Inhibition studies of bovine xanthine oxidase by luteolin, silibinin, quercetin, and curcumin

Article abstract of DOI:10.1021/np8007123

Xanthine oxidoreductase (XOR) is a molybdenum-containing enzyme that under physiological conditions catalyzes the final two steps in purine catabolism, ultimately generating uric acid for excretion. Here we have investigated four naturally occurring compounds that have been reported to be inhibitors of XOR in order to examine the nature of their inhibition utilizing in vitro steady-state kinetic studies. We find that luteolin and quercetin are competitive inhibitors and that silibinin is a mixed-type inhibitor of the enzyme in vitro, and, unlike allopurinol, the inhibition is not time-dependent. These three natural products also decrease the production of superoxide by the enzyme. In contrast, and contrary to previous reports in the literature based on in vivo and other nonmechanistic studies, we find that curcumin did not inhibit the activity of purified XO nor its superoxide production in vitro.

Full text of DOI:10.1021/np8007123

J. Nat. Prod. 2009, 72, 725–731
725
Inhibition Studies of Bovine Xanthine Oxidase by Luteolin, Silibinin, Quercetin, and Curcumin
†
,‡
,†
James M. Pauff and Russ Hille*
Department of Biochemistry, UniVersity of California, RiVerside, California 92521, and Medical Scientist Program, The Ohio State UniVersity,
Columbus, Ohio 43210
ReceiVed NoVember 7, 2008
Xanthine oxidoreductase (XOR) is a molybdenum-containing enzyme that under physiological conditions catalyzes the
final two steps in purine catabolism, ultimately generating uric acid for excretion. Here we have investigated four naturally
occurring compounds that have been reported to be inhibitors of XOR in order to examine the nature of their inhibition
utilizing in Vitro steady-state kinetic studies. We find that luteolin and quercetin are competitive inhibitors and that
silibinin is a mixed-type inhibitor of the enzyme in Vitro, and, unlike allopurinol, the inhibition is not time-dependent.
These three natural products also decrease the production of superoxide by the enzyme. In contrast, and contrary to
previous reports in the literature based on in ViVo and other nonmechanistic studies, we find that curcumin did not
inhibit the activity of purified XO nor its superoxide production in Vitro.
Xanthine oxidoreductase (XOR) is a 290 kDa molybdenum-
containing enzyme that has been studied extensively from a
biochemical perspective for more than 80 years. In human physiol-
ogy, XOR catalyzes the final two steps of purine catabolism,
transforming hypoxanthine to xanthine and then xanthine to uric
acid by sequential oxidative hydroxylations at C-2 and C-8 of the
Table 1. Steady-State Kinetic Parameters for the Inhibition of
Xanthine Oxidase
cat (s-1
(µM-1 s-1
k
)
K
m
(µM)
k
cat/K
m
)
i
K (µM)
Luteolin
1.9 ( 0.7
1
0 µM
6.2 ( 0.2
31 ( 5
0.20
0.03
0.02
0.05
3.5
2
5 µM
3.8 ( 0.2 127 ( 20
2.6 ( 0.2 164 ( 30
5.8 ( 0.5 126 ( 30
12.1 ( 0.6 3.5 ( 0.8
1
purine ring, respectively (Figure 1). Xanthine oxidoreductase exists
50 µM
in two forms. The protein normally exists as a dehydrogenase
incubation
control
Quercetin
+
(
xanthine dehydrogenase, XDH) and utilizes NAD as its final
1.2 ( 0.7
electron acceptor in catalysis. Under certain conditions, most notably
10 µM
25 µM
50 µM
5.9 ( 0.2 3.7 ( 0.3
4.5 ( 0.4
1.9 ( 0.2 106 ( 30
4.3 ( 0.8 104 ( 50
10.5 ( 0.4 1.2 ( 0.3
1.6
0.10
0.02
0.04
8.8
ischemia and/or hypoxia, XDH can be converted to an oxidase form
43 ( 10
+
(
XO), which can no longer reduce NAD and instead utilizes O
2
exclusively as the terminal electron acceptor in the course of
turnover. This conversion may occur either by oxidation of
incubation
control
Curcumin
2
,3
sulfhydryl groups and/or by limited proteolysis. Once in the
oxidase form, the enzyme generates significant amounts of H
and superoxide radicals, although XDH can also react with O and
generate these reactive oxygen species (ROS). Under normal
1
2
5
0 µM
5 µM
0 µM
13.3 ( 0.6 2.8 ( 0.6
11.8 ( 0.4 1.8 ( 0.3
12.2 ( 0.5 2.4 ( 0.5
12.4 ( 0.8 3.2 ( 0.9
12.4 ( 0.4 2.1 ( 0.4
4.8
6.5
5.1
3.9
5.9
2
O
2
2
3
incubation
control
Silibinin
+
conditions, however, NAD effectively competes with O
2
and limits
6
,7
the generation of ROS by the dehydrogenase.
