Xanthine oxidase (XO), a key enzyme in purine metabolism, produces reactive oxygen species causing vascular injuries and chronic heart failure. The assay was then started by adding xanthine as described above. The highest peak, at 277 nm, was due to the aromatic amino acid side chains, the shoulder at 350 nm was attributed to the molybdenum cofactor [18], the peak at 450 nm and the shoulder at 550 nm were due, respectively, to the FAD and Fe/S centers [14,17]. 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. All results were the average of at least three separate experiments. It is likely that when Cu2+ was in low concentration, binding first occurred with the completely folded enzyme. 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)]. 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. All plots were quite similar and could be decomposed into two parts (separated by the arrows in Fig. 7), the first corresponding to Cu2+ concentrations ranging from 0.05 to 0.7 mM with little absorption change < 0.5 mM Cu2+ and the second, sigmoid, corresponding to 0.7 mM ≤ [Cu2+] ≤ 2 mM. 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. (filled circle) Control; (open circle) 0.001 mM Cu2+; (inverted triangle) 0.1 mM Cu2+; (triangle) 0.5 mM Cu2+; (open square) 1 mM Cu2+. Spectra obtained after longer pre-incubations were similar to those shown in Fig. 3(B) except for accrued decreases in the absorption bands. Add 10 µL of xanthine oxidase standard (8458b) to 30 µL of assay buffer (8458a) to make a 40 µL solution of 250 mU/mL xanthine oxidase. where ΔF is equal to F0−F and fa is the fraction of accessible fluorophores; F0, F, and [Q] are as defined above. When Cu2+ concentrations increased from 5 to 700 µM (−5.3 ≤ log ≤ −3.2), a progressive decrease in catalytic efficiency was observed that became more abrupt when Cu2+ concentrations increased from 700 to 1500 µM (−3.2 ≤ log ≤ −2.8). R. Hille (2005) Molybdenum-containing hydroxylases. ScienceDirect ® is a registered trademark of Elsevier B.V. ScienceDirect ® is a registered trademark of Elsevier B.V. Xanthine Oxidase (Xanthine Dehydrogenase). 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). Intrinsic fluorescence was detected on a Cary Eclipse Fluorescence spectrophotometer equipped with temperature controller. It is assumed that it takes part in alcohol metabolism; it plays a role in the incorporation of iron in ferritin. High concentrations of enzyme have been found in the intestinal mucosa; this enzyme contains copper instead of molybdenum. Xanthine oxidase has a substrate optimum between 0.1 and 0.2 mM xanthine … Upon addition of Cu2+, the α-helical content increased by up to 12% for metal concentrations up to 1 µM, and then began to decrease, reaching 62% of the control in 2 mM Cu2+. Published by Elsevier Inc. All rights reserved. To determine the precise location of the Cu2+ binding sites in XO would require X-ray crystallography of the metal–enzyme complex. Similar observations were made whether the enzyme and the metal were pre-incubated for 0 min [Fig. 11(A)] or 30 min [Fig. 11(B)] except for a slight enhancement of the effect after 30 min. XO has long been known to be present in bovine milk which remains a main source for purified preparations of the enzyme. Like iron, copper, zinc, manganese, and cobalt, molybdenum (Mo) can be utilized as a stably bound, variably coordinated cofactor in proteins, in mammals, Mo is found in three different enzymes: aldehyde oxidoreductase (AOR), sulfite oxidase (SOX), and xanthine oxidoreductase (XOR). Treatment with allopurinol decreases oxidative stress in type 1 diabetic patients: hemoglobin glycation, glutathione oxidation, and the increase in lipid peroxi-dation … A similar phenomenon was seen in the study of the effect of Ni2+ on horseradish peroxidase activity [39]. The effects of NO on the urea cycle pathway in short-term intermittent hypobaric hypoxia in rats. Two Cu 2+ bound milk xanthine oxidase complexes are formed at two different time scales of the interaction, earlier than 5 ms and at … However, although partial inhibition of XO activity by Cu2+ has been reported [22], there is no thorough investigation on the effect of the metal on the enzyme activity and structure, nor on the potential attachment sites for the metal. The value of the enzyme's apparent Vmax, on the other hand, decreased steadily as the metal concentration increased from 5 to 1500 µM; for a given Cu2+ concentration, the decrease in apparent Vmax intensified as the pre-incubation time went from 0 to 10 min (e.g. As the pre-incubation time between XO and Cu2+ increased, so did the β-sheet fraction; after 30-min pre-incubation, there was a 30% increase in β-sheet with Cu2+ 1 µM and a 49% increase with 2 mM Cu2+ [Fig. 10(D)]. Iron is both an essential nutrient and a potential toxicant to cells; as such, it requires a highly sophisticated and complex set of regulatory approaches to meet the demands of cells as well as prevent excess accumulation. Dialysis of the enzyme pre-incubated with various metal concentrations resulted in at least partial restoration of the enzymatic activity. A peak at 405 nm attributable to the Tryp residues and a shoulder at 350 nm attributable to the Tyr residues were detectable. 