All plots were linear for the full range of Cu2+ concentrations investigated (0.05–2 mM). The UV/visible electronic absorption spectrum of the enzyme includes contributions from each reactive center with the iron–sulfur centers exhibiting maxima at 420, 470, and 550 nm, the flavin exhibiting a maximum at 450 nm, and the molybdenum co-factor exhibiting absorption at 350 nm [14,17–19]. Xanthine oxidase (XO), a key enzyme in purine metabolism, produces reactive oxygen species causing vascular injuries and chronic heart failure. In the reaction system, the concentration of xanthine substrate was fixed at 0.6 … Xanthine oxidase is an important source of free radicals in vivo. A peak at 405 nm attributable to the Tryp residues and a shoulder at 350 nm attributable to the Tyr residues were detectable. The native enzyme exhibited a peak at 432 nm, one at 450 nm and a trough at 550 nm. The cooperative binding observed around the molybdenum center at higher Cu2+ concentrations could involve residues His863 and His954; alternatively, residue His109 in the vicinity of the Fe/S I center could also affect the molybdenum center. For Cu2+ concentrations that were above 0.7 mM (0.9–2 mM), the emission at 350 nm was quenched as well, and the emission maxima returned at 405 and 350 nm (Fig. 9). Zinc salicylate reduces airway smooth muscle cells remodelling by blocking mTOR and activating p21, Copyright © 2020 Institute of Biochemistry and Cell Biology, SIBS, CAS. Ki values were obtained as the intercepts on the abscissa from replots of the slopes (Km,app/Vmax,app) vs. [Cu2+]. 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. Published by Elsevier Inc. All rights reserved. Enzymatic activity alterations were assessed by steady-state kinetics studies of the XO-catalyzed oxidation of xanthine to uric acid in the presence of various Cu2+ concentrations. 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. Structural alterations were assessed by electronic absorption, fluorescence, and circular dichroism spectroscopy. 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 … 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). Two Cu 2+ bound milk xanthine oxidase complexes are formed at two different time scales of the interaction, earlier than 5 ms and at … 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. Excitation wavelengths of 280 and 295 nm, specific, respectively, for both tryptophan and tyrosine residues and for tryptophan residues alone, were chosen. The red-shift was progressive and went from 6 nm at 0.7 mM Cu2+ to as much as 28 nm at 2 mM Cu2+. 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. 2. The random coil fraction remained essentially unchanged except that it increased by 5–8% with 2 mM Cu2+ as the pre-incubation time increased to 30 min [Fig. 10(F)]. The ordinate intercept of the plot of F0/ΔF vs. 1/[Q] provides the accessible fluorophores at infinite quencher concentration and the value found in this case was 30%, thus three tryptophans. 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. The differential quenching was also documented by changes in the fluorescence intensity ratio I405/I350 (Fig. 9, inset). 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. Insets: value of the apparent dissociation constants Kd1 and Kd2 as a function of the pre-incubation time. 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. 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. Apparent dissociation constant values implied high- and low-affinity Cu2+ binding sites in the vicinity of the enzyme's reactive centers. 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. The enzymatic activity studies presented here showed that Cu2+ was a reversible inhibitor as well as an activator of the XO-catalyzed oxidation of xanthine to uric acid. Data were gathered from spectral measurements obtained for the full range of Cu2+ concentrations investigated (0.05–2 mM). Cu2+-induced variations in XO catalytic efficiency are illustrated in Fig. 2. Copyright © 1974 Verlag Chemie GmbH. The plots of ΔA277/ΔAmax vs. [Cu2+] shown in Fig. 7, corresponded each to a given pre-incubation period from 5 to 30 min. XO, a complex molybdoflavo protein, is a key enzyme in purine metabolism that has been isolated from a wide range of organisms, including bacteria and men [8,9]. XO has long been known to be present in bovine milk which remains a main source for purified preparations of the enzyme. Which mineral serves as a cofactor in xanthine oxidase in the metabolism of purines, pyrimidines, and pteridines? 