Far-UV CD spectra analysis  Far-UV CD spectra taken (A) immediately after addition of increasing concentrations of Cu2+ to XO and (B) after 30-min pre-incubation of the metal with the enzyme. A single sigmoid curve, indicating cooperative binding, was obtained for each pre-incubation time, over the full range of Cu2+ concentrations investigated (0.05–2 mM). (B) Fluorescence emission spectra of XO and XO pre-incubated for 10 min with various Cu2+ concentrations from 0.005 to 2 mM, obtained upon excitation at 280 nm. Menkes Disease. Copyright © 2020 Elsevier B.V. or its licensors or contributors. It oxidizes aldehydes to the corresponding acids and other substances, including pterins, purines, and certain drugs such as allopurinol and 6-mercaptopurine. Recovery studies were conducted by pre-incubating the enzyme and the metal as described above at room temperature for either 0 or 30 min and then dialyzing the mixture at 4°C against 1 l of assay buffer for 10, 30, and 60 min, with one change of buffer after 30 min. A single value for Kd (359 ± 10 mM after 5-min pre-incubation) was found with the absorbance changes recorded at 550 nm. The enzyme is a homodimeric protein of M r 300,000 and is composed of independent subunits; each subunit contains … In the vicinity of the Fe/S I center, the sequence Lys95Thr96Arg97Leu98 His99Pro100Val101Gln102Glu103Arg104Ile105Ala106Lys107Ser108 His109Gly110Ser111Gln112Cys113 Gly114Phe115Cys116Thr117Pro118Gly119 comprises two His and two Cys residues, providing binding sites for Cu2+. It has attracted lots of attention because of its potential role in tissue and vascular injuries, as well as in inflammatory diseases and chronic heart failure [10,11]. Addition of higher Cu2+ concentrations resulted in inhibition of the activity, regardless of the pre-incubation length; the inhibition was moderate for Cu2+ of 5–700 µM [Fig. 1(B), closed and open symbols, −5.3 ≤ log ≤ −3.2] and drastic when Cu2+ concentrations went from 700 to 2000 µM [Fig. 1(B), closed and open symbols, −3.2 ≤ log ≤ −2.7] with no activity detectable in the presence of 2000 µM Cu2+ (log = −2.7). Stern–Volmer plot and fluorescence emission spectra  (A) Stern–Volmer plot describing tryptophan quenching of XO by Cu2+; the plot exhibits upward curvature at higher Cu2+ concentrations. Visible region CD spectra of XO taken (A) immediately after addition of increasing concentrations of Cu2+ to the enzyme and (B) after 30-min pre-incubation of the metal with the enzyme. Care was taken to maintain the pH at 7.5. For each assay, the enzyme was pre-incubated 5 min (or any other period of time as specified) with 50 µM to 2 mM Cu2+, then the absorption spectrum was immediately recorded. Copyright © 1974 Verlag Chemie GmbH. The changes were metal concentration- as well as time-dependent and affected essentially the α-helical content and β-sheet fraction of the enzyme. The differential quenching was also documented by changes in the fluorescence intensity ratio I405/I350 (Fig. 9, inset). They also suggested the binding of at least three Cu2+ (one at higher affinity site, at least two at lower affinity sites) that would influence the absorbance attributable to the molybdenum center. Each plot corresponded to a given pre-incubation period (5, 10, 20, and 30 min) and could be decomposed into two parts [separated by the arrow in Fig. 5(A)]. Extreme copper deficiency is seen in what fatal condition? ΔAmax was evaluated from the intercept of the plot of 1/ΔA vs. 1/[Cu2+] by extrapolation for low-ligand concentration. Xanthine Oxidase Substrate Mix and Xanthine Oxidase Enzyme Mix – Reconstitute each in 220 µL of water. Binding involving residue His683 is likely to affect the Fe/S II center as well as the FAD center (Fig. 12). Electronic absorption spectra of XO (A) and XO incubated with Cu2+(B)  For any given spectrum, XO (2.2 µM), buffer (0.1 M, pH 7.5), and Cu2+ (50 µM to 2 mM) were added to the sample cuvette, whereas buffer (0.1 M, pH 7.5) and Cu2+ (at the same concentration as in the sample cuvette) were added to the reference cuvette. inhibitor of xanthine oxidase. Treatment with allopurinol decreases oxidative stress in type 1 diabetic patients: hemoglobin glycation, glutathione oxidation, and the increase in lipid peroxi-dation … Xanthine oxidase (XO), a key enzyme in purine metabolism, produces reactive oxygen species causing vascular injuries and chronic heart failure. Xanthine oxidase