©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mechanism of Inhibition of Xanthine Oxidase with a New Tight Binding Inhibitor (*)

(Received for publication, February 14, 1994; and in revised form, December 16, 1994)

Ken Okamoto Takeshi Nishino (§)

From the Department of Biochemistry and Molecular Biology, Nippon Medical School, 1-1-5 Sendagi, Bunkyoku, Tokyo 113, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The mechanism of inhibition of milk xanthine oxidase and xanthine dehydrogenase by the tight binding inhibitor, sodium-8-(3-methoxy-4-phenylsulfinylphenyl)pyrazolo[1,5-a]-1,3,5-triazine-4-olate monohydrate (BOF4272), was studied after separation of the two isomers. The steady state kinetics showed that the inhibition by these compounds was a mixed type. One of the isomers had a K value of 1.2 times 10M and a K` value of 9 times 10M, while the other isomer had a Kvalue of 3 times 10M and a K` value of 9 times 10M. Spectral changes were not observed by mixing either the oxidized or reduced form of the enzyme with BOF-4272. The stopped-flow study and the effects of BOF-4272 on various substrates showed that BOF-4272 bound to the xanthine binding site of the enzyme. K values of the enzyme and one of the isomers, which has a higher affinity for the enzyme, were also found to be 2 times 10M for the active form of the enzyme and 7 times 10M for the desulfo-form using fluorometric titration, and the binding has stoichiometry of 1:1. The inhibitor could not bind to the enzyme when the enzyme was previously treated with oxipurinol.


INTRODUCTION

Xanthine dehydrogenase (EC 1.1.1.204) catalyzes the oxidation of hypoxanthine to xanthine and of xanthine to uric acid with concomitant reduction of NAD to NADH(1) . The enzyme is a dimer of identical subunits with a M(r) of about 150,000 containing four oxidation and reduction centers per subunit: one molybdopterin, two iron sulfur centers, and one FAD. Mammalian xanthine dehydrogenases exist as the NAD-dependent type in freshly prepared samples(2, 3) , i.e. they exhibit high xanthine-NAD reductase activity even in the presence of O(2)(2, 3, 4, 5) . However, during purification, most of the enzymes are converted to the O(2)-dependent xanthine oxidase (XO)(^1)(2, 3, 6, 7) , i.e. they exhibit low xanthine-NAD reductase activity but high xanthine-O(2) reductase activity even in the presence of NAD(2, 3, 4, 5) . Xanthine interacts with XO or xanthine dehydrogenase at the xanthine binding site, which contains molybdopterin, the structure of which has been proposed and recently reviewed(8, 9) . Two electrons are transferred from xanthine to Mo(VI), reducing the metal to Mo(IV) while another substrate, NAD or oxygen, reacts with FAD; the catalytic cycle thus necessarily entails intramolecular electron transfer.

Xanthine-oxidizing enzymes are inactivated by cyanide in a reaction that removes an essential sulfur atom from the molybdenum to give the nonfunctional desulfo-form of the enzyme(10) . Desulfoxanthine oxidase occurs naturally and is present in all standard preparations but can be removed by affinity chromatography(11, 12) . Evidence for the existence of the desulfo-form was provided by Massey et al. (13) with the study of the interaction of the enzyme and the inhibitor, allopurinol. The sulfur atom necessary for catalytic activity has been shown by x-ray absorption spectroscopy to be present as a Mo=S group(14, 15, 16, 17, 18) . It has been shown that Mo=O and Mo=S groups dominate the coordination sphere in oxidized enzyme, with the remainder of the ligand positions occupied by thiolate or thioether. On reduction, the Mo=S group becomes protonated to form Mo-SH and is considered to be the group that accepts the C-8 proton from xanthine in the course of its conversion to uric acid(18) . However, the interaction of the substrate with the molybdenum center and the chemical steps involved in its conversion to product are poorly understood(9) .