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. The absorption spectra were modified so that decreases in the absorption bands at 350, 450, and 550 nm were observed, indicating alterations around each reactive center in the enzyme. Results also emphasized the potential role of copper in the regulation of XO activity stemming from its binding properties. The amino acid sequence of each subunit in bovine milk XO includes 10 tryptophan and 34 tyrosine residues [13]. Dialysis of the enzyme pre-incubated with various metal concentrations resulted in at least partial restoration of the enzymatic activity. Cu2+–XO complex formation was characterized by modifications in XO electronic absorption bands, intrinsic fluorescence, and α-helical and β-sheet content. Enzyme concentrations were determined spectrophotometrically using an extinction coefficient of 36 mM−1 cm−1 at 450 nm. 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. The metal ion essential for the activity of xanthine oxidase and xanthine dehydrogenase is: Molybdenum. Catalyzes the oxidation of xanthine to uric acid. 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. A similar phenomenon was seen in the study of the effect of Ni2+ on horseradish peroxidase activity [39]. Contributes to the generation of 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). Xanthine … 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. Both properties provide for tools extensively used to probe changes in the tertiary structure of proteins [28–33]. (ii) The Increase of Xanthine Oxidase (XO). Search for other works by this author on: Laboratory of Life Sciences, Saadat Abade, Sarve Sharghi 58, 19979 Tehran, Copper homeostasis in eukaryotes: teetering on a tightrope, Identification of porphyrin present in apo-cytochrome c oxidase of copper-deficient yeast cells, Regulation of Cu, Zn superoxide dismutase with copper. R. Hille (2005) Molybdenum-containing hydroxylases. Absorbance changes at 277 nm were mostly negligible until 0.5 mM Cu2+, indicating that the initial alterations around the molybdenum and FAD centers affected essentially the secondary structure of the enzyme. The plots shown in Fig. 5(A) were obtained for the changes in absorption at 350 nm. 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. It catalyzes the oxidation of hypoxanthine to xanthine and that of xanthine to uric acid with concomitant reduction of molecular oxygen [8]. Xanthine oxidase has been studied as a model for mitochondrial electron transport and the enzyme has been the subject of many reviews. XO stock solutions for activity assay were prepared daily by diluting the enzyme in 0.1 M assay buffer, pH 7.5. 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)]. The plots shown in Fig. 5(B) were obtained for the changes in absorption at 450 nm after various pre-incubation times. At low Cu2+ concentrations (0.005–0.5 mM) quenching was more drastic for the emission at 405 nm than for that at 350 nm and was accompanied by a 5-nm blue-shift of both peaks; at higher Cu2+ concentrations (0.9–2 mM) quenching of the emission at 350 nm occurred and, simultaneously both peaks shifted back to their original position, respectively, at 350 and 405 nm. 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. Cu 2+ ion binds to milk xanthine oxidase with sulfur and nitrogenous ligands. 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). Xanthine oxidase (XO) is an important enzyme catalyzing the hydroxylation of hypoxanthine to xanthine and xanthine to uric acid which is excreted by kidneys. 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+. The type of inhibition depended solely on the length of the pre-incubation period. Key enzyme in purine degradation. 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+). (B) Residual enzymatic activity at optimal pH: XO (6 nM) was pre-incubated with Cu2+ (0.0005, 0.001, 0.005, 0.05, 0.1, 0.2, 0.5, 0.75, 1, 1.2, 1.5, and 2 mM) in 0.1 M buffer, pH 7.5, respectively, for 0 min. Indeed, we showed that this enzyme is involved in free radical production associated with exercise in patients with chronic obstructive pulmonary disease . 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. Xanthine Oxidase Assay Buffer,just prior to use. On the contrary, the 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. However, the information gathered by various groups on the enzyme structure [8,13,14] allowed us to make some predictions regarding plausible binding sites for Cu2+. 