenase:oxidase ratios (typically between 5 and 6) than do other xanthine dehydrogenase preparations so far reported in the literature. Maximum dehydrogenase:oxidase ratios, greater than 10, could be obtained from this procedure if only peak dehydrogenase fractions from the chromatography columns were saved. The present small-scale purification method, which can be completed in 48-60 h, utilizes ammonium sulfate fractionation, Sephadex G-200 column chromatography, Blue Dextran-Sepharose column chromatography, and preparative gel electrophoresis. o 1987 Academic
Prear,
Inc.
Xanthine dehydrogenase (XDH4; EC 1.1.1.204, formerly 1.2.1.37) is one of a group of molybdenum, iron-sulfur flavin hydroxylases that act on a wide variety of purine, pteridine, and pyrimidine substrates (1). In mammalian tissues this enzyme is thought to catalyze the oxidation of hypoxanthine and xanthine to uric acid, with a concomitant reduction of NAD+ (2-6). In vitro, i.e., in crude tissue preparations, XDH is readily converted to its oxidase form, xanr This work was supported by BRSG No. 2-SO7RR 05448, awarded to the University of Minnesota School of Public Health by the Biomedical Research Grant Program, Division of Research and Resources, National Institutes of Health, Bethesda, MD. r Present address: Department of Biological Sciences, Yarmouk University, Irbid, Jordan. 3 To whom correspondence should be addressed. ’ Abbreviations used: XDH, xanthine dehydrogenase; X0, xanthine oxidase; NBT, nitroblue tetrazolium; PMS, phenazine methosulfate; PMSF, phenylmethylsulfonyl fluoride.
thine oxidase (X0; EC 1.1.3.22, formerly 1.2.3.2) by oxidation in air at 37°C or by proteolysis with trypsin (2). When this enzyme was isolated initially from rat liver, it was described as xanthine oxidase (7). The air-oxidized enzyme, but not the proteolytically digested enzyme, can be reconverted to xanthine dehydrogenase upon exposure to thiols such as dithioerythritol (6, 8-10). Under the current IUPAC nomenclature xanthine oxidase is called the type 0 form of xanthine oxidoreductase, whereas xanthine dehydrogenase is designated the type D form. Both types have been purified from various mammalian and nonmammalian tissue sources (9,11,12,13). Most of these purification procedures, however, are either laborious or require harsh procedures, e.g., acetone or heat precipitation. Thus, relatively low yields of enzyme are usually obtained. In the present study we report a simple, quick, and gentle procedure for the 219
0003-9861/87 $3.00 Copyright All rights
0 1987 by Academic Press. Inc. of reproduction in any foorm reserved.
220
SULEIMAN
small-scale purification of xanthine dehydrogenase from rat liver. This procedure offers a high yield of enzyme (>20% of the starting activity) with a relatively high type D/type 0 ratio (typically in the range 5-6). MATERIALS
AND
METHODS
Adult male Sprague-Dawley rats (ZOO-250 g) were purchased from Biolab (Madison, Wisconsin) and were used as liver donors. Bovine serum albumin, horse ferricytochrome c, lactate dehydrogenase, 2-mercaptoethanol, milk xanthine oxidase, NAD+, nitroblue tetrazolium (NBT), phenazine methosulfate (PMS), phenylmethylsulfonyl fluoride (PMSF), ethylenediaminetetraacetic acid, disodium salt (EDTA), reactive blue 2-Sepharose (Blue Dextran-Sepharose), trypsin inhibitor, and xanthine were all obtained from the Sigma Chemical Co. (St. Louis, MO). Acrylamide, bisacrylamide, ammonium persulfate, and N,N,N’,N’tetramethylethylenediamine were purchased from Bio-Rad Laboratories (Richmond, CA). Other chemicals were of reagent quality. Purification proceduv-e. Each animal was initially anesthetized by an ip injection of pentobarbital (60 mg/kg) before the abdomen was opened. The liver was then perfused with