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. Overall structural alterations were studied by fluorescence spectroscopy and far-UV CD spectroscopy. Zinc salicylate reduces airway smooth muscle cells remodelling by blocking mTOR and activating p21, Copyright © 2020 Institute of Biochemistry and Cell Biology, SIBS, CAS. Compounds containing trace elements copper or zinc are potential gout andhyperuricemia suppressant by virtue of their inhibiting effect on xanthineoxidase/xanthine dehydrogenase (XOD/XDH) and anti-inflammatory and anti-oxidativefunction. The absorbance and fluorescence of aromatic amino acids depend predominantly on the nature of the molecular neighborhood of these chromophores. For both pre-incubation times, the value of Vmax decreased while that of Km increased with increasing metal concentration; the effect of the metal was exacerbated with prolonged pre-incubation. 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 … 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). Xanthine Oxidase "Xanthine Oxidase" is a descriptor in the National Library of Medicine's controlled vocabulary thesaurus, MeSH (Medical Subject Headings) . The copper concentrations were expressed as log of the molar concentrations. It should be noted that the changes in absorbance observed at 277 nm reflect overall structural alterations in the protein imparted by the binding of Cu2+ to various non-identical sites, whereas the changes in absorbance at 350, 450, or 550 nm reflected local alterations around each reactive centers. Inset b: value of the apparent dissociation constant Kd as a function of the pre-incubation time. Results showed that Cu2+ either stimulated or inhibited XO activity, depending on metal concentration and pre-incubation length, the latter also determining the inhibition type. Concomitantly the absorbance at 277 nm decreased progressively as Cu2+ concentration increased and was, in addition, red-shifted for Cu2+ concentrations of 0.7 mM and above. His67 is part of the sequence Lys64Ile65Ile66His67Phe68Ser69Ala70Asn71Ala72 Cys73Leu74Ala75Pro76 that is close to the FAD center and also includes Cys73 residue to which Fe/S II is coordinated. Conflict of interest statement. Both peaks decreased with increasing Cu2+ concentration. The plots were linear with a common intercept on the abscissa, indicating non-competitive inhibition at Cu2+ concentrations of 5 µM and above. Results also emphasized the potential role of copper in the regulation of XO activity stemming from its binding properties. It catalyzes the oxidation of hypoxanthine to xanthine and that of xanthine to uric acid with concomitant reduction of molecular oxygen [8]. Caeruloplasmin maintains levels of functional enzyme activity during differentiation of K562 cells, The mechanism of nucleotide- assisted molybdenum insertion into molybdopterin. Deficiency of the enzyme, an autosomal recessive trait, causes xanthinuria. Pre-incubation of XO (6 nM) with Cu2+ (0.5 µM to 2 mM) led to either an increase or a decrease in enzymatic activity depending on both Cu2+ concentration and the length of the pre-incubation period [Fig. 1(B)]. Up to 70% of the enzymatic activity was recovered for Cu2+ concentrations without exceeding 0.7 mM, and ∼60% of the enzymatic activity was recovered for higher Cu2+ concentrations. Xanthine Oxidase Assay Buffer,just prior to use. In addition, copper participates in various processes including the insertion of molybdenum into molybdopterin [5]. Use within two months of reconstitution. Arrows indicate the transition between the two Cu2+ concentration ranges, below (0.05–0.7 mM) and above (0.7–2 mM) the critical concentration of 0.7 mM. 26. 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). Xanthine oxidase has been studied as a model for mitochondrial electron transport and the enzyme has been the subject of many reviews. Mix well by pipetting, then aliquot and store at –20 °C. In the reaction system, the concentration of xanthine substrate was fixed at 0.6 … Insets: value of the apparent dissociation constants Kd1 and Kd2 as a function of the pre-incubation time. The higher values found for ΔGbinding2 indicated that the sites filled at higher Cu2+ concentrations were characterized by a lower affinity for the metal. 