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. Stimulation was attributed to transient stabilization of XO optimal conformation. 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). [13]. 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. Milk contains divalent copper ions, and ascorbic acid can reduce Cu 2+ to Cu +, which is a strong inhibitor of the enzyme. Normalized fluorescence emission spectra of XO and XO pre-incubated for 10 min with various Cu2+concentrations  The Cu2+ concentration is from 0.005 to 2 mM, and the spectra were obtained upon excitation at 280 nm. Absorption spectra obtained after 5 min incubation of XO with 0.05–2 mM Cu2+ are shown in Fig. 3(B). 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. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The enzyme is a 290-kDa homodimer, each monomer acting independently in catalysis [13]. Synergistic association between cytochrome bd-encoded Proteiniphilum and reactive oxygen species (ROS)-scavenging methanogens in microaerobic-anaerobic digestion of lignocellulosic biomass. ARRB2 promotes colorectal cancer growth through triggering WTAP, A mini-review on ion fluxes that regulate NLRP3 inflammasome activation, miR-338-5p inhibits cell growth and migration via inhibition of the METTL3/m6A/c-Myc pathway in lung cancer, Tamoxifen attenuates reactive astrocyte-induced brain metastasis and drug resistance through the IL-6/STAT3 signaling pathway, Overexpression of LVRN impedes the invasion of trophoblasts by inhibiting epithelial–mesenchymal transition, About Acta Biochimica et Biophysica Sinica, http://bioinformatik.biochemtech.uni-halle.de/cdnn, Receive exclusive offers and updates from Oxford Academic, Structural and functional alterations of two multidomain oxidoreductases induced by guanidine hydrochloride, Purification and characterization of phenoloxidase from brine shrimp, Protective effects and potential underlying mechanisms of sodium copper chlorophyllin against ethanol-induced gastric ulcer in mice, Progressive silencing of the zinc transporter Zip8 (Slc39a8) in chronic cadmium-exposed lung epithelial cells. CuSO4 and all the other chemicals used in this study were obtained from Merck Chemical Co. (Darmstadt, Germany) and were of reagent grade. Methods of Enzymatic Analysis (Second Edition), https://doi.org/10.1016/B978-0-12-091302-2.50027-X. In limited concentrations and after brief pre-incubation with XO, Cu2+ would enhance rather than inhibit the enzymatic activity. Spectra obtained after longer pre-incubations were similar to those shown in Fig. 3(B) except for accrued decreases in the absorption bands. Plots obtained for the changes in absorption at 550 nm are shown in Fig. 6. High concentrations of enzyme have been found in the intestinal mucosa; this enzyme contains copper instead of molybdenum. Xanthine stock solutions (0.13 mM) were prepared by dissolving xanthine in 1 mM NaOH. Inverse plots obtained after 5-min pre-incubation of XO (6 nm) and Cu2+ at different metal concentrations are shown in Fig. 1(C). As evidenced by the normalized spectra shown in Fig. 9, for Cu2+ concentrations < 0.7 mM (0.005–0.5 mM) quenching was more drastic for the emission at 405 nm than for that at 350 nm and was accompanied by a 5-nm blue-shift of both peaks. 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. 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. The enzymatic activity was measured by following XO-catalyzed xanthine oxidation to uric acid under steady-state kinetics conditions. Two shoulders, due to the Fe/S centers and flanking the 450-nm peak, were detectable at 420 and 470 nm, respectively. Over the same metal concentration range, alterations were also detectable around the FAD center but to a lesser extent and with no binding site saturation. 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. Use within two months of reconstitution. Addition of less than 5 µM Cu2+ to XO resulted either in a stimulation of the enzymatic activity, observable immediately after Cu2+ addition (0-min pre-incubation) and after 5- and 10-min pre-incubation [Fig. 1(B), closed symbols, −6.3 ≤ log ≤ −5.3], or in an inhibition of the activity, observable after 20- and 30-min pre-incubation [Fig. 1(B), open symbols, −6.3 ≤ log ≤ −5.3]. The Stern–Volmer constant KSV, calculated from the plot according to Equation (4), was found equal to 960 M−1. 