lo-15 ml of cold (0-4°C) 0.25 M sucrose solution. The liver was removed from the animal, washed with the 0.25 M sucrose solution, weighed, and then homogenized with a Potter-Elvehjem apparatus in 10 vol of 0.1 M phosphate buffer, pH 7.8, containing 100 mM 2-mercaptoethanol, 3 mM PMSF, 1 mM EDTA, and 80 mg/liter trypsin inhibitor (buffer A). The homogenate was centrifuged at 15,000g for 20 min in a Sorvall RC-5B refrigerated centrifuge. All of the subsequent operations were performed at 0-4°C. Solid ammonium sulfate was added with stirring to the clear, reddish supernatant to obtain a final concentration of 20 g/100 ml. After 30 min of gentle stirring, the suspension was fractionated by centrifugation at 15,OOOgfor 20 min. The pellet was discarded, and an additional 20 g/100 ml ammonium sulfate was added to the supernatant. After a second centrifugation at 15,OOOgfor 20 min the supernatant was decanted, and the pellet was dissolved in 25-30 ml of buffer A. The enzyme was then dialyzed overnight against several volumes of 0.1 M phosphate (pH 7.8,l mM EDTA, 20 mM 2-mercaptoethanol, and 1 mM PMSF (buffer B). The dialyzed enzyme solution was concentrated in an Amicon ultrafiltration cell fitted with a Diaflo PM10 ultrafiltration membrane (Amicon Corporation, Lexington, MA) and then applied to a Sephadex G-200 column (2.5 X 45 cm) pre-equilibrated with buffer B. The enzyme was eluted from the column in the same buffer. Three-milliliter fractions were collected. Fractions containing xanthine dehydrogenase
AND
STEVENS
activity were pooled, concentrated and then reapplied to the G-200 column. After this second elution, the fractions containing active enzyme were again pooled and concentrated. The enzyme solution was then applied to a 1.5 X 14-cm Blue Dextran-Sepharose column (reactive blue 2-Sepharose) and eluted with buffer B. Three-milliliter fractions were collected. The final step in the purification procedure was polyacrylamide gel elution. The fractions from the Blue Dextran-Sepharose column that contained active enzyme were pooled and concentrated in an Amicon filtration cell to a volume of 1 ml. The enzyme preparation was then placed on 7% native polyacrylamide gel discs and electrophoresed for 6 h at 250 V (see below for details). After electrophoresis one of the polyacrylamide gels was stained for xanthine dehydrogenase activity to locate the enzyme. The corresponding sections on the other nine gels were excised and saved. The gel slices were diced with scissors to approximately 1 mm’ and placed in a tube (0.5 X 12 cm) plugged with 1.5 cm of stacking polyacrylamide gel prepared according to the procedure of Laemmli (14). The tube was then filled to near capacity with stacking gel, according to a modification of the procedure by Weber and Kuter (15), with extraordinary care to avoid air bubbles. A dialysis bag was fitted to the bottom end. The tube was filled with 0.1 M phosphate buffer, pH 7.8, and placed back into the electrophoresis unit. The gel containing the enzyme was again electrophoresed at 250 V for 6 h, or until the enzyme was out of the gel and into the dialysis bag. The dialysis bag containing the enzyme (yellow color) was then removed from the tube, and the enzyme was dialyzed for several hours against buffer B. Gel electrophoresis. For the last step in the purification procedure and for determining the purity of the final enzyme preparation, polyacrylamide discontinuous (disc) gel electrophoresis was performed on 7% native gel (under nondenaturing conditions) polymerized with ammonium persulfate according to Laemmli (14). Twenty to fifty microliters of a 1:l mixture of sample buffer (10% glycerol and 0.5 mM bromphenol blue in 100 mM Tris buffer, pH 6.8) and enzyme solution (2.5 mg/ml