The specific
1
0 µM
5.0 ( 0.3
4.0 ( 0.4
4.3 ( 0.3
3.8 ( 0.1
36 ( 8
30 ( 10
39 ( 10
53 ( 7
0.14
0.13
0.11
0.07
5.6
conformational change responsible for the D to O conversion
2
5 µM
involves modification of the access channel to the flavin site (with
3,4
its FAD cofactor) of XOR, eliminating NAD+ binding. Once
50 µM
incubation
control
generated, these ROS can interfere with a multitude of cellular
functions and processes. Two of the most extensively studied are
the integrity of cellular membrane lipids and the interactions of
superoxide with the vasodilator NO in circulation. In the latter case,
superoxide from xanthine oxidase has been shown to degrade
S-nitrothiols, which are considered to be a storage form of NO, as
10.9 ( 0.4 1.9 ( 0.3
Allopurinol
10 µM
8.1 ( 0.6
6.5 ( 0.6
6.9 ( 0.5
25 ( 7
43 ( 20
63 ( 20
0.32
0.15
0.11
25 µM
5
0 µM
incubation
control
1.3 ( 0.3 189 ( 90
10.1 ( 0.8 3.1 ( 1.0
0.007
3.3
8,9
well as converting NO to the vaso-inactive compound peroxynitrite.
Superoxide has also been shown to reduce H to form destructive
hydroxyl radicals, and even carbonate radicals.
O
2 2
1
0
attacks and stroke and in spinal cord injury, as well as being a
11,16,17
destructive force in myocardial or renal hypoxia and infarctions.
XOR is thus potentially a main player in many pathological
conditions and, additionally, is also thought to be involved in the
pathogenesis of and secondary complications associated with other
The mechanism of substrate hydroxylation by the molybdenum
center of XOR is depicted in Figure 1, being initiated by abstraction
of a proton from the Mo-OH group by an active site glutamate
residue that is universally conserved in this family of molybdenum-
11-13
diseases.
Hyperuricemia resulting from XOR activity is central
to the pathogenesis of gout and gouty arthritis, as well as being
causative in cases of high serum urate following (tumor) cellular
5
containing enzymes. Subsequent to deprotonation, nucleophilic
1
4,15
attack on substrate and concomitant hydride transfer to the metal
center leads to an intermediate having product coordinated to the
now reduced Mo center via the newly introduced hydroxy group.
From the molybdenum center, electrons are passed sequentially via
two [2Fe-2S] clusters to an FAD site, where they are finally passed
necrosis in tumor lysis syndrome.
XOR in its oxidase form is
considered to be a main source of oxidative stress and destructive
free radicals in ischemia-reperfusion injury associated with heart
*
To whom correspondence may be addressed. Tel: (951) 827-6345. Fax:
to NAD+ or O
2
.
(
951) 827-4294. E-mail: russ.hille@ucr.edu.
†
Inhibition of XOR is a primary objective in treating any case of
11,18
University of California, Riverside.
The Ohio State University.
‡
hyperuricemia.