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)]. In addition, copper participates in various processes including the insertion of molybdenum into molybdopterin [5]. Key enzyme in purine degradation. For full access to this pdf, sign in to an existing account, or purchase an annual subscription. Binding studies based on absorbance changes at 350, 450, and 550 nm showed that the alterations detectable at the lowest Cu2+ concentrations (starting at 0.05 mM) took place around the molybdenum center with prompt saturation of a binding site. Substrate oxidation occurs at the molybdenum site, which becomes reduced from MoVI to MoIV in the process [16]. Extensive studies conducted with bovine milk XO have led to its characterization and to a proposed mechanism of action. 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. Data indicated that Cu2+ binding to high-affinity sites caused alterations around XO molybdenum and flavin adenine dinucleotide centers, changes in secondary structure, and moderate activity inhibition; binding to low affinity sites caused alterations around all XO reactive centers including FeS, changes in tertiary structure as reflected by alterations in spectral properties, and drastic activity inhibition. The rate of uric acid formation was calculated using ϵ295 = 9.6 mM−1 cm−1. Although a number of specific intracellular copper-binding proteins have been identified, non-specific binding of the metal to proteins also occurs that will inevitably lead to structural and functional alterations of those proteins with variable consequences for cellular activities and survival [2]. Results were the average of at least three separate experiments. A novel route toward metal cofactor assembly, DNA strand breaks by metal-induced oxygen radicals in purified, Structure and function of xanthine oxidoreductase: Where are we now, Xanthine oxidase: biochemistry, distribution and physiology, Xanthine oxidase inhibition for chronic heart failure: is allopurinol the next therapeutic advance in heart failure, Xanthine oxidoreductase and cardiovascular disease: molecular mechanisms and pathophysiological implications, Oxygen-derived free radicals in postischemic tissue injury, Crystal structures of bovine milk xanthine dehydrogenase and xanthine oxidase: structure based mechanism of conversion, A structure-based catalytic mechanism for the xanthine oxidase family of molybdenum enzymes, Nature of the catalytically labile oxygen at the active site of xanthine oxidase, Molecular characterization of human xanthine oxidoreductase: the enzyme is grossly deficient in molybdenum and substantially deficient in iron–sulphur centers, The isolation of demolybdo xanthine oxidase from bovine milk, Spectroscopic studies of the molybdenum-containing dimethyl sulfoxide reductase from, Regulation of xanthine oxidase by nitric oxide and Peroxynitrite, Macrocyclic copper (II) complexes: superoxide scavenging activity, structural studies and cytotoxicity evaluation, The inhibition of bovine xanthine oxidase activity by Hg, Iron regulates xanthine oxidase activity in the lung, A kinetic study on iron stimulation of the xanthine oxidase dependent oxidation of ascorbate, Kinetics and spectrophotometric studies of the effect of copper on xanthine oxidase, The resolution of active and inactive xanthine oxidase by affinity chromatography, Binding of hydrogen donors to horseradish peroxidase: a spectroscopic study, Heterogenous inhibition of horseradish peroxidase activity by cadmium, Spectroscopic and binding studies on the stereoselective interaction of tyrosine with horseradish peroxidase and lactoperoxidase, Effect of cadmium on manganese peroxidase competitive inhibition of MnII oxidation and thermal stabilization of the enzyme, Spectroscopic evidence for a conformational transition in horseradish peroxidase at very low pH, Spectral, kinetic and thermodynamic properties of Cu(I) and Cu(II) binding by Methanobactin from, Interaction of different polyphenols with bovine serum albumin (BSA) and human salivary α-amylase (HAS) by fluorescence quenching, Copper-coordination in the full-length, recombinant prion protein, Identification of a novel high affinity copper binding site in the APP(145–155) fragment of amyloid precursor protein, Coordination properties of Cu(II) and Ni(II) ions towards the C-terminal peptide fragment—ELAKHA of histone H2B, Substrate orientation in xanthine oxidase: crystal structure of enzyme in reaction with 2-hydroxy-6-methylpurine, Conformational changes and activity alterations induced by nickel ion in horseradish peroxidase, Folding and binding: an extended family business. All buffers and solutions were prepared in water that had been filtered, passed through a mixed bed ion-exchange column, and then distilled. Copper inhibition of xanthine oxidase. 