protein) was added to each tube. The reservoir buffer of the electrophoresis system consisted of 0.05 M Tris and 0.38 M glycine, pH 8.9, according to Jovin et al (16). Electrophoresis was performed at 250 V. For determining enzyme purity the gels were stained after electrophoresis for protein with amido black or for enzyme activity by immersion for 15 min in the following staining mixture (17): 0.1 M phosphate buffer (pH 7.8), 0.05 M xanthine, 0.37 my NBT, 0.75 mM NAD+, and 0.13 mM PMS. Xanthine oxidase (type 0) activity was detected by means of the above staining solution without NAD+ (17,18). Standards for the molecular mass determination included milk xanthine oxidase (-300,000 Da), lactate dehydrogenase (~100,000 Da), bovine serum albumin
PURIFICATION
OF RAT
LIVER
(60,000 Da) and horse ferricytochrome c (14,000 Da). The molecular mass of the purified xanthine dehydrogenase was estimated from a best-fit line obtained with the Rf values of the molecular mass standards above. Enzyme assays. Xanthine oxidoreductase activity (type D + type 0) was monitored in homogenates, supernates, and the chromatography fractions at various steps along the purification procedure by following the amount of uric acid formed at 293 nm, as described by Stirpe and Della Corte (2). All enzyme assays were performed in a Beckman DU-7HS spectrophotometer at 25°C. One unit of enzyme activity represents 1 nmol uric acid produced/min. The assay mixture contained 0.1 M potassium phosphate (pH 7.8), 0.2 mM xanthine, 0.4 mM NAD+, and 20-100 ~1 of the enzyme sample in a final volume of 2.5 ml. Xanthine oxidase activity (type 0 enzyme) was determined in an identical manner, although in the absence of NAD+. Aldehyde oxidase (EC 1.2.3.1) activity was assayed according to the procedure of Rajagopalan and Handler (11). The assay mixture contained 0.05 M KPOI (pH 7.8), 0.005% EDTA, 6 mru N-methylnicotinamide chloride, and enzyme in a final volume of 2.5 ml. After mixing, the reaction was followed at 300 nm in a Beckman DU-7HS spectrophotometer. One unit of enzyme activity represents an absorbancy change of l/cm/min at 25°C. Protein was determined by the method of Lowry et al. (19), with bovine serum albumin as the standard.
Fraction
XANTHINE
DEHYDROGENASE
221
RESULTS
Pur(ficatim
Procedure
Figure 1 shows a typical elution profile obtained when the second 20 g/100 ml ammonium sulfate precipitate is fractionated on Sephadex G-200 (first passage). Both the type D and the type 0 enzyme activity patterns (0 - 0 and 0 - 0, respectively) are shown, along with the total protein pattern (0 - - - 0). It should be noted that the two enzyme activity profiles are not congruent. The type 0 activity peak lags slightly behind the type D activity peak, suggesting that the type 0 activity exhibits a slightly smaller molecular mass. This finding is consistent with the observation that this type 0 contaminating activity cannot be converted to the type D activity with 2mercaptoethanol or dithioerythritol. Thus, this type 0 enzyme activity is most likely associated with the proteolytically digested form of xanthine oxidoreductase. Interestingly, the addition of five times the concentration of trypsin inhibitor and/ or PMSF to buffer A had no effect on the amount of the type 0 contaminating en-
Number
FIG. 1. Total protein and xanthine oxidoreductase activity profiles from a Sephadex G-200 column. This figure represents data obtained during the first passage of the second ammonium sulfate precipitate through this column. Three-milliliter fractions were collected in buffer B. Enzyme activity for type D and type 0 xanthine oxidoreductase was assayed as stated in the text. l - - - 0, Total protein; 0 - 0, xanthine dehydrogenase activity; 0 - 0, xanthine oxidase activity.