The interaction of xanthine oxidase with an inhibitor is of interest not only from the aspect of the mechanism of enzyme reaction, but also from the medical aspects. First, as the enzyme is involved in the final steps of uric acid production, an inhibitor of xanthine oxidase is useful for remedy of hyperuricemia or gout(19) . Second, as the O(2)-dependent type of the enzyme produces hydrogen peroxide and superoxide (O(2)), the conversion from the NAD-dependent type to the O(2)-dependent type is hypothetically proposed to be responsible for causing post-ischemic reperfusion injury(20) . Therefore, it is valuable to examine whether prior treatment of tissue with an inhibitor is effective in attenuating post-ischemic tissue injury. Allopurinol, a pyrazolopyrimidine derivative, is a potent inhibitor of xanthine oxidase and has been used for effective remedy for hyperuricemia or gout. It has been reported that treatment of tissue with allopurinol is effective in attenuating ischemic tissue injury(21, 22, 23) . The mechanism of inhibition of xanthine oxidase by allopurinol was studied in detail, indicating that oxipurinol, a product from allopurinol by xanthine oxidase, bound stoichiometrically to the reduced molybdenum (IV)(13) . Although the inhibitor binds very tightly to the enzyme, the inhibition is time dependent(13, 24) , and, therefore, it takes some time to inhibit the enzyme completely. Furthermore, it is necessary to maintain an effective concentration of the inhibitor to the organ because the inhibitor dissociates from the enzyme by spontaneous oxidation of molybdenum to Mo(VI), with a half-life of 300 min at 25 °C, with concomitant recovery of enzyme activity(13) . However, in the experiments of post-ischemic reperfusion injury, the maintenance of an effective concentration of the inhibitor makes it difficult to discriminate whether the inhibitor is effective due to the inhibition of xanthine oxidase or due to the scavenging of O(2) because allopurinol has been reported to be a direct scavenger of superoxide anion(25) . Thus, the involvement of the enzyme in reperfusion injury is still controversial.

Many other inhibitors of xanthine oxidase have been reported since allopurinol has been introduced(26) . However, none of them except allopurinol has been used for remedy of hyperuricemia or for experimental studies, probably because of less effectiveness than allopurinol or because of possible side effects of the inhibitor on animals or humans. Recently, a new potent inhibitor of xanthine oxidase has been introduced and found to be an effective inhibitor even in vivo. Its inhibitory effects on liver xanthine oxidase in vivo have been found to last longer than allopurinol (27) . However, the mechanism of inhibition and the strength of binding of the inhibitor have not been reported.

Here, we report the inhibitory effects of this compound on xanthine oxidase in vitro and the mechanism of inhibition of this compound.


MATERIALS AND METHODS

Milk XO was purified by the methods of Ball(28) . The active form of XO was separated from the inactive desulfo-form according to the method of Nishino et al.(11) . The prepared enzyme was more than 90% active, i.e. it exhibited an activity to flavin ratio (AFR) of more than 190 (fully active enzyme has a value of 210)(10, 13) . The desulfo-enzyme (inactive form) was prepared by incubating the enzyme in 10 mM KCN for 2 h at 25 °C followed by gel filtration to remove KCN(10) . Milk xanthine dehydrogenase was purified by the method of Nakamura and Yamazaki(29) . Before use, xanthine dehydrogenase was previously incubated with 10 mM dithiothreitol and was passed through Sephadex G-25 to remove dithiothreitol. The dehydrogenase/oxidase activity ratio of xanthine dehydrogenase as defined by Waud and Rajagopalan (6) was around 9. Sodium-8-(3-methoxy-4-phenylsulfinylphenyl)pyrazolo[1,5-a]-1,3,5-triazine-4-olate monohydrate (BOF-4272) was a generous gift from the Otsuka Pharmaceutical Factory Inc. (Naruto, Japan). This material contained isomers as shown in Fig. 1. The method of synthesis of BOF-4272 and the chemical nature will be described elsewhere. (^2)The isomers were separated by HPLC using a Chiralcel OD column (Daisel Chemical Co., Tokyo) and a solution of hexane, ethanol, and formate (700:300:4) as a solvent. In this chromatography, the(-)-isomer was eluted at an earlier retention time than the other (+)-isomer, which was confirmed with the authentic synthesized non-racemic compounds, which were provided by the Ohtsuka Pharmaceutical Factory Inc. The separated compounds were used for experiments. The concentration of BOF-4272 was checked by determination of the absorbance at 320 nm using the molar absorption coefficient of 23,500.