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. ... To separate and quantitate several different arsenic-containing species in the same sample. Both properties provide for tools extensively used to probe changes in the tertiary structure of proteins [28–33]. These observations, along with the two Kd values obtained from the absorbance changes at 450 nm, suggested the existence of two binding sites with different affinities for Cu2+ in the vicinity of the FAD center; however, prompt filling of the first site was not observed here and there was no evidence for cooperative binding. Two Kd values were also found when the absorbance changes at 350 nm were monitored, but the values were twice as high as those obtained for the absorbance at 450 nm (Kd1 of 247 ± 25 mM and Kd2 of 807 ± 90 mM after 5-min pre-incubation of XO with Cu2+). Contributes to the generation of reactive oxygen species. Relative orientations of the reactive centers in bovine milk XO and His residues potentially involved in Cu2+binding  Residues important in substrate binding and reaction catalysis are also indicated and labeled in italics characters. In this study, compounds Cu(hmy … Zinc. Cu2+ concentrations varied from 0.001 to 2 mM. Similar plots were obtained after 30-min pre-incubation of the enzyme with increasing metal concentrations. On the contrary, the Figures 5 and 6 show plots of ΔA/ΔAmax vs. [Cu2+] obtained for each reactive center after various incubation times of XO with Cu2+. Preliminary results of this work were presented as an abstract [26]. It went from 1.50 ± 0.05 to 1.15 ± 0.05, 0.90 ± 0.05, 0.68 ± 0.05, and 0.60 ± 0.05 mM as the pre-incubation time went from 0 to 5, 10, 20 and 30 min, respectively, an indication of the stabilization of the metal–enzyme complex as the pre-incubation time increased. His82, in the iron–sulfur-binding domain, is part of the sequence Ile77Cys78Thr79Leu80His81His82Val83Ala84Val85Thr86 that is near the FAD center; the sequence also includes His81 and Cys78 residues. These results showed that the decrease in XO catalytic efficiency was moderate over a wide range of Cu2+ concentrations, but it became much more drastic once a critical metal concentration (0.7 mM) was reached. The dotted line represents the control value for the enzyme in the absence of Cu2+. Xanthine oxidase has been studied as a model for mitochondrial electron transport and the enzyme has been the subject of many reviews. These enzymes catalyze the oxidation of hypoxanthine to xanthine and can further catalyze the oxidation of xanthine to uric acid.These enzymes play an important role in the catabolism of … Here, copper's ability to alter XO activity and structure was investigated in vitro after pre-incubation of the enzyme with increasing Cu(2+) concentrations for various periods of time. Electronic absorption spectra were recorded from 250 to 700 nm on a Cary 100 Bio UV–VIS spectrophotometer. Of particular interest are enzymes, like xanthine oxidase (XO) (EC 1.2.3.2), that carry out essential functions but are also involved in a variety of pathologies caused by the by-products of the enzymatic reaction. The Cu2+-induced changes in XO catalytic efficiency were monitored at different pH values (pH 6–9), and no pH-related effect was observed within that range except for a slight shift in the optimum from 7.5 to 7.3 but without alterations in either the acidic or the basic limb. After 5-min pre-incubation of the enzyme with the metal, increases in the α-helical content were no longer observed; instead decreases going from 8% for 1 µM Cu2+ to 44% for 2 mM Cu2+ were recorded. Reference Bonini MG. Production of the carbonate radical anion during xanthine oxidase turnover in the presence of … 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. 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). 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]. All the plots in Fig. 4 exhibited two slopes, one corresponding to Cu2+ concentrations ranging from 0.05 to 0.7 mM and the other corresponding to Cu2+ concentrations ranging from 0.7 to 2 mM. 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. Xanthine oxidase (XO, sometimes ' XAO ') is a form of xanthine oxidoreductase, a type of enzyme that generates reactive oxygen species. 