222
SULEIMAN I
AND
I
Fraction
Number
FIG. 2. Total protein and xanthine oxidoreductase activity profiles from the Blue Dextran-Sepharose column. Three-milliliter fractions were collected in buffer B. Enzyme activity for type D and type 0 xanthine oxidoreductase was assayed as stated in the text. 0 - - - 0, Total protein; 0 - 0, xanthine dehydrogenase activity; 0 - 0, xanthine oxidase activity.
zyme activity present. Removal of these protease inhibitors from buffer A, however, yielded preparations that contained a much greater amount of oxidase activity. This additional increase in oxidase activity in the absence of the protease inhibitors also was not convertible to the dehydrogenase activity by 2-mercaptoethanol. Figure 2 shows the elution profile of enzyme activity from the Blue Dextran-
STEVENS
Sepharose column. Again, the activity profiles of the type D enzyme and type 0 enzyme are not congruent. It also should be noted in this figure that the type D:type 0 ratio (D/O) of enzyme activity remains high, and correlates well with the data observed in Figure 1. The maximum D/O found in the peak fraction of xanthine dehydrogenase in this figure was 8.0, although the pooled sample applied to the polyacrylamide disc gels exhibited a D/O of 5.5 (see Table I). If the peak dehydrogenase fractions from these columns were collected exclusively, the final enzyme preparation would exhibit a D/O greater than 10. When optimal D/O of the final enzyme preparation was sought, however, much less enzyme was recoverable (6-10% of the starting activity vs. 21% of the starting activity as reported here). Table I describes a complete set of results that is obtained typically with this purification scheme. As can be seen, the final enzyme preparation has a specific activity of 2470 units/mg protein, indicating that an approximately lOOO-fold purification had taken place. Also noted is the fairly high yield of pure enzyme, 21% of the starting activity. The D/O ratio of pooled enzyme of each step is also provided in this table. These latter data indicate that the buffers used throughout this procedure were able to maintain the enzyme in the type D form.
TABLE
I
PURIFICATION OF RAT LIVER XANTHINE OXIDOREDUCTASEa
Purification
step
15,000~ supernatant Ammonium sulfate Sephadex G-200 Blue Dextran-Sepharose Gel elution
Volume (ml) 135 35 24 12 1.6
Total dehydrogenase activity (units&) 8970 5480 4810 3604 1853
Total protein bg) 3654 218 23 4 0.75
Specific activity (units/mg) 2.5 25.1 209 901 2470
Recovery (%I 100 61 54 40 21
D/O’ 6.1 5.8 5.6 5.5 5.5
a Rat liver was perfused initially and then washed and homogenized as described in the text. The data in this table depict a representative experiment of the purification scheme. Liver tissue from a single animal was used. b One unit of enzyme activity is expressed as 1 nmol uric acid produced per minute. ‘Type D:type 0 enzyme activity ratio.
PURIFICATION
OF RAT
LIVER
XANTHINE
DEHYDROGENASE
223
The final enzyme preparation was found to be devoid of detectable aldehyde oxidase activity (data not shown). This latter enzyme exhibits purification characteristics very similar to those of xanthine oxidoreductase and has been reported by other investigators to be present in purified xanthine oxidoreductase preparations.
Enzyme Characteristics The absorption spectra of purified xanthine dehydrogenase are shown in Fig. 3. Both the oxidized and the reduced enzyme spectra are shown. Each spectrum closely resembles that of milk xanthine oxidase, as reported by Massey et al. (20), Nelson and Handler (21), and Kielley (22). A flavin peak at 460 nm is clearly evident in these spectra. The spectra also exhibit absorption in the region, 500-650 nm, characteristic of many iron-containing flavoproteins. Shown in Fig. 4 are the nondenaturing disc gels of purified xanthine dehydrogenase. The gel on the right was stained for protein; the gel on the left for enzyme activity. As can be seen, one major band was present when the gels were stained either for enzyme activity or for protein. No other bands were detected on either gel. In addition, the activity band corresponded identically to the protein band. When compared against molecular mass standards
FIG. 4. Polyacrylamide electrophoresis gels of purified xanthine oxidoreductase (type D). (A) Purified xanthine dehydrogenase stained for enzyme activity; (B) purified xanthine dehydrogenase stained for protein. Staining procedures are described in the text.
run simultaneously on polyacrylamide gels, a molecular mass of approximately 300,000 Da was estimated for this protein (data not shown). DISCUSSION
Wavelength
(nm)
FIG. 3. Absorption spectra of purified xanthine oxidoreductase (type D). The enzyme solution contained 0.25 mg/ml. The solid line represents the oxidized enzyme, while the dotted line represents enzyme reduced with xanthine (1.0 mM).