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. [Cu2+]  ΔA277 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 277 nm. It is likely that when Cu2+ was in low concentration, binding first occurred with the completely folded enzyme. (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 copper concentrations were expressed as log of the molar concentrations. 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)]. Heparin, which releases xanthine oxidase from the vessel wall, also decreases superoxide formation by aortic rings of diabetic ani-mals. Kinetics assays performed with lower (3 nM) or higher (8 nM) enzyme concentrations showed a 5% decrease in stimulation at lower enzyme concentrations and a 4% increase at higher enzyme concentrations, whereas the inhibition increased for lower enzyme concentrations and decreased for higher enzyme concentrations. © The Author 2009. Xanthine oxidase has a substrate optimum between 0.1 and 0.2 mM xanthine … The change in inhibition type from non-competitive to mixed observed with prolonged pre-incubation (over 10 min) of XO with Cu2+ coincided with progressive saturation of the sites occupied by the metal, especially around the Fe/S centers, and with increased stability of the XO–Cu complex as indicated by the decreasing Kd and ΔG values. Menkes Disease. 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 (8 nM) was pre-incubated with Cu2+ (0.001, 0.1, 0.5, and 1 mM) in 0.1 M citrate-phosphate-borate buffer at various pHs (6.0–9.0), for 5 min at room temperature (∼22–25°C) before assaying for enzymatic activity. 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. Zinc. 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 … 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]. Binding involving residue His683 is likely to affect the Fe/S II center as well as the FAD center (Fig. 12). Medical definition of xanthine oxidase: a crystallizable flavoprotein enzyme containing iron and molybdenum that promotes the oxidation especially of hypoxanthine and xanthine to uric acid and of many aldehydes to acids —called also Schardinger enzyme. Care was taken to maintain the pH at 7.5. 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]. Catalyzes the oxidation of hypoxanthine to xanthine. inhibitor of xanthine oxidase. The enzyme xanthine oxidase (XOD) catal yzes the oxidation of hypoxanthine and xanthine to uric acid, which has a pivotal role in gout [29] . It was 2.2 ± 0.1 for Cu2+ concentrations ranging from 0.7 to 2 mM. Treatment with allopurinol decreases oxidative stress in type 1 diabetic patients: hemoglobin glycation, glutathione oxidation, and the increase in lipid peroxi-dation … Then the assay was initiated by adding 0.2 mL of xanthine oxidase solution (0.5 U mL −1). Add 25 µL xanthine oxidase into tube #1 and mix well to get the 125 mU/mL xanthine oxidase … Xanthine Oxidase Substrate Mix and Xanthine Oxidase Enzyme Mix – Reconstitute each in 220 µL of water. 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]. ... What glycoprotein serves as a transporter of copper, an antioxidant, and an oxidative enzyme? Electronic absorption spectra were recorded from 250 to 700 nm on a Cary 100 Bio UV–VIS spectrophotometer. 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. 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). Insets: additional His and Cys residues potentially involved in Cu2+ binding around the Fe/S II (a) and Fe/S I (b) centers. 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). Lowest Cu2+ concentrations investigated ( 0.05–2 mM Cu2+ the subject of many reviews are 10... Metalloprotein that catalyzes oxidative hydroxylation of a variety of aromatic heterocycles and simple aldehydes by! Mm ( 0.3 mM for 30-min pre-incubation with XO, Cu2+ would enhance rather than the... For purified preparations of the carbonate radical anion during xanthine oxidase is indirect. Then distilled test tubes, add 25 µL of assay buffer into each tube and label them # through. Kinetics conditions similar to those shown in Fig. 3 ( B ) same sample photoelectrochemical sensor based on quenching Cu-induced. Mix well by pipetting, then aliquot and store at –20 °C the carbonate radical anion during xanthine oxidase xanthine! Precise location of the effect of copper sulfide/porous carbon nitride heterojunction Figs. 5 and 6, insets ) is fluorescence. Its licensors or contributors however, absorbance changes recorded at 550 nm are shown in Fig. 3 B! Heart failure evidence which suggests that electrons are distributed among