Allopurinol was the first mechanism-based
1
0.1021/np8007123 CCC: $40.75

2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/16/2009

7
26 Journal of Natural Products, 2009, Vol. 72, No. 4
Pauff and Hille
Figure 1. Catalytic mechanism of xanthine oxidoreductase conversion of xanthine to uric acid at the molybdenum-containing active site.
inhibitor of the enzyme to be developed and is still the primary
drug for combating hyperuricemia. Allopurinol is hydroxylated by
the enzyme to alloxanthine (oxypurinol), which, once formed,
coordinates tightly to the reduced form of the molybdenum center
specifically (Figure 2), replacing the Mo-OH group of native
Vitro. Mechanistic details of the inhibition or for the lack of
inhibition are discussed.
Results and Discussion
For each compound, absorbance spectra were taken each minute
for 10 min to demonstrate stability of the stock solutions. Each
compound was also monitored at 295 nm in the presence of 50
nM XO, and additionally at 550 nm with 75 µM cytochrome c as
well as XO. No change in absorbance was observed at 295 nm for
any of the compounds tested, although a very slow increase in A550
in the cases of quercetin and luteolin was observed, indicating slow
reduction of oxidized cytochrome c. None of the four compounds
tested here caused any reduction of anaerobic enzyme, as monitored
by the absorbance at 450 nm, indicating that none are hydroxylated
2
0
enzyme. Although allopurinol has longstanding use in pharma-
cotherapy and is efficacious in both lowering urate levels in the
body and retarding the metabolism of chemotherapeutic agents such
as 6-mercaptopurine, some individuals exhibit hypersensitivity to
the drug; in particular, side effects such as vasculitis (especially in
1
1
those with already compromised renal function) may occur. The
development of alternative XOR inhibitors thus remains desirable.
One such inhibitor that is currently in clinical trials is febuxostat,
whose crystal structure in complex with the enzyme has been
21-23
determined.
This structure shows the (hydroxylated) inhibitor
40
by the enzyme (at least not on the time scale of the present assays).
bound at the molybdenum center and blocking access to the active
site of the enzyme in a manner reminiscent of alloxanthine
Allopurinol at concentrations of 10, 25, and 50 µM was used as
a control for comparison to any inhibition exhibited by each of the
natural products to be tested. Consistent with previous work,
allopurinol inhibited XO in an uncompetitive manner, reflecting
binding of the hydroxylated product alloxanthine to the reduced
1
9
binding.
Investigations of the structure and function of xanthine oxi-
doreductase have most often utilized the enzyme isolated from
bovine milk, although the enzyme from human, avian, and bacterial
sources has also been characterized, and all have been found to be
homologous with, for example, the identical constitution of redox-
IV
20
(
k
Mo ) form of the enzyme. Allopurinol was shown to reduce
seen with xanthine (reflecting the reaction of free enzyme
cat/K
m
and free xanthine at low [xanthine]) some 30-fold at a concentration
of 50 µM and >400-fold at 25 µM following a 10 min preincubation
with the enzyme. Figure 5 compares the ∆A295 over 10 min in the
presence of each compound at 50 µM with 100 µM xanthine and
3
2-35
active centers.
investigated XO from bovine milk as well as XDH from the purple
In our own laboratory we have extensively
3
6-39
bacterium Rhodobacter capsulatus.
progress on such biochemical studies of the enzyme are available
Reviews of the current
5
nM XO, showing time-dependent inhibition of XO by allopurinol
1
,2
42
elsewhere.
as noted previously by others. Other kinetic data for the inhibition
of XO by allopurinol are given in Table 1.
Although many previous reports have suggested the existence
of naturally occurring XOR inhibitors ranging from flavonoids to
a host of other natural plant products, and several of these studies
have utilized the readily available enzyme from bovine milk, little
work has focused on the detailed mechanism by which such
The reduction of cytochrome c by superoxide generated by XO
in the course of turnover was significantly decreased at each
concentration of allopurinol, as shown in Table 2. The decrease
was most pronounced following incubation of the enzyme with 25
µM allopurinol, as expected, and was proportionate to the decrease
in xanthine consumption following incubation as monitored at 295
nm in the above experiments.