The enzyme xanthine oxidoreductase is believed to exist predominantly, if not exclusively, in the type D form (as xanthine dehydrogenase) in mammalian tissues. However, it gradually changes to an oxidase during most purification procedures or on standing after purification. This phenomenon has been reported by many investigators (2, 3, 8, 9, 23, 24). In our procedure, it was discovered that the addition of protease inhibitor PMSF, trypsin inhibitor, and 2-mercaptoethanol to the homogenization buffer (buffer A) and of PMSF and 2-mercaptoethanol to buffer B were able to minimize the conversion of xanthine dehydrogenase to xanthine oxidase during the purification procedure. Battelli et al (5) have reported that the addition of soybean trypsin inhibitor to the 100,OOOgsuperna-
224
SULEIMAN
tant of various rat tissues was also effective in maintaining xanthine dehydrogenase activity throughout their purification procedure. Ikegami and Nishino recently reported essentially no conversion of the Wistar rat liver enzyme to the proteolytitally digested oxidase form, even though protease inhibitors were absent from their buffers (25). An explanation for this result in view of our findings, as well as those reported by Battelli et cd. (5), is not apparent. In the present study, the rat liver xanthine dehydrogenase was purified at a considerably higher D/O ratio (typically between 5 and 6) for the pure enzyme than the highest ratio (3.6) reported by other investigators (9). The failure of early investigators to obtain such a high D/O ratio in their purification procedures may have been the result of their use of an acetone or heat precipitation step, since either of these steps might have caused partial loss of the dehydrogenase activity (9,13,20,26). Our procedure specifically avoids these steps and, as shown in Table I, is able to maintain a D/O similar to that observed in fresh tissue homogenates. This D/O, however, is a pooled fraction value and not an optimal value. As can be seen in Figs. 1 and 2, a considerably higher D/O value, in the range of 8-12, could be obtained if only the peak xanthine dehydrogenase fractions from the chromatography columns are saved. This practice, however, severely limits the yield of enzyme by this procedure. Only 6-10% of the starting activity was recoverable when maximal D/O was sought. A small amount of type 0 enzyme (the proteolytically digested form of xanthine oxidoreductase) was always observed in the starting tissue homogenates in this investigation. The level of this enzyme form could not be diminished any further than that reported in this article by the addition of up to five times the amount of proteolytic inhibitors to buffer A. Therefore, it is our conclusion that rat liver tissue may actually contain some type 0 enzyme activity in vivo. Other investigators have also suggested that type 0 xanthine oxidoreductase
AND STEVENS
may be present in mammalian tissues, especially in the small intestine (5). The present purification procedure yields a relatively high percentage of the starting xanthine dehydrogenase activity, 21% .This yield is much better than that reported in previous procedures [2.7% (8,9)-10% (25)]. This high yield again may be due to the fact that fewer steps are required in our purification procedure, as well as the fact that only mild purification steps were utilized. The reported specific activity (2470 units/mg protein) obtained with this procedure also compares favorably to the best preparations recorded in the literature. Irie (13) has reported a specific activity of 2500 units/mg protein from chicken liver, and Krenitsky et al. (27) have reported a specific activity of 3000 units/mg protein from human liver. These findings represent the highest specific activities reported to date. By comparison, Murison (28) obtained enzyme exhibiting a specific activity of 1710 units/mg protein from chicken liver. Ikegami and Niskino (25) reported purifying xanthine oxidoreductase from Wistar rat liver, but their final preparation contained enzyme with a relatively low specific activity, 1390 units/mg protein. This low specific activity corresponds with their finding of a significant amount of desulfoxanthine oxidoreductase in their purified sample. We did not find this inactive form of xanthine dehydrogenase in our preparation. Therefore, perhaps the desulfonated enzyme exists naturally in the Wistar rat liver (Ikegami and Niskino preparation) and not in Sprague-Dawley rat liver (our preparation), or perhaps the desulfonated enzyme was produced from the native enzyme during their purification procedure. No other enzyme purification procedure, however, has mentioned this latter possibility. In conclusion, a mild and rapid procedure for the purification of rat liver xanthine dehydrogenase on a small scale has been developed. The enzyme was purified approximately lOOO-fold and exhibits a high state of purity, as evidenced spectrophotometrically by its high specific activity and by disc gel electrophoresis.