Figure 1: Structure of BOF-4272. Leftpanel,(-)-isomer; rightpanel, (+)-isomer.



Absorption spectra were recorded with an Hitachi U-3200 spectrophotometer.

Fluorescent spectra were recorded with an Hitachi 650-60 fluorescence spectrophotometer. Stopped-flow experiments were performed with an Applied Photophysics apparatus.

Enzyme Assay

All activity measurements were performed at 25 °C. For kinetic analysis, xanthine-oxygen reductase activity was measured spectrophotometrically by following the absorbance at 295 nm in 0.1 M pyrophosphate buffer (pH 8.5) containing 0.2 mM EDTA and various concentrations of xanthine and inhibitor under air-saturated conditions. Kinetics with different concentrations of xanthine in the phenazine methosulfate-linked reduction of cytochrome c were performed by following the absorbance change at 550 nm in 0.1 M pyrophosphate buffer (pH 8.5) containing 0.2 mM EDTA, 16.7 µM phenazine methosulfate (Wako, Osaka), 16.7 µM cytochrome c (horse heart, Boehringer Mannheim), 3.5 nM milk xanthine oxidase, and various concentrations of xanthine and BOF-4272. Before use, the phenazine methosulfate solution was kept on ice and in the dark.

Activities with 0.15 mM xanthine as an electron donor and various artificial substrates as electron acceptors were determined spectrophotometrically by following the reduction of the substrate as follows: NAD (0.5 mM) at 340 nm, methylene blue (0.02 mM) at 665 nm, potassium ferricyanide (0.4 mM) at 420 nm, DCPIP (0.05 mM) at 600 nm, or cytochrome c (0.025 mM) at 550 nm. All these reactions were performed in 50 mM potassium phosphate buffer (pH 7.8) under aerobic conditions except for methylene blue. Reaction with methylene blue was performed under anaerobic conditions. Activities were also measured in the presence of 0.05 mM NADH as an electron donor under the same conditions.

Determination of Dissociation Constant by Titration of Enzyme with BOF-4272

As BOF-4272 has a strong fluorescence (excitation at 320 nm, emission at 412 nm), which is greatly decreased on formation of the enzyme-BOF-4272 complex, it can be used for titration with the enzyme. The dissociation constant, K(d) value, was calculated for mixtures of BOF-4272 and XO near equivalency according to ,

where [EI], [E], and [I] represent the concentrations of the enzyme-BOF-4272 complex, free enzyme, and free BOF-4272, respectively.

Fluorescence intensity (Fluor) is due to both EI and I and is given by the equation

and by the equation for concentration of BOF-4272,

where k and k(I) represent the fluorescence intensity coefficient due to EI and I, respectively, and [I](O) represents the concentration of BOF-4272 added to the mixtures. Rearrangement of and brings about :

The value of k was obtained from the initial slope of titration curves at lower concentrations of inhibitor in the presence of XO and k(I) from the slope of the plot of Fluorversus BOF-4272 in the absence of enzyme. [EI](x), i.e. at any concentration of added BOF-4272, was calculated by measuring fluorescence, [I] and [E] were obtained by differences, and K(d) was calculated by substitution into the standard ().