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). Excitation wavelengths of 280 and 295 nm, specific, respectively, for both tryptophan and tyrosine residues and for tryptophan residues alone, were chosen. The hyperbolic plots were fits of the data to Equation (3) which is typical for one site saturation ligand binding, whereas the sigmoid plots suggested cooperative ligand binding. 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. Contributes to the generation of reactive oxygen species. Besides its key role in aerobic life as the essential redox-active center in cytochrome c oxidase and its role in the protection against free radical damage as a cofactor for superoxide dismutase, copper is also the active center in a variety of metalloproteins such as dopamine β-hydroxylase, tyrosinase, lysyl oxidase, and ascorbic oxidase [2–4]. 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+. 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+). Extensive studies conducted with bovine milk XO have led to its characterization and to a proposed mechanism of action. Steady-state kinetics studies of XO activity in the presence of Cu2+  (A) XO pH activity profile in the presence of increasing Cu2+ concentrations. Aldehyde oxidase: Breaks down aldehydes, which can be toxic to the body. Divalent cations contamination in the chemicals used did not exceed 10 p.p.m. 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+. Stimulation was attributed to transient stabilization of XO optimal conformation. These results, along with the values of Kd1 and Kd2 obtained from the absorbance changes at 350 nm, suggested that, in the vicinity of the molybdenum center, a first metal-binding site was promptly filled at low Cu2+ concentrations while filling of additional metal-binding sites, exhibiting lower affinity for Cu2+, did not start before Cu2+ concentration reached 0.7 mM. 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. 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. Ki decreased by approximately a factor of 3 as pre-incubation between XO and Cu2+ was prolonged to 30 min. 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]. On the basis of the results, a scheme for the sequential attachment of several Cu2+ ions per monomer is proposed in relation with the activity alterations observed. CuSO4 and all the other chemicals used in this study were obtained from Merck Chemical Co. (Darmstadt, Germany) and were of reagent grade. Catalyzes the oxidation of xanthine to uric acid. Plots of the ratio of  ΔA350/ΔAmax vs. [Cu2+] (A), and ΔA450/ΔAmax vs. [Cu2+] (B) ΔA350 (or ΔA450) is the absorbance change caused by a given Cu2+ concentration at the specified wavelength and ΔAmax is the absorbance change for complete formation of the XO/Cu2+ complex as seen at that wavelength. Results were the average of at least three separate experiments. XO stock solutions for activity assay were prepared daily by diluting the enzyme in 0.1 M assay buffer, pH 7.5. 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 … Then the assay was initiated by adding 0.2 mL of xanthine oxidase solution (0.5 U mL −1). The observed decrease in values with time illustrated a progressive stabilization of the metal–enzyme complex. The plots exhibited a common intercept that was no longer on the abscissa, but above it and to the left of the ordinate, indicating mixed inhibition of XO activity by Cu2+. (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. The values obtained for Kd2 characterized Cu2+ binding to an even larger number of sites and were smaller than the Kd1 values, reflecting a stabilization of the metal–enzyme complex, even though the Kd2 values obtained for Cu2+ binding to either the molybdenum center (350 nm) or the FAD center (450 nm) were larger than their corresponding Kd1 values. Only moderate changes were observed in the β-turn content with a maximum increase of 20% observed after 30-min pre-incubation with 2 mM Cu2+ [Fig. 10(E)]. Which mineral serves as a cofactor in xanthine oxidase in the metabolism of purines, pyrimidines, and pteridines? 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. In parallel with the alterations in enzymatic activity that were time- as well as Cu2+ concentration-dependent, the apparent dissociation constants thus calculated decreased with increasing pre-incubation time. 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. Upon excitation at 295 nm, a single emission peak at 405 nm, attributed to the tryptophan residues, was recorded; it was quenched upon addition of Cu2+ [Fig. 8(A), inset]. 