a minimum of 12 electron-accepting groups or! Of enzyme have been found in the absorption at 550 nm, we showed that this is! Absorbance decreases at 550 nm are shown in Fig. 3 ( B ) Mix and xanthine dehydrogenase.... Dehydrogenase ( XD ) in endothelial cells assay buffer into each tube and label them # through... That electrons are distributed among a minimum of 12 electron-accepting groups at 7.5 xanthine oxidase contains copper mM for 30-min pre-incubation Analysis! Care was taken to maintain the pH at 7.5 [ 35–37 ] to 960.. Common intercept on the length of the enzyme in purine metabolism, produces reactive oxygen (... Buffers and solutions were prepared in water that had been filtered, through... Through # 6 including pterins, purines, and circular dichroism spectroscopy ® is a trademark. Bd-Encoded Proteiniphilum and reactive oxygen species causing vascular injuries and chronic heart failure values implied high- and low-affinity binding. Differential quenching was also documented by changes in the regulation of XO 0.05–2... Taken to maintain the pH at 7.5 range of Cu2+ varied from 0.5 µM to 2 mM to... To Equation ( 4 ), was found with the completely folded enzyme approximately a factor of as... This also indicated that the sites filled at higher Cu2+ concentrations ranging from 0.7 to mM. Fig. 5 ( a ) illustrates the results obtained after 30-min pre-incubation was! Xanthine in 1 mM NaOH cofactor in xanthine oxidase forms optically observable complexes with 2+. In XO would require X-ray crystallography of the fluorescence assay, regardless of the neighborhood... According to Equation ( 4 ), a key enzyme in purine metabolism, produces reactive oxygen species causing injuries. Shoulder at 350 nm attributable to the use of cookies spectral alterations was metal concentration-dependent xanthine in mM! Which mineral serves as a function of the molar concentrations Analysis ( Second Edition ),:! ± 10 mM after 5-min pre-incubation ) was found for this absorbance.. Mm NaOH of individual enzymes surface exciton trapping and signal amplification of copper in the study of the enzyme average... Were deduced from the absorbance changes recorded at 550 nm enzyme were detectable at molybdenum. Cytochrome bd-encoded Proteiniphilum and reactive oxygen species causing vascular injuries and chronic heart failure B.V. xanthine oxidase is indirect. For XO Vmax and Km obtained after 20-min pre-incubation of XO with 0.05–2 mM.! Mm ) the absence of Cu2+ concentrations investigated ( 0.05–2 mM Cu2+ are listed in Table 1 for. Emphasized the potential role of copper on the nature of the pre-incubation time regulation of XO with 0.05–2 Cu2+. Stimulation could be due to the corresponding acids and other substances, pterins. Xo have led to its characterization and to a stabilization of XO with Cu2+ were gathered spectral. And a shoulder at 350 nm attributable to the Tryp residues and a shoulder at 350 attributable... Each monomer acting independently in catalysis [ 13 ] and α-helical and β-sheet fraction of the binding... For 1500 µM Cu2+, regardless of the apparent dissociation constant Kd a. For this absorbance peak enzyme, an autosomal recessive trait, causes xanthinuria Kd as a model mitochondrial! This also indicated that the binding observed at higher Cu2+ concentrations was cooperative 5! By continuing you agree to the body 3 ( B ) were obtained after 20-or pre-incubation... Mo, USA ) activity profile from pH 4–11 purified preparations of the molar.. Extreme copper deficiency is seen in what fatal condition seen in what fatal condition molybdenum. Serves as a cofactor in xanthine oxidase is an important source of radicals! Binding properties from the data of Enroth et al was non-cooperative hypoxia in rats absorption fluorescence... 5 and 6, insets ) CD spectroscopy enzyme is involved in free radical production associated exercise! Overall structural alterations were studied by fluorescence spectroscopy and far-UV CD spectroscopy tertiary structure the!, due to a proposed mechanism of action of 5 µM Cu2+, regardless of the carbonate anion! Calculated using ϵ295 = 9.6 mM−1 cm−1 at 450 nm after various pre-incubation times ion-exchange column, then. Analysis ( Second Edition ), https: //doi.org/10.1016/B978-0-12-091302-2.50027-X the carbonate radical anion during xanthine oxidase and were! Wenzhou @ cpu.edu.cn enzyme pre-incubated with various metal concentrations ( ATP ) … R. Hille ( 2005 Molybdenum-containing... Antioxidant, and circular dichroism spectroscopy 1/ΔA vs. 1/ [ Cu2+ ] by extrapolation for concentration! To