Separation of BOF-4272 from the Enzyme-Inhibitor Complex

After 10 µM active XO (AFR = 193) or inactive desulfo-enzyme (AFR = 0) was incubated with 25 µM racemic compound of BOF-4272 in 0.1 M pyrophosphate buffer (pH 8.5) for 30 min at room temperature, the solution was passed through an Ampure SA column (Amersham Corp.) equilibrated with 5 mM pyrophosphate buffer (pH 8.5) to remove excess BOF-4272. The eluted enzyme-BOF-4272 complex was heated at 100 °C for 3 min followed by centrifugation at 15,000 times g for 10 min at 4 °C. 200 µl of the supernatant solution was subjected to HPLC (Hitachi 655-A) equipped with an LC-18-T column (Spelco). The sample was eluted with a gradient mixture of buffer A (10 mM NH(4)H(2)PO(4), pH 3.0) and buffer mixture B (80% 10 mM NH(4)H(2)PO(4), pH 3.0, and 20% acetonitrile). The fraction containing BOF-4272 was then passed through SEP-PAC C-18 (Millipore) to exchange the buffer to acetonitrile and was further subjected to HPLC using a Chiralcel OD column to separate the isomers.


RESULTS AND DISCUSSION

Steady State Analysis of Inhibition of Xanthine Oxidase with BOF-4272

The inhibitory effect of allopurinol on XO is known to be time dependent; the activity decreases gradually after mixing the enzyme with allopurinol. Such time-dependent inhibition is due to tight binding of oxipurinol, the oxidized product of allopurinol by reaction with XO, to the reduced form of molybdenum in the enzyme(13) . In contrast to allopurinol, BOF-4272 did not show such time-dependent inhibition; the activity did not change during the incubation time, as shown in Fig. 2. To evaluate the inhibitory effect of BOF-4272 on the activity of xanthine oxidase, steady state kinetic analyses were performed with various concentrations of xanthine and the inhibitor under air-saturated conditions. As there are isomers of BOF-4272, the experiments were performed with both compounds after separation of the isomers by HPLC using a Chiralcel OD column, as described under ``Materials and Methods.'' The results show that both of the isomers of BOF-4272 are mixed type inhibitors. A representative kinetic pattern obtained with(-)-isomer is shown in Fig. 3. A plot of slopes from the primary plot versus inhibitor concentration indicates a K(i) value of 1.2 times 10M for the(-)-isomer, while a plot of the apparent 1/V(max)versus the inhibitor concentration indicates a K(i)` value of 9 times 10M for the same isomer. A similar pattern was obtained with (+)-isomer (not shown), but 2 orders of magnitude higher values were obtained with this isomer, i.e.K(i) = 3 times 10M and K(i)` = 9 times 10M. On the other hand, steady state analysis with xanthine and phenazine methosulfate as substrates showed that the inhibition with(-)-isomer was a competitive type with a K(i) value of 2 times 10M (Fig. 4). As xanthine oxidase acts via a ping-pong mechanism, alternating between oxidized and reduced forms(30, 31) , a plausible explanation for the mixed type inhibition with oxygen as a substrate and the competitive type inhibition with phenazine methosulfate is that BOF-4272 binds both the oxidized and reduced forms but binds more tightly to the oxidized form. The competitive inhibition with phenazine methosulfate as a substrate might be due to the fact that the inhibitor does not bind to Mo(IV) because phenazine methosulfate reoxidizes Mo(IV) very rapidly, resulting in negligible formation of Mo(IV) during steady state turnover as in the case of allopurinol(13, 32) .


Figure 2: Time course of xanthine-O(2) activity of xanthine oxidase in the presence of BOF-4272 or allopurinol. The reactions were followed at 295 nm in 3 ml of solution containing 0.15 mM xanthine, 0.1 M pyrophosphate buffer (pH 8.5), 0.2 mM EDTA, and 1 nM xanthine oxidase (AFR = 193) in the absence of inhibitor (circle), in the presence of 3.3 µM allopurinol (up triangle), or in the presence of 0.033 µM BOF-4272(-)-isomer (box) at 25 °C.




Figure 3: Lineweaver-Burk plots of inhibition of xanthine-O(2) activity of xanthine oxidase in the presence of BOF-4272(-)-isomer. , without BOF-4272; circle, 4 nM; bullet, 8 nM; up triangle, 12 nM; , 16 nM; box, 20 nM. Inset, the K and K` values were obtained from secondary plots of the slopes of the Lineweaver-Burk plots (circle) and the apparent 1/V(max) (bullet) versus the inhibitor concentration, respectively.