Inhibition of Xanthine Oxidase by Luteolin. As shown in
Table 1, among the natural products examined here luteolin had
m i
the greatest effect on kcat/K , with a K of 1.9 ( 0.7 µM. At 10
µM, the inhibitory effect of luteolin when presented to XO with
24-31
inhibition may occur.
Here we have examined the mechanism
of inhibition exhibited by four such compounds: silibinin, quercetin,
curcumin, and luteolin. We find that luteolin, silibinin, and quercetin
are inhibitors of the enzyme, as manifested in their reduction in
the initial rate of catalysis of xanthine to urate, and that luteolin
and quercetin also proportionately reduce the rate of superoxide
generation by XO with xanthine. In contrast, we find that curcumin
does not inhibit the production of urate or superoxide by XO in
xanthine resulted in a nearly 20-fold reduction in turnover of the

Effect of Luteolin, Silibinin, and Quercetin on XOR
Journal of Natural Products, 2009, Vol. 72, No. 4 727
Table 2. Initial Rates of Reduction for Cytochrome c by
Xanthine Oxidase + Xanthine in the Presence of Natural
a
Product
rate of cyt c reduction (nM s-1)
Luteolin
control
160 ( 0.6
100 ( 3
82 ( 9
10 µM
25 µM
50 µM
71 ( 3
incubation
control)
Quercetin
110 ( 10
(170 ( 20)
(
control
170 ( 1
140 ( 20
100 ( 7
80 ( 6
10 µM
25 µM
50 µM
incubation
control)
Curcumin
110 ( 7
(170 ( 20)
(
control
160 ( 20
160 ( 7
10 µM
25 µM
50 µM
150 ( 6
150 ( 6
incubation
control)
Silibinin
150 ( 10
(170 ( 20)
(
control
160 ( 0.6
160 ( 7
10 µM
25 µM
50 µM
150 ( 17
140 ( 13
120 ( 4
incubation
control)
(
(170 ( 20)
Allopurinol
control
170 ( 10
140 ( 10
140 ( 6
10 µM
25 µM
50 µM
120 ( 9
incubation
control)
28 ( 1
(170 ( 20)
(
a
Also shown are rates following 10 min incubations of enzyme with
5 µM natural products and cytochrome c prior to the addition of
2
xanthine. Final reaction mixtures were monitored over several minutes
at 550 nm and contained 50 nM XO, 100 µM xanthine, natural product,
and 75 µM cytochrome c. Error expressed as standard deviations from
triplicate trials.
Figure 2. Active site of XDH from Rhodobacter capsulatus (PDB
code 1JRP) showing the coordination of alloxanthine (reacted
allopurinol) directly to the molybdenum center (modified from 37).
Numbering in parentheses is for the bovine enzyme. Panel B is
rotated 60° to the right from panel A.
quercetin, and this value decreases further to less than 20% of the
uninhibited kcat at 50 µM. The effects on K
with a 100-fold increase in the presence of 50 µM quercetin and a
>400-fold reduction in kcat/K from a value of 8.8 to 0.04. The K
m
are also pronounced,
m
i
for competitive inhibition by quercetin was determined to be 1.2
( 0.7 µM. As shown in Figure 4, the double-reciprocal plot for
quercetin again resulted in linear fits that intersected near the origin
at x ) 0, and the secondary plot of these slopes versus [quercetin]
free enzyme as assessed by kcat/K
turnover by the free enzyme at luteolin concentrations of 25 and
0 µM, culminating in a 170-fold reduction in kcat/K ; observed
rate constants were reduced up to 6-fold compared to control at
these three concentrations. The double-reciprocal plot of [XO]/Vobs
versus 1/[xanthine] showed a series of linear relationships at each
m
. There was further inhibition of
5
m
crossed at a value for y ) 0 to give the value above for K
Preincubation of XO with 25 µM quercetin did not result in any
additional decrease in turnover rate, although the K for xanthine
i
.
m
[
luteolin], which intersected near the origin at x ) 0, as shown in
Figure 4. The secondary plot of the slopes versus [luteolin] resulted
in a linear relationship and the value for K above. Any additional
was slightly more than twice that seen without preincubation. The
production of superoxide by XO with xanthine as substrate was
reduced at 25 and 50 µM quercetin, with resulting decreases
analogous to those with luteolin and most significant at 50 µM
quercetin. As for the case of luteolin, the inhibition of superoxide
production showed the same inhibitor concentration dependence
as for the turnover of xanthine.
i
effect following incubation of the enzyme with luteolin prior to
the introduction of xanthine was negligible compared to that seen
whentheenzymewasexposedtoxanthineandluteolinsimultaneously.