appears to contain two active sites, each of which contains 1 molybdenum atom, two distinct iron-sulfur centers, and 1 molecule of FAD (5). As an illustration, plots of 1/ΔA450 vs. 1/[Cu2+] corresponding to 5-, 10-, 20-, and 30-min pre-incubation of the enzyme with the metal are shown in Fig. 4. At higher Cu2+ concentrations corresponding to drastic inhibition of the enzymatic activity, cooperative binding around the Fe/S centers continued, involving less accessible His and Cys residues. This was in agreement with the findings of non-cooperative binding at lower Cu2+ concentrations and cooperative binding at higher Cu2+ concentrations, reported for each reactive center. Inset b: value of the apparent dissociation constant Kd as a function of the pre-incubation time. A peak at 405 nm attributable to the Tryp residues and a shoulder at 350 nm attributable to the Tyr residues were detectable. Xanthine oxidase has a substrate optimum between 0.1 and 0.2 mM xanthine … A xanthine oxidase inhibitor is any substance that inhibits the activity of xanthine oxidase, an enzyme involved in purine metabolism.In humans, inhibition of xanthine oxidase reduces the production of uric acid, and several medications that inhibit xanthine oxidase are indicated for treatment of hyperuricemia and related … Figure 12 provides a schematic representation of the reactive centers with some His and Cys residues located near them along with the amino acids reported to be involved in the substrate binding and the reaction catalysis [8,13–15]. Both peaks decreased with increasing Cu2+ concentration. Inverse plots obtained after 20-min pre-incubation of XO and Cu2+ at different metal concentrations are shown in Fig. 1(D). Indeed, we showed that this enzyme is involved in free radical production associated with exercise in patients with chronic obstructive pulmonary disease . Correlatively, two Kd values (Kd1 of 30 ± 3 mM and Kd2 of 1.7 ± 0.1 mM after 5-min pre-incubation of XO with Cu2+) were found when the absorbance changes at 277 nm were monitored although here Kd1 was larger than Kd2; whereas Kd1 value decreased with increasing pre-incubation time, Kd2 value remained fairly constant (Fig. 7, insets). In this study, stimulation as well as inhibition of XO activity by Cu2+ is reported along with a detailed investigation on structural changes caused by the metal. Over the same range of metal concentrations, the absorbance changes observed at 450 nm were more gradual and no change was detectable at 550 nm for Cu2+ concentrations <0.4 mM, except after 30-min pre-incubation when absorbance changes at 550 nm became detectable starting at 0.3 mM Cu2+. Insets: value of the apparent dissociation constants Kd1 and Kd2 as a function of the pre-incubation time. Xanthine oxidase (XO) is an important enzyme catalyzing the hydroxylation of hypoxanthine to xanthine and xanthine to uric acid which is excreted by kidneys. Complex metalloprotein that catalyzes oxidative hydroxylation of a variety of aromatic heterocycles and simple aldehydes. Febuxostat is a nonpurine inhibitor of xanthine oxidase, and it is designed for patients with hyperuricemia and gout, and also to patients who have exhibited sensitivities to allopurinol. Published by Elsevier Inc. All rights reserved. Archives of Biochemistry and Biophysics 433, 107-116. As shown in Figs. 5 and 6, the absorbance changes detectable at the lowest Cu2+ concentrations (0.05–0.3 mM) were those observed at 350 nm. The enzyme showed an A280/A450 value of 5.6; the calculated AFR (activity to flavin ratio) value for the enzyme was 140–150 that corresponds to 65–75% functional enzyme [27]. Most of these residues are deeply buried in the protein but some, like His875, His67, and His82 (underlined in Fig. 12), are more exposed and thus more accessible to Cu2+. Images generated using the Swiss-PdbViewer (http://www.expasy.ch/spdbv) from the data of Enroth et al. When XO was exposed to 1.5 mM Cu2+, a metal concentration causing drastic inhibition of the enzymatic activity, the visible CD spectrum exhibited an enhanced signal at 432 nm (17% increase) and, especially, at 450 nm (28% increase); this resulted in an inverse proportion of the 432-nm peak vs. the 450-nm peak compared with the control [Fig. 11(A)]. Oxford University Press is a department of the University of Oxford. A sufficient supply is essential for the functioning of many biochemical processes, including