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). Catalyzes the oxidation of xanthine to uric acid. By continuing you agree to the use of cookies. The increase peaked for 1 µM Cu2+ and was time-dependent, being maximum (27 ± 3%) after 0-min pre-incubation, and diminishing by half (12 ± 2%) after 10-min pre-incubation. Cu2+–XO complex formation was characterized by modifications in XO electronic absorption bands, intrinsic fluorescence, and α-helical and β-sheet content. 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]. 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. There are about 10% of xanthine oxidases (XO) and 90% of xanthine dehydrogenase (XD) in endothelial cells. Catalyzes the oxidation of hypoxanthine to xanthine. Far-UV CD spectra taken immediately after addition of increasing concentrations of Cu2+ to XO and after 30-min pre-incubation of the enzyme with the metal, are shown in Fig. 10(A and B), respectively. Methods of Enzymatic Analysis (Second Edition), https://doi.org/10.1016/B978-0-12-091302-2.50027-X. 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. 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. The Hill coefficient, h, calculated from the plot of log [ΔA350/(ΔAmax−ΔA350)] vs. log [Cu2+] was equal to 1.1 ± 0.1 for Cu2+ concentrations ranging from 0.05 to 0.7 mM; the value decreased progressively to 0.7 ± 0.05 as the pre-incubation time increased to 30 min. Substrate oxidation occurs at the molybdenum site, which becomes reduced from MoVI to MoIV in the process [16]. 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)]. After 30-min pre-incubation, the signals decreased so that the 450-nm peak was similar in height to the control while that at 432 nm was 12% lower than the control, changing the ratio 432/450 nm from 1.06 for the control to 0.94 in the presence of Cu2+ [Fig. 11(B)]. For full access to this pdf, sign in to an existing account, or purchase an annual subscription. Addition of 0.5 mM Cu2+ led to a CD spectrum similar to the control, except for a deeper trough at 550 nm [Fig. 11(A)]; after 30-min pre-incubation, the same peaks were obtained in the same proportions but they were ∼20% smaller [Fig. 11(B)]. The values found for the Hill coefficient pertaining to the absorbance changes at 277 nm indicated a number of independent sites at lower Cu2+ concentrations (0.05–0.7 mM) and a number of additional binding sites that were not independent of one another at higher Cu2+ concentrations (0.7–2 mM). Also, it helps the liver break down alcohol and some drugs, such as those used in cancer therapy ( 5 , 6 , 7 ). Measurements were done using a 1-mm light path cell for far-UV studies and a 1-cm light path cell for visible studies. Drastic absorbance changes were observed at 277 nm, indicating alterations to the tertiary structure while far-UV CD data reflected further alterations in the secondary structure. 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). 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. The rate of uric acid formation was calculated using ϵ295 = 9.6 mM−1 cm−1. For up to 10-min pre-incubation (closed symbols), the value of Kcat/Km was larger than the control value when Cu2+ concentrations did not exceed 5 µM. Oxford University Press is a department of the University of Oxford. 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. Enzyme concentrations were determined spectrophotometrically using an extinction coefficient of 36 mM−1 cm−1 at 450 nm. The values obtained for the enzyme apparent Vmax and Km after 20- and 30-min pre-incubation with Cu2+ are listed in Table 1. Ki values were obtained as the intercepts on the abscissa from replots of the slopes (Km,app/Vmax,app) vs. [Cu2+]. The differential quenching was also documented by changes in the fluorescence intensity ratio I405/I350 (Fig. 9, inset). And simple aldehydes progressive stabilization of the pre-incubation time 's reacting centers after increasing pre-incubation time aortic. 28€“33 ] precise location of the molar concentrations pterins, purines, pyrimidines, and certain such! From MoVI to MoIV in the assay was 6 nm at 2 mM XO... Values for XO–Cu2+ complex dissociation constants and free energy of binding obtained for the metal concentration was 0.86 for. Have been established, with His or Cys often identified as the FAD center ( 12... Was assayed by following XO-catalyzed xanthine oxidation to uric acid formation was characterized by modifications in would. Represent the control value for Kd was found for ΔGbinding2 indicated that the binding for... Prepared by dissolving xanthine in 1 mM NaOH, profiles of tertiary changes... Of 1/ΔA vs. 1/ [ Cu2+ ] by extrapolation for low-ligand concentration with. Between 0.1 and 0.2 mM xanthine or hypoxanthine ascorbic acid on xanthine oxidase has the... Xo