At 100 µM xanthine, 5 nM XO, and 50 µM luteolin, significant
inhibition was observed with a linear increase in A295 over 10 min
Inhibition of Xanthine Oxidase by Silibinin. Inhibition of
XO by silibinin in the steady state appeared to be independent of
the silibinin concentration. At silibinin concentrations of 10, 25,
and 50 µM, the kcat for xanthine turnover is reduced by ap-
proximately 50% in each case, and the value of k /K is reduced
(Figure 5). Preincubation of the enzyme with luteolin did not result
in any additional effect of the inhibition, a very similar case to
that with quercetin discussed below. Also, the extent of inhibition
of superoxide generation was proportionate to inhibition of xanthine
turnover (Table 2).
Inhibition of Xanthine Oxidase by Quercetin. Inhibition
of XO by quercetin was next examined. The rate for overall turnover
is reduced by approximately 60% at a concentration of 10 µM
cat
m
50-fold. Preincubation of the enzyme with 25 µM silibinin resulted
in no additional decrease in kcat, but rather a significantly increased
K
m
m
, and the value for kcat/K was further decreased from 5.6 to
0.07, reflecting an 80-fold reduction when compared to the control.

7
28 Journal of Natural Products, 2009, Vol. 72, No. 4
Pauff and Hille
Figure 3. Natural compounds in the current study, previously reported to be inhibitors of xanthine oxidase. Also shown are allopurinol,
xanthine, and lumazine.
No value for K
i
could be obtained for inhibition by silibinin, as the
superoxide by the reaction of xanthine oxidase with xanthine, as
followed by a ∆A550 associated with the reduction of cytochrome
c. A modest decline in superoxide is apparent only at the highest
concentration of silibinin, and the significant declines in the cases
of luteolin and quercetin are nearly proportionate to the inhibition
of urate production at each concentration (see Tables 1 and 2). This
observation, that the decreases in superoxide production are
proportional to the decreased turnover of xanthine, strongly suggests
that each inhibitor here acts at the molybdenum-containing active
site of XOR to competitively inhibit the reduction of the enzyme
by xanthine, rather than acting directly at the FAD-containing flavin
linear fits of double-reciprocal plots were nearly identical for the
three silibinin concentrations, and thus a secondary plot of the slopes
versus [silibinin] did not cross the x-axis at a meaningful point.
In the presence of 50 µM silibinin, the production of superoxide
by XO was decreased by approximately 20% relative to the control
at 100 µM xanthine. The reaction of silibinin alone with XO under
anaerobic conditions did not yield any apparent enzyme reduction,
indicating that silibinin does not reduce the enzyme. The reduction
of cytochrome c by XO was also not appreciably affected by
silibinin.
Effect of Curcumin on Xanthine Oxidase. Curcumin did
not demonstrate any appreciable inhibition of purified bovine
xanthine oxidase at 10, 25, 50, or 100 µM in our steady-state assays.
Neither did preincubation of XO with 25 µM curcumin for 10 min
2
site that interacts with O . This results in a slower time course of
superoxide production at a given xanthine concentration. Thus any
decrease in the reduction of cytochrome c via the scavenging of
superoxide radicals by the inhibitor itself (acting as an antioxidant),
or by direct interaction (i.e., interposition) at the flavin site of XO
m
result in a significant change in either kcat or K . Superoxide
production was not significantly altered by any concentration of
curcumin, either initially or following preincubation. These results
contrast with other reports, principally involving in ViVo studies,
which suggest that curcurmin inhibits xanthine oxidase in this
concentration range.
2
to inhibit final electron transfer to O , is unlikely here.