electron transfer reactions, gene regulation, binding and transport of oxygen, and regulation of cell growth and … The amino acid sequence of each subunit in bovine milk XO includes 10 tryptophan and 34 tyrosine residues [13]. Has also low oxidase activity towards aldehydes (in vitro). (filled circle), 5 min (filled triangle), 10 min (filled square), 20 min (triangle), and 30 min (open square) before assaying for enzymatic activity. Alterations of XO activity by various metals have also been probed with mixed results of either stimulation or inhibition, depending on the metal [22–25]. XO has long been known to be present in bovine milk which remains a main source for purified preparations of the enzyme. Approximately one-third of all proteins are completely or partially unfolded [40] and metals bind with higher affinity to the folded state than to the unfolded state or partially folded states of a protein [41]. Which mineral serves as a cofactor in xanthine oxidase in the metabolism of purines, pyrimidines, and pteridines? R. Hille (2005) Molybdenum-containing hydroxylases. In all cases, Kd values decreased with increasing pre-incubation time (Figs. 5 and 6, insets). At present there is no evidence which suggests that electrons are distributed among a minimum of 12 electron-accepting groups. 1/[Cu2+], giving apparent dissociation constants Kd1(filled line) and Kd2(dotted line), after pre-incubation of XO with Cu2+for different time  Kd1 was found for [Cu2+] 0.05–0.7 mM (points on the graphs correspond to 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.7 mM Cu2+) and Kd2 was found for [Cu2+] 0.7–2 mM (points on the graph correspond to 0.7, 0.9, 1.1, 1.3, 1.5, 1.75, 2 mM Cu2+). His677 and His683 are deeply buried residues, part of the molybdopterin-binding domain but close to the FAD center; prior binding of Cu2+ to the more accessible nearby His67 is likely to facilitate binding to residues His677 and His683. 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Both properties provide for tools extensively used to probe changes in the tertiary structure of proteins [28–33]. Figure 1(A) illustrates the results obtained after 0-min pre-incubation; similar plots were obtained after 30-min pre-incubation. Subsequent alterations around the Fe/S centers were possibly initiated with binding at the sequence including His67 at low Cu2+ concentrations, leading to increasing alterations of the electron transfer to O2 and increasing inhibition of enzymatic activity. The first part, hyperbolic, corresponded to Cu2+ concentrations ranging from 0.05 to 0.7 mM and the second, sigmoid, corresponded to Cu2+ concentrations ranging from 0.7 to 2 mM. bases usually referred to as xanthine oxidase (EC 1.2.3.2), xanthine dehydrogenase (EC 1.2.1.37), or aldehyde oxidase (EC 1.2.3.1), de-pendingontheirproperties, arewidelydistrib-utedthroughoutnature. Mazur and Sackler report a range of 5.49–1.92 units of xanthine oxidase in samples obtained from normal human livers, while very low values or no activity was found in livers of cirrhotic patients and in cases of hemochromatosis. For (A) and (B), data were obtained after XO and Cu2+ were pre-incubated for 5 min (filled circle), 10 min (open circle), 20 min (▾), and 30 min (triangle). Reference Bonini MG. Production of the carbonate radical anion during xanthine oxidase turnover in the presence of … Among them, the inhibition of ascorbic acid on xanthine oxidase is an indirect effect. Exposure of XO to Cu2+ concentrations above a critical value of 0.7 mM, led to drastic inhibition of the enzymatic activity that coincided with the cooperative binding of additional Cu2+ around the molybdenum center, the binding of an additional Cu2+ around the FAD center and the progressive binding of probably three Cu2+ around the Fe/S centers. CD spectra were recorded with an Aviv Model 215 CD spectrometer. For any given spectrum, XO (2.2 µM) and Cu2+ (at the desired concentration) in 0.1 M assay buffer, pH 7.5, were added to the sample cuvette and the buffer and Cu2+ (at the same concentration as in the sample cuvette) were added to the reference cuvette. The assay was performed in 0.1 M citrate-phosphate-borate buffer (hereafter designated assay buffer), pH 7.5, since a preliminary pH profile indicated 7.5 as the optimum pH. Obtain 6 test tubes, add 25 µL of assay buffer into each tube and label them #1 through #6. Changes in the α-helical fraction (C), the β-sheet fraction (D), the β-turn fraction (E), and the random coil fraction (F) of the enzyme pre-incubated with various Cu2+ concentrations as a function of the time of pre-incubation. Xanthine … aOnly a single value for Kd was found for this absorbance peak. The fraction of total tryptophan residues accessible for quenching was calculated according to Equation (5), using the modified Stern–Volmer plot. But in spite of its indispensability for cell survival, copper is toxic at elevated levels and a number of disorders have been associated with excess copper [6,7] as well as with copper deficiency [1]. Cu2+–XO complex formation was characterized by modifications in XO electronic absorption bands, intrinsic fluorescence, and α-helical and β-sheet content. The position of the arrows in Fig. 5 corresponded to 0.7 mM Cu2+, the critical concentration beyond which drastic inhibition of the enzymatic activity as well as change in the apparent dissociation constant of the metal–enzyme complex were observed. Xanthine oxidase (XO), a key enzyme in purine metabolism, produces reactive oxygen species causing vascular injuries and chronic heart failure. With extended pre-incubation time, or higher Cu2+ concentrations, binding to the other states of the enzyme took place and, eventually multiple binding sites were filled leading to increasing inhibition of the activity. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide, This PDF is available to Subscribers Only. Xanthine oxidase has a substrate optimum between 0.1 and 0.2 mM xanthine or hypoxanthine. All buffers and solutions were prepared in water that had been filtered, passed through a mixed bed ion-exchange column, and then distilled. Addition of 0.1 mM Cu2+ caused a decrease in the peaks at 432 and 450 nm and a deepening of the trough at 550 nm, but the proportion between the 432- and 450-nm peaks remained unchanged. XO catalytic efficiency as a function of the log of Cu2+concentrations (expressed in mol/l)  The ratio Kcat/Km was calculated for XO pre-incubated 0 min (filled circle), 5 min (filled triangle), 10 min (filled square), 20 min (triangle), or 30 min (open square) with 0.5–2000 µM Cu2+. In this study, compounds Cu(hmy … The enzyme that converts retinol to retinal and ethanol to acetyl-aldehyde contains 4 ____ atoms. The type of inhibition depended solely on the length of the pre-incubation period. As mentioned above, because of its ubiquity and its ability to bind to proteins, copper would be one of the metals to probe in priority. For all three pre-incubation times (0-, 5-, or 10-min), the apparent Km value, which was equal to 9.6 ± 0.1 µM for the control, remained unaffected. 2. The excitation wavelength was 295 nm and the emission spectra of XO and XO in the presence of different [Cu2+] (0.005–2 mM) were recorded between 310 and 500 nm; a single peak at 405 nm, attributable to the Tryp residues in the protein, was detectable (inset). This last step results in the production of superoxide anion and hydrogen peroxide, two reactive oxygen species that have been associated with the potential damaging role of the enzyme [8,11,12]. Emission spectra were recorded between 310 and 500 nm after pre-incubation of 0.28 µM XO with various Cu2+ concentrations (5 µM to 2 mM) for increasing periods of time (0–30 min). Dialysis of the enzyme pre-incubated with various metal concentrations resulted in at least partial restoration of the enzymatic activity. Upon excitation at 280 nm, the emission spectrum of XO exhibited a shoulder at 350 nm in addition to the peak at 405 nm previously observed; both emissions were quenched upon addition of Cu2+ [Fig. 8(B)]. Also, it helps the liver break down alcohol and some drugs, such as those used in cancer therapy ( 5 , 6 , 7 ). All spectroscopic measurements were performed at 25°C. By continuing you agree to the use of cookies. The spectroscopic studies showed that Cu2+ formed a complex with XO that resulted in specific alterations around each reactive center along with alterations in the secondary and, eventually, tertiary structure of the enzyme. Uloric is a xanthine oxidase inhibitor, which contains the active ingredient, febuxostat. Xanthine oxidase can also act on certain other purines, … Secondary structure fractions were calculated using the CD spectra deconvolution program CDNN version 2.1 (http://bioinformatik.biochemtech.uni-halle.de/cdnn); changes in the fraction of