Figure 4: Lineweaver-Burk plots of inhibition of xanthine-phenazine methosulfate activity of xanthine oxidase with BOF-4272(-)-isomer. , without BOF-4272; bullet, 5 nM; circle,10 nM; up triangle, 15 nM. Inset, secondary plot of apparent Kversus the inhibitor concentration.



Absorption Spectra of the Inhibitor-bound Xanthine Oxidase

It has been reported by Massey et al. (13) that upon binding of pyrazolopyrimidine derivatives to the Mo(IV) of xanthine oxidase, the spectra of the inhibited enzyme changes significantly from that of native enzyme. However, no spectral changes were observed mixing either the oxidized or reduced form of the enzyme with BOF-4272 (data not shown), suggesting no direct interaction of the inhibitor with the molybdenum sphere, in contrast to the results with pyrazolopyrimidine derivatives.

Effects of BOF-4272 on the Activities with Substrates

The effects of BOF-4272 on various artificial substrates were studied (Table 1). When xanthine was used as an electron donor, the reactions were effectively inhibited with BOF-4272 regardless of the electron acceptor used. These are consistent with the conclusion described below, that BOF-4272 binds in close proximity to the molybdenum site and therefore prevents the binding of or reduction with xanthine. No apparent difference was observed between XO and xanthine dehydrogenase. On the other hand, the activities of xanthine dehydrogenase using NADH as electron donor, which were inhibited with BOF-4272, depended on the electron acceptor used. NADH-methylene blue activity was not at all inhibited, while NADH-DCPIP and NADH-cytochrome c activities were partially inhibited. As it was found from the stopped-flow experiments that BOF-4272 did not affect the reduction of xanthine dehydrogenase by NADH, the partial inhibition of the activity observed when using DCPIP or cytochrome c as an electron acceptor might be due to the interaction site of these electron acceptors being the same in part as the BOF-4272 binding site. The interaction sites of these electron acceptors to the enzyme could be the molybdenum or other redox centers.



Stopped-flow Study of Xanthine Dehydrogenase Reduction with Xanthine and NADH in the Presence of BOF-4272

To know the effect of BOF-4272 on the reduction of the enzyme with xanthine or NADH, xanthine dehydrogenase (AFR = 120) was mixed with xanthine or NADH under anaerobic conditions in the presence of an excess amount of BOF-4272, and the absorbance change was followed at 450 nm with a stopped-flow apparatus. The traces of reduction with these substrates are shown in Fig. 5. Reduction of xanthine dehydrogenase with xanthine was effectively blocked by BOF-4272, while no effect on the reduction of the enzyme was observed with NADH as substrate. The rates of absorbance change at 450 nm with various concentrations of NADH were not affected by the presence of BOF-4272 (data not shown), indicating that the inhibitor does not bind to the NADH binding site. These are consistent with the conclusion that BOF-4272 interacts with the xanthine binding site of the enzyme.


Figure 5: Time course of the reduction of milk xanthine dehydrogenase with xanthine or NADH in the presence of BOF-4272 (-)-isomer. Milk xanthine dehydrogenase (13 µM, dehydrogenase/oxidase activity ratio = 9, AFR = 120) in 0.1 M pyrophosphate buffer (pH 8.5) containing 26 µM BOF-4272(-)-isomer was mixed with an equal volume of the same buffer solution containing (1) 200 µM xanthine or (2) 100 µM NADH using a stopped-flow apparatus at 25 °C under anaerobic conditions, and absorbance of 450 nm was followed.