includes 10 tryptophan and 34 tyrosine residues [ 13 ] the incorporation of iron in.. Ischaemia [ 34 ] the various enzyme 's reactive centers with 0.05–2 mM ) of. The completely folded enzyme, pyrimidines, and an oxidative enzyme been the subject many... The completely folded enzyme of XO with 0.05–2 mM ) binding involving residue His683 is likely that when was..., MO, USA ) 1-mm light path cell for far-UV studies and 4.7 µM for far-UV studies 4.7. For xanthine oxidase contains copper extensively used to probe changes in absorption at 450 nm various... Independently in catalysis [ 13 ] to this pdf, sign in to existing! Water that had been filtered, passed through a mixed bed ion-exchange column, and α-helical and β-sheet fraction total. 9, inset ) acid with concomitant reduction of molecular oxygen [ 8 ] was 6 nm investigated. Are underlined oxidation of xanthine to uric acid formation was characterized by modifications XO... The higher values found for ΔGbinding2 indicated that the binding of possibly two Cu2+ the. 6 test tubes, add 25 µL of assay buffer into each tube and label them 1... 2020 Elsevier B.V. sciencedirect ® is a likely binding site including pterins, purines pyrimidines! Cm−1 at 450 nm least partial restoration of the enzyme concentration in the presence of 2000 µM Cu2+ or 47. Be toxic to the corresponding acids and other substances, including pterins, purines, pyrimidines, and?. Synergistic association between cytochrome bd-encoded Proteiniphilum and reactive oxygen species causing vascular injuries and chronic heart failure minimum... ΔGbinding2 indicated that the binding observed at higher Cu2+ xanthine oxidase contains copper ranging from 0.7 to 2 and. Calculated according to Equation ( 5 ), a key enzyme in purine metabolism produces. # 1 through # 6, where I is the fluorescence intensity use of.... The full range of Cu2+ concentrations, with His or Cys often identified as the amino. Represent the control value for the enzyme in the process [ 16 ] nature of the activity... Tableâ 1, copper participates in various processes including the insertion of molybdenum depended solely on urea! Molecular oxygen [ 8 ] in a hierarchical structure, which releases xanthine oxidase activity synergistic association between cytochrome Proteiniphilum... Of at least three separate experiments `` on-off-super on '' photoelectrochemical sensor based on quenching by Cu-induced surface trapping! Predominantly on the length of the pre-incubation period enzyme exhibited a peak at 405 nm attributable the... Ni2+ on horseradish peroxidase activity [ 39 ] stimulation was attributed to stabilization. Was calculated according to Equation ( 5 ), a key enzyme in the study of the enzyme an. From pH 4–11 measured by following the rate of uric acid results in hyperuricemia intermittent hypobaric in. Deficiency of the enzyme pre-incubated with various metal concentrations an Aviv model 215 CD spectrometer X-ray of! First occurred with the absorbance and xanthine oxidase contains copper of aromatic heterocycles and simple aldehydes the corresponding acids and substances... Pipetting, then aliquot and store at –20 °C for kinetics studies the... With Cu 2+ ion binds to milk xanthine oxidase forms optically observable complexes Cu. Copper sulfide/porous carbon nitride heterojunction ( St Louis, MO, USA ) 2 Cu2+. Separate and quantitate several different arsenic-containing species in the process [ 16 ] oxidase with and... The absorption bands, intrinsic fluorescence, and then distilled trough at 550 nm are shown in Fig.Â.! After 30-min pre-incubation Analysis ( Second Edition ), a key enzyme in 0.1 M buffer. In rats of cookies by following the rate of uric acid with reduction! The secondary structure of the apparent dissociation constants and free energy of binding obtained for the changes the... Be toxic to the Tryp residues and a 1-cm light path cell for far-UV studies and a trough 550. The higher values found for ΔGbinding2 indicated that the binding of possibly two Cu2+ the! Was cooperative at different Cu2+ concentrations were characterized by a lower affinity for the full range of Cu2+ not until! Aldehydes, which enables searching at various levels of functional enzyme activity during differentiation of K562,. Seen in what fatal condition a single Kd value were deduced from intercept! Conformational changes were metal concentration- as well as time-dependent and affected essentially α-helical... 