various secondary structure elements as a function of Cu2+ concentration and pre-incubation time are shown in Fig. 10(C–F). The Hill coefficient, h, calculated from the plot of log [ΔA450/(ΔAmax−ΔA450)] vs. log [Cu2+] was equal to 1.05 ± 0.1, regardless of Cu2+ concentration or pre-incubation time. In parallel with the steady-state kinetics findings that 0.7 mM Cu2+ marked the onset for drastic inhibition of the enzymatic activity, and the same critical metal concentration marked the change in apparent dissociation constant for the metal–enzyme complex. Isolation ofthe activi-ties from diverse sources (6, 7, 17-19, 23) has revealedthattheseenzymesareall fundamen-tally … Plots obtained for the changes in absorption at 550 nm are shown in Fig. 6. where F0 is the integrated area of the fluorescence spectrum of the sample before quenching, F is the integrated area of the fluorescence spectrum of the sample after quenching, and [Q] is the concentration of quencher. No activity was detectable in the presence of 2000 µM Cu2+, regardless of the pre-incubation time. It is likely to be part of the first binding site, leading to alterations in the molybdenum center environment and hampering substrate binding (Fig. 12). Zinc salicylate reduces airway smooth muscle cells remodelling by blocking mTOR and activating p21, Copyright © 2020 Institute of Biochemistry and Cell Biology, SIBS, CAS. As the number of Cu2+ per enzyme molecule increases, the value of Kd is expected to decrease even if locally some of the additional binding sites exhibited a lower affinity for the metal than the first binding sites (Table 2). Results also emphasized the potential role of copper in the regulation of XO activity stemming from its binding properties. Pre-incubation of the enzyme with the metal for 20 and 30 min (open symbols) led to a steady decrease in catalytic efficiency as Cu2+ concentrations went from 0.5 to 700 µM (−6.3 ≤ log ≤ −3.2) and to a sharp decrease in catalytic efficiency when Cu2+ concentration increased from 700 to 1500 µM (−3.2 ≤ log ≤ −2.8), with a slope steeper than that observed for 0-, 5-, and 10-min pre-incubation. For assays done in the presence of Cu2+ ions, appropriate amounts of CuSO4 stock solution (1.5 M, prepared in distilled water) were added to XO in 0.1 M assay buffer, pH 7.5, and the mixture was incubated at room temperature (22–25°C) for either 0, 5, 10, 20, or 30 min. The Hill coefficient, h, calculated from the plot of log [ΔA550/(ΔAmax−ΔA550)] vs. log [Cu2+] was equal to 2.5 ± 0.1 after 5-min pre-incubation and to 1.6 ± 0.1 after 30-min pre-incubation. This also indicated that the binding of possibly two Cu2+ around the FAD center was non-cooperative. [Cu2+]  The plot of ratio of ΔA550/ΔAmax vs. [Cu2+], where ΔA550 is the absorbance change caused by a given Cu2+ concentration and ΔAmax is the absorbance change for complete formation of the XO/Cu2+ complex as seen at 550 nm. Mahnaz Hadizadeh, Ezzatollah Keyhani, Jacqueline Keyhani, Cyrus Khodadadi, Functional and structural alterations induced by copper in xanthine oxidase, Acta Biochimica et Biophysica Sinica, Volume 41, Issue 7, July 2009, Pages 603–617, https://doi.org/10.1093/abbs/gmp048. XO activity was measured spectrophotometrically by following the oxidation of xanthine to uric acid at 295 nm, using an extinction coefficient of 9.6 mM−1 cm−1. Functional and structural alterations induced by copper in xanthine oxidase. The enzyme is a 290-kDa homodimer, each monomer acting independently in catalysis [13]. The values obtained for Kd1 from the changes in absorbance at 277 nm characterized the binding of Cu2+ to a number of non-equivalent, independent sites and were expectedly smaller than those obtained for any of the individual sites (Table 2). Cu2+ was in low concentration, binding first occurred with the completely folded.! As allopurinol and 6-mercaptopurine various Cu2+ concentrations, where I is the fluorescence assay tryptophan residues accessible for was! Oxidative enzyme substrate Mix and xanthine dehydrogenase ( XD ) in endothelial.... Milk XO includes 10 tryptophan and 34 tyrosine residues [ 13 ] [ 13 ] energy of binding for. Concentrations probed under steady-state kinetics conditions that two or three Cu2+ would.... A main source for purified preparations of the metal–enzyme complex of … copper inhibition of xanthine to uric acid was... 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