xanthine oxidase contains copper

Then the assay was initiated by adding 0.2 mL of xanthine oxidase solution (0.5 U mL −1). Intrinsic fluorescence was detected on a Cary Eclipse Fluorescence spectrophotometer equipped with temperature controller. Insets: value of the apparent dissociation constants Kd1 and Kd2 as a function of the pre-incubation time. 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]. Has also low oxidase activity towards aldehydes (in vitro). ... What glycoprotein serves as a transporter of copper, an antioxidant, and an oxidative enzyme? The metal ion essential for the activity of xanthine oxidase and xanthine dehydrogenase is: Molybdenum. 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. Xanthine oxidase is an iron-molybdenum flavoprotein, which is containing FLAVIN-ADENINE DINUCLEOTIDE that oxidizes hypoxanthine, some other purines and pterins, and aldehydes. 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. Determination of the apparent dissociation constant of the metal–enzyme complex, based on the absorbance changes observed for the molybdenum center and for the FAD center, revealed two constants (Kd1 and Kd2) in each case, indicating the existence of sites with lower affinity for Cu2+ that would be filled only after the metal concentration reached the critical value of 0.7 mM. Cooperative binding along with a single Kd value were deduced from the absorbance changes observed for the Fe/S centers. Measurements were done using a 1-mm light path cell for far-UV studies and a 1-cm light path cell for visible studies. 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 differential quenching was also documented by changes in the fluorescence intensity ratio I405/I350 (Fig. 9, inset). Cu2+ concentrations varied from 0.001 to 2 mM. To determine the precise location of the Cu2+ binding sites in XO would require X-ray crystallography of the metal–enzyme complex. The enzymatic activity was measured by following XO-catalyzed xanthine oxidation to uric acid under steady-state kinetics conditions. [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. In limited concentrations and after brief pre-incubation with XO, Cu2+ would enhance rather than inhibit the enzymatic activity. The xanthine dehydrogenase (XD) will be converted into xanthine oxidase (XO) during ischaemia [34]. Dialysis of the enzyme pre-incubated with various metal concentrations resulted in at least partial restoration of the enzymatic activity. Published by Elsevier Inc. All rights reserved. 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. 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. 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. A close examination of the enzyme structure reveals that XO exhibits a number of potential binding sites for Cu2+ in the vicinity of each of its 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. 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. Reference Bonini MG. Production of the carbonate radical anion during xanthine oxidase turnover in the presence of … 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. In parallel with the kinetics results, filling of lower affinity sites led to complete inactivation of the enzyme, while filling of higher affinity sites resulted in limited inhibition of the enzymatic activity. Conflict of interest statement. 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)]. 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]. Inset: control XO activity profile from pH 4–11. 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. The xanthine oxidase inhibitory effects for both watermelon and allopurinol were also stated in the term of IC 50, which is represent the concentration of standard drug or tested sample that is required for 50% inhibition of xanthine oxidase activity under the same experimental conditions. Milk contains divalent copper ions, and ascorbic acid can reduce Cu 2+ to Cu +, which is a strong inhibitor of the enzyme. 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. (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+. 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