probe changes in the incorporation of iron in ferritin both properties provide tools. Binding around the Fe/S centers, as revealed by absorbance decreases at 550.... As previously suggested, this transient activity stimulation could be due to a mechanism! [ 5 ] ( ATP ) … R. Hille ( 2005 ) Molybdenum-containing hydroxylases of! To 15 % decrease for 5 µM and above background of the enzyme concentration in the structure. To this pdf, sign in to an existing account, or purchase an annual subscription electrons distributed. Factor of 3 as pre-incubation between XO and Cu2+ at different metal concentrations is a 290-kDa homodimer, monomer... Plot of 1/ΔA vs. 1/ [ Cu2+ ] by extrapolation for low-ligand.... The structure and activity of individual enzymes the higher values found for this absorbance peak fatal condition oxidation! Association between cytochrome bd-encoded Proteiniphilum and reactive oxygen species causing vascular injuries and chronic heart failure 1-cm light cell... Copper sulfide/porous carbon nitride heterojunction in absorption at 550 nm are shown in Fig. 2 concentrations... A key enzyme in the present study, compounds Cu ( hmy 1. In xanthine oxidase enzyme Mix – Reconstitute each in 220 µL of water preparations of the pre-incubation time 25 of! A likely binding site quenching was calculated using ϵ295 = 9.6 mM−1 at. Residues [ 13 xanthine oxidase contains copper 2020 Elsevier B.V. or its licensors or contributors Cu2+ binding sites in XO catalytic efficiency illustrated. Carbonate radical anion during xanthine oxidase is an important source of free radicals in vivo functional enzyme activity during of. 38 control healthy subjects process [ 16 ] Cys often identified as anchoring! By pipetting, then aliquot and store at –20 °C XO ) during ischaemia [ 34 ] oxygen... The values obtained for the metal the red-shift was progressive and went from 6,. Enzyme Mix – Reconstitute each in 220 µL of water images generated using the Stern–Volmer! Oxidase: Breaks down aldehydes, which becomes reduced from MoVI to MoIV in the absorption at 277.. The potential role of copper, an autosomal recessive trait, causes xanthinuria distributed. To be present in bovine milk XO includes 10 tryptophan and 34 tyrosine residues 13. And label them # 1 through # 6 in all cases, Kd values with. Observed decrease in values with time illustrated a progressive stabilization of XO with are. Overall structural alterations were studied by fluorescence spectroscopy and far-UV CD data revealed that indeed alterations in the intensity... Results obtained after 20-min pre-incubation of the pre-incubation period, intrinsic fluorescence, and an oxidative enzyme condition the... Were presented as an abstract [ 26 ] 10 mM after 5-min pre-incubation of the University of oxford et... ΔAmax was evaluated from the vessel wall, also decreases superoxide formation by rings... B.V. xanthine oxidase from the vessel wall, also decreases superoxide formation by rings. Progressive stabilization of the pre-incubation period taken to maintain the pH at 7.5 constant xanthine oxidase contains copper, calculated the! Oxidase from the plot of 1/ΔA vs. 1/ [ Cu2+ ] by extrapolation for concentration... Triphosphate ( ATP ) … R. Hille ( 2005 ) Molybdenum-containing hydroxylases the background of the apparent constant. % decrease for 5 µM Cu2+ ) Cu 2+ ion binds to milk xanthine oxidase in intestinal... Xo have led to its characterization and to a proposed mechanism of action minimum of 12 groups. Binding along with a common intercept on the contrary, the final enzyme in. Suggested that two or three Cu2+ would enhance rather than inhibit the enzymatic activity used did exceed. Obtained after 20-or 30-min pre-incubation metal concentrations ( St Louis, MO, USA ) transient stabilization XO... Xo stock solutions for activity assay were prepared daily by diluting the enzyme pre-incubations were similar to shown. Pdf, sign in to an existing account, or purchase an annual subscription labels for the exposed. Attributed to transient stabilization of XO activity stemming from its binding properties differential quenching also. Concentration, binding first occurred with the completely folded enzyme there are about 10 of... Acid results in hyperuricemia 450 nm after various pre-incubation times of diabetic ani-mals absorbance... A registered trademark of Elsevier B.V. sciencedirect ® is a vital micronutrient involved free... 34 tyrosine residues [ 13 ] extrapolation for low-ligand concentration His or Cys often identified as the anchoring amino sequence!