Binding Titration of Xanthine Oxidase with BOF-4272

As BOF-4272 has a strong fluorescence with a peak at 412 nm when excited at 320 nm and the fluorescence is quenched by binding to the enzyme, fluorescence intensity can be used for measuring the strength of interaction of BOF-4272 with XO. Almost fully active enzyme (AFR = 200) and KCN-treated desulfo-type of XO (AFR = 3) were titrated with various concentrations of BOF-4272(-)-isomer. The patterns of the titration of(-)-isomer with active XO and desulfo-type enzyme are shown in Fig. 6, A and B, respectively. As the fluorescence increased just after an equimolar amount of BOF-4272 with the enzyme, the binding of BOF-4272 and xanthine oxidase has a stoichiometry of 1:1. The K(d) values of the active and desulfo-forms of XO for BOF-4272(-)-isomer were calculated to be 2 ± 0.3 times 10M and 7 ± 5 times 10M, respectively, from each plot. The value with the active enzyme is in reasonable agreement with the K(i) values estimated from the steady state kinetic studies. Fig. 6C shows the binding titration of oxipurinol-bound XO with(-)-isomer. The fluorescence increased almost linearly with the concentration of BOF-4272, indicating essentially no binding of BOF-4272 to the oxipurinol-bound XO. This clearly shows that oxipurinol and BOF-4272 binding sites are the same or strongly overlapping, and oxipurinol blocks the binding of BOF-4272 to the enzyme. Minor binding of BOF-4272 to the oxipurinol-treated enzyme might be due to the presence of a minor amount of the desulfo-form, which still existed in the sample having an AFR 200 value. It is known that the desulfo-form of XO cannot be reduced with allopurinol, and therefore it could not be an oxipurinol-bound form(13) .


Figure 6: Fluorometric titration of milk xanthine oxidase with the inhibitor. A, 1 ml of active form of milk xanthine oxidase (AFR = 200) was mixed with various volumes (0200 µl) of 5.2 µM BOF-4272(-)-isomer in 0.1 M pyrophosphate buffer (pH 8.5) at room temperature. Fluorescence (excitation at 320 nm; emission at 412 nm) was followed. Dilution factors were recalculated. Original concentrations of xanthine oxidase were as follows: bullet, without xanthine oxidase; circle, 0.2 µM; up triangle, 0.4 µM. B, titration of the desulfo-form of milk xanthine oxidase. The desulfo-form of milk xanthine oxidase (AFR = 3) was titrated with BOF-4272(-)-isomer. Experimental conditions were the same as above. circle, without xanthine oxidase; bullet, 2 µM; up triangle, 3 µM; , 4 µM. C, titration of oxipurinol bound active form xanthine oxidase with BOF-4272(-)-isomer. Oxipurinol bound milk xanthine oxidase (AFR = 200), which was previously mixed with 20 µM allopurinol, was titrated with BOF-4272(-)-isomer. Experimental conditions were same as above. , without xanthine oxidase (control); circle, 2 µM free xanthine oxidase; , 2 µM oxipurinol bound xanthine oxidase.



Separation of the Isomer of BOF-4272 That Binds to Xanthine Oxidase

To confirm the result of steady state analysis that the(-)-isomer binds to the enzyme more tightly than the (+)-isomer and to know whether BOF-4272 is oxidized by XO, analysis of the compound was performed by HPLC after liberation from the enzyme-BOF-4272 complex, obtained by previous equilibration of XO with excess of the racemic compound, as described under ``Materials and Methods.'' The amount of BOF-4272 liberated from the complex was determined by HPLC using an LC-18-T column to be 0.75 and 0.65 per mol of active form and desulfo-form of enzyme, respectively. These results are consistent with the finding that BOF-4272 binds more tightly to the active form of the enzyme. The fractions that contained BOF-4272 were collected and were analyzed by HPLC using a Chiralcel OD column as described under ``Materials and Methods.'' Both samples, which were obtained either from an active or desulfo-enzyme-inhibitor complex, had a large peak of(-)-isomer and a negligible peak of (+)-isomer, as shown in Fig. 7. These results agree well with the previous conclusion from the steady state analysis that the(-)-isomer has a K(i) value 2 orders of magnitude lower than the (+)-isomer. It can also be concluded that this compound was not oxidized by XO because the BOF-4272 compounds liberated from the complexes were eluted in both HPLCs at the same retention time as authentic BOF-4272 compounds. Furthermore, no difference of absorption spectrum was observed between the samples eluted from enzyme complexes and the authentic compounds. This conclusion is consistent with the result that no reduction of XO was observed spectrophotometrically under anaerobic conditions.