Reports from various in ViVo studies have suggested that luteolin,
quercetin, and silibinin may approach the effectivenesss of allopu-
2
4-31
rinol as inhibitors of xanthine oxidase.
The present results
demonstrate that these three compounds are each inhibitors in Vitro,
although there is no time-dependent increase in inhibition analogous
to that with allopurinol and the effective K ’s determined here do
i
not approach those for allopurinol. Given the unknown pharma-
cokinetics of these natural products, the effective dose could
therefore be considerably higher than for either allopurinol or the
more recently approved febuxostat.
The present in Vitro study was undertaken given the conflicting
literature reports concerning the inhibition of XO by the compounds
investigated. The issue is particularly important given the desir-
ability of developing novel clinical inhibitors of XO due to the
adverse events sometimes associated with acute and chronic
administration of allopurinol. We find that xanthine oxidase is
inhibited by luteolin, quercetin, and silibinin, but not curcumin.
The mode of inhibition by luteolin and quercetin was competitive,
while that by silibinin was a mixed type of inhibition. Inhibition
by luteolin and quercetin is not surprising given the resemblance
to the known substrate lumazine, a pteridine (Figure 3) known to
In the case of the nonflavonoid curcumin, the lack of inhibition
is at variance with previous reports that did not utilize purified
3
1
enzyme. We note that curcumin is not planar like xanthine,
lumazine, or other known inhibitors of XO and does not otherwise
structurally resemble any of these compounds. Given that the
solvent access channel to the deeply buried active site of XO
4
3
be hydroxylated to violapterin. On the other hand, it is perhaps
surprising that the bulkier silibinin is indeed an inhibitor. This
natural compound does possess a benzopyran ring, however, which
resembles the analogous moieties of quercetin and luteolin.
With regard to superoxide production by XO, three of the four
compounds tested appear to decrease the initial production of
3
narrows to <10 Å approximately halfway in, the central sp -
hybridized carbon of curcumin (Figure 3), it is likely that curcumin
is unable to access the substrate binding site. Similarly, the physical
shape of curcumin, with a bend of approximately 110°, makes it
unlikely that it simply occludes the solvent access channel as seen

Effect of Luteolin, Silibinin, and Quercetin on XOR
Journal of Natural Products, 2009, Vol. 72, No. 4 729
Figure 4. Double-reciprocal plots of [XO]/Vobs versus 1/[xanthine] for each concentration of luteolin (panel A) and quercetin (panel B).
Also shown is the resulting plot of slopes versus 1/[inhibitor] used to determine the K for each respective compound. The intersection of
i
the linear fits at x ) 0 for these two inhibitors indicates that the mode of inhibition is competitive. Panel C depicts the analogous double-
reciprocal plot for silibinin, with linear fits that are not consistent with competitive inhibition of the enzyme.
with febuxostat. Finally, we note that curcumin lacks a reactive
carbon center that would constitute a possible site of hydroxylation
by XO. Thus with the lack of a plausible mechanistic or structural
basis for inhibition, our results showing a lack of XO inhibition by
curcumin are not surprising. Previous reports demonstrating inhibi-
tion of xanthine oxidoreductase by curcumin using other methods
may indicate that suppression of superoxide production or the
production of uric acid in these experiments is an indirect
consequence of curcumin’s presence. Our results clearly indicate
that curcumin does not directly inhibit the activity of bovine
xanthine oxidase, and thus further investigations are warranted.
Our work here demonstrates that luteolin, silibinin, and quercetin
inhibit xanthine oxidase and suggests that these compounds, or
derivatives of them, may be useful leads in the development of
24-30
clinically useful inhibitors of XO.
The nature of this inhibition,
particularly the somewhat stronger effect of quercetin and luteolin
relative to that of silibinin, is interesting and merits further
characterization. It seems likely that luteolin, silibinin, and quercetin
position in the active site of XO with the dihydroxybenzene
functionality of their benzopyran moiety directed toward the
molybdenum cofactor, their benzopyran intercalated between
Phe914 and Phe1009, their C1 carbonyl groups directed toward

7
30 Journal of Natural Products, 2009, Vol. 72, No. 4
Pauff and Hille
Figure 5. Time course of the inhibition of XO by allopurinol, luteolin, silibinin, and quercetin. Reactions were monitored by A295 at 30 s
intervals over 10 min. Each point represents an average of three trials.