PURIFICATION
OF RAT
LIVER
REFERENCES 1. COUGHLAN, M. P. (1980) in Molybdenum and Molybdenum-Containing Enzymes (Coughlan, M. P., Ed.), pp. 119-185, Pergamon, Oxford. 2. STIRPE, F., AND DELLA CORTE, E. (1969) J. Bill Chem. 244,3855-3863. 3. DELLA CORTE, E., GOZZETTI, G., NOVELLO, F., AND STIRPE, F. (1969) Biochim Biquhvs. Acta 191, 164166. 4. JOYCE, P., AND DUKE, E. J. (1971) Biochem. J. 125, 111P. 5. BATTELLI, M. G., DELLA CORTE, E., AND STIRPE, F. (1972) B&hem. J. 126, ‘747-749. 6. SCHOUTSEN, B., DEJONG, J. W., HARMSEN, E., DE TOMLO, P., AND ACHTERBERG, P. W. (1983) Biochim Biophys. Acta 762,519-524. 7. ROWE, P. B., AND WYNGAARDEN, J. B. (1966) J. Biol. Chem 241,5571-5576. 8. DELLA CORTE, E., AND STIRPE, F. (1972) Biochem J. 126,739-745. 9. WAUD, W. R., AND RAJAGOPALAN, K. V. (1976) Arch Biochem Biophys. 172,354-364. 10. BA~ELLI, M. G., LORENZONI, E., AND STIRPE, F. (1973) BiocAem J. 131,191-198. 11. RAJAGOPALAN, K. V., AND HANDLER, P. (1967) J. Biol. Chem 242,4097-4107. 12. VALENTINE, R. C., JACKSON, R. L., AND WOLFE, R. S. (1962) Biochewz. Biophys. Res. Commun 7,453-456. 13. IRIE, S. (1984) J. B&hem. 95,405-412.
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14. LAEMMLI, U. K. (1970) Nature @ondon) 227,680685. 15. WEBER, K., AND KUTER, D. J. (1971) J. Biol. Chem. 246,4504-4509. 16. JOVIN, T., CHRAMBACH, A., AND NAUGHTON, M. A. (1964) And Biochem 9,350-369. 17. LYERLA, T. A., AND FOURNIER, P. C. (1983) Comp. Biochem Physiol. 76B, 497-502. 18. SMYTH, M. J., AND DUKE, E. J. (1976) in Flavins and Flavoproteins (Singer, T. P., Ed.), pp. 572575, Elsevier, Amsterdam. 19. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 20. MASSEY, V., BRUMBY, P. E., KOMAI, H., AND PALMER, G. (1969) J. BioL Chew. 244,1682-1691. 21. NELSON, C. A., AND HANDLER, P. (1968) J. Biol. Chem 243,5360-5373. 22. KIELLEY, R. K. (1955) J. BioZ. Chem. 216,405-412. 23. WAUD, W. R., AND RAJAGOPALAN, K. V. (1976) Arch. Biochem. Biophys. 172,35&379. 24. BRAY, R. C. (1975) in The Enzymes (Boyer, B. D., Ed.), Vol. 12, pp. 300-419, Academic Press, New York. 25. IKEGAMI, T., AND NISHINO, T. (1986) Arch. Biochem, Biophys. 247,254-260. 26. CLEERE, W. F., AND CAUGHLAN, M. P. (1975) Cmp. B&hem. Physiol. 50B, 311-322. 27. KRENITSKY, T. A., SPECTOR, T., AND HALL, W. W. (1986) Arch. Biochem Biophys. 247,108-119. 28. MURISON, G. (1969) Dev. BioL 20,518-543.