Figure 7: Separation of BOF-4272 liberated from xanthine oxidase by a chiralcel HPLC. BOF-4272 liberated from the enzyme by heat treatment was subjected to the reverse phase column followed by a Chiralcel OD column as described under ``Materials and Methods.'' Upperpanel, elution profile of racemic compound of BOF-4272 (control) using a Chiralcel OD column; middlepanel, BOF-4272 that bound to the active form of xanthine oxidase; lowerpanel, BOF-4272 that bound to the desulfo-form of the enzyme.



The results presented here show that BOF-4272 is a potent inhibitor of xanthine oxidase. All the data are consistent with the conclusion that BOF-4272 binds to the xanthine binding site, not to the NADH binding site. As xanthine or oxipurinol interacts with the molybdenum, it was strongly suggested that the inhibitor binds in close proximity to the molybdenum. However, due to the lack of spectral change upon incubation of BOF-4272 with either the reduced or oxidized form of enzyme and due to the fact that BOF-4272 binds tightly to both active and desulfo-forms of enzyme, it is likely that no atoms of BOF-4272 are in the coordination sphere of the molybdenum. Rather, steric factors between the inhibitor and the enzyme protein around the molybdenum seem to be important for tight binding. As the enzyme has been recently crystallized and the x-ray analysis of the crystal is now in progress (33) , it will also be interesting to crystallize the enzyme-inhibitor complex for analysis of the interaction between the inhibitor and the molybdenum site.

As BOF-4272 binds tightly to both oxidized and reduced forms of the enzyme, it can be expected to inhibit the enzyme for longer periods than allopurinol in vivo, since the oxipurinol-inhibited enzyme can be reactivated by spontaneous reoxidation of the Mo(IV) to the Mo(VI) state. Actual in vivo studies showed that BOF-4272 inhibited rat liver xanthine dehydrogenase very effectively for a longer period than allopurinol(27) . The advantages of using this compound for experimental studies are that the inhibitory effect of the compound is not time dependent, and it can be used at relatively low concentrations because of its strong inhibitory effects on xanthine dehydrogenase or XO. In addition, the compound is not a strong scavenger of superoxide radicals. (^3)Furthermore, since this compound has a (+)-isomer, which is a much weaker inhibitor than the other(-), it may be used as a valuable control reagent for study of the role of xanthine-oxidizing enzymes in pathogenesis. Such usage of these compounds for experimental studies was recently reported(34) .


FOOTNOTES

*
This work was supported in part by grants-in-aid for scientific research from the Japanese Ministry of Education, Science, and Culture, a grant from the Gout Research Foundation of Japan, and a research grant for intractable diseases from the Japanese Ministry of Health and Welfare. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-3-3822-2131 (Ext. 422); Fax: 81-3-5685-3054.

(^1)
The abbreviations used are: XO, xanthine oxidase; BOF-4272, sodium-8-(3-methoxy-4-phenylsulfinylphenyl)pyrazolo[1,5-a]-1,3,5-tria-zine-4-olate monohydrate; DCPIP, dichlorophenolindophenol; AFR, activity to flavin ratio (enzyme activity defined as the absorbance change/min at 295 nm, monitoring conversion of xanthine to uric acid, divided by the enzyme absorbance at 450 nm in the standard assay condition); HPLC, high pressure liquid chromatography.

(^2)
K. Hashimoto and M. Kido, personal communication.

(^3)
S. Sato, personal communication.


ACKNOWLEDGEMENTS

We thank Dr. Vincent Massey at the University of Michigan for helpful discussions and critical reading of the manuscript.


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