Arg880, and their additional moiety projecting backward into the
solvent channel. Given our recent success at obtaining crystal
structures of bovine XO with substrate species bound in the
enzyme’s active site, it should be possible to obtain structures of
XO with luteolin, quercetin, and possibly silibinin. This would
allow direct observation of each compound’s interaction(s) with
active site residues, and thus allow further insight not only into the
role that these residues may be playing in normal catalysis but also
into possibly new means of inhibiting the enzyme.
prior to the addition of xanthine to initiate the assay. Controls were
run in the absence of any inhibitor in the reaction mix, both before
and after testing each respective compound in the assays above.
Superoxide production was monitored indirectly by monitoring the
nonenzymatic reduction of cytochrome c by O . The capacity for each
2
compound to influence superoxide production was determined by
•-
4
4
including 75 µM oxidized cytochrome c to the reaction mix, monitoring
-1
-1 45
the reaction at 550 nm (∆ꢀ550 ) 19.6 mM cm ). Stock solutions
of cytochrome c were prepared by incubating concentrated solutions
for 10 min with 2-3 mM ferricyanide, followed by passage through a
Sephadex G-25 column; final concentrations were determined from
-
1
-1 46
Experimental Section
serial dilutions of the stock, using ꢀ410 nm ) 106 mM cm
.
Assays
were conducted in the presence of 10, 25, and 50 µM of each compound
Xenobiotics and Enzyme. Silibinin, quercetin, luteolin, curcumin,
and xanthine were purchased from Aldrich (Sigma-Aldrich, St. Louis,
MO). All were at >95% purity and were used without further
purification. Stock solutions of luteolin, quercetin, silibinin, and
curcumin were prepared at a concentration of 5 mM in 0.1-0.2 M
aqueous KOH, as were 33.3 and 1 mM xanthine stock solutions. All
other reagents were purchased from either Aldrich or Fisher (Thermo
Fisher Scientific, Waltham, MA) and were of the highest purity
commercially available.
tested with 50 nM XO and 100 µM xanthine.
To ascertain whether each compound could be hydroxylated by
enzyme, 2 µM XO was titrated under anaerobic conditions with 25
µM of each compound. Reduction of the enzyme, as reflected in a
bleaching throughout the visible region, was monitored over 10 min
and quantified at 450 nm as described previously using ∆ꢀ450 ) 37.8
-
1
-1 40
mM cm
.
Each reaction was run at 25 °C in 0.1 M MOPS, 0.2 mM EDTA,
and 0.1 M KCl (for ionic strength) at a pH of 7.4, with a final reaction
volume of 1 mL (3 mL in the case of anaerobic titrations). All assays
were conducted using a Hewlett-Packard 8452A diode array spectro-
photometer interfaced with the Hewlett-Packard Chemstation (Palo Alto,
CA).
Xanthine oxidase was isolated and purified from bovine milk
40,41
accordingtothepreviouslyreportedmethodofMasseyandco-workers.
The enzyme as isolated was routinely approximately 70% active based
on the activity-to-flavin ratio, consistent with previous literature, the
nonfunctional enzyme lacking a catalytically essential ModS ligand
4
0
in the active site. The enzyme was stored in buffer containing 1 mM
salicylate at -80 °C and passed down a Sephadex G-25 column to
remove the salicylate prior to use.
Acknowledgment. The authors would like to thank Dr. M. Dunn
for the use of his laboratory facilities at UCR. This work was supported
by NIH grant GM 075036.
Steady-State Kinetics and Absorption Spectra. Inhibition of XO
was determined as follows. Each assay contained 50 nM XO, with
xanthine concentrations ranging from 1 to 800 µM in the presence or
absence of a known concentration of each natural compound in question
or in the presence of allopurinol as a standard reference. Each of the
four compounds in question was assayed at 10, 25, 50, and 100 µM
over this concentration range of xanthine, monitoring the ∆A295
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