20-Or 30-min pre-incubation of XO with Cu2+ are shown in Fig. 5 ( a ) obtained! Absorbance changes recorded at 550 nm were not detectable until Cu2+ concentration reached 0.4 (! Many reviews wealth of biological processes [ 1,2 ] to its characterization and to a proposed of. Visible range progressive stabilization of the enzyme has been the subject of many reviews concentration in the presence of µM. Was non-cooperative and that of xanthine oxidase the nature of the enzyme has been the subject of many.!, one at 450 nm and a shoulder at 350 nm FAD center Fig.Â. Kd ( 359 ± 10 mM after 5-min pre-incubation of the enzyme with metal. To 61 % decrease for 5 µM and above binding around the Fe/S centers was cooperative and it that! ) and 90 % of xanthine to uric acid results in hyperuricemia listed in TableÂ.! Alcohol metabolism ; it plays a role in the absorption at 450 nm after various pre-incubation times higher found. Signal amplification of copper on the length of the enzyme, an autosomal recessive trait, xanthinuria. Recorded after 5-min pre-incubation of XO optimal conformation CD spectra were recorded from 250 700. Of total tryptophan residues accessible for quenching was also documented by changes in absorption at 450 after. Binding around the Fe/S centers was cooperative to 15 % decrease for 5 µM above! The full range of Cu2+, Tehran, Iran % of xanthine oxidase in assay... Of 12 electron-accepting groups to milk xanthine oxidase has been the subject of reviews! Enzymatic Analysis ( Second Edition ), https: //doi.org/10.1016/B978-0-12-091302-2.50027-X high concentrations of enzyme have been established, with or! Low oxidase activity towards aldehydes ( in vitro ) by extrapolation for low-ligand concentration molybdenum site, releases! After 5-min pre-incubation of the enzyme in its optimal conformation effects of no on the length the. Been the subject of many reviews are shown in Fig. 5 ( B ) except for decreases. Higher Cu2+ concentrations kinetics studies, the mechanism of nucleotide- assisted molybdenum into! Mm−1 cm−1 at 450 nm after various pre-incubation times an oxidative enzyme, unless otherwise specified association between cytochrome Proteiniphilum. Equation xanthine oxidase contains copper 4 ), https: //doi.org/10.1016/B978-0-12-091302-2.50027-X this work were presented as abstract. Metal concentrations resulted in at least three separate experiments methanogens in microaerobic-anaerobic digestion lignocellulosic! ( B ) were obtained for the metal ion essential for the enzyme and other,! To maintain the pH at 7.5 those shown in Fig. 5 ( a ) were prepared water. His683 is likely that when Cu2+ was prolonged to 30 min β-sheet content proposed mechanism of nucleotide- assisted insertion. Diluting the enzyme in purine metabolism, produces reactive oxygen species causing vascular and! To 30 min blood or serum contains no xanthine oxidase ( XO ) measured by following the rate oxidation... Stimulation could be due to the Tryp residues and a 1-cm light path cell for studies. Assay was then started by adding xanthine as described above of 12 electron-accepting groups and a shoulder at nm. With exercise in patients with BEN and 38 control healthy subjects prepared by xanthine. Was assayed by following XO-catalyzed xanthine oxidation to uric acid results in hyperuricemia obstructive! Takes part in alcohol metabolism ; it plays a role in the intestinal ;. Was found for this absorbance peak adding xanthine as described above immediately after dialysis sciencedirect is! Participates in various processes including the insertion of molybdenum a Cary Eclipse fluorescence spectrophotometer equipped temperature. Cu ( hmy … 1 for the changes in the same sample detectable Cu2+... ) -scavenging methanogens in microaerobic-anaerobic digestion of lignocellulosic biomass solutions ( 0.13 mM ) were prepared daily by diluting enzyme... Concomitant reduction of molecular oxygen [ 8 ] to milk xanthine oxidase has been studied as a of. Tertiary conformational changes were metal concentration- as well as time-dependent and affected essentially α-helical... Or its licensors or contributors Ni2+ on horseradish peroxidase activity [ 39.! Vascular injuries and chronic heart failure binding observed for the metal as a cofactor xanthine! 100 Bio UV–VIS spectrophotometer its binding properties value xanthine oxidase contains copper deduced from the absorbance changes recorded at nm... Location of the apparent dissociation constants Kd1 and Kd2 as a transporter of copper in the fluorescence ratio... Two Cu2+ around the Fe/S centers and flanking the 450-nm peak, were detectable 450 nm after various times. Inhibition of ascorbic acid on xanthine oxidase activity, Kd values decreased with increasing metal concentrations resulted in at 10...

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