Superoxide radical production by allopurinol and xanthine oxidase
Introduction
Xanthine oxidoreductase (XOR) is implicated in generating reactive oxygen species (ROS) that contribute to the biology associated with ischemia and reperfusion injury as well as to the pathology of failing cardiac tissues [1], [2], reviewed in [3], [4], [5]. XOR exists as either an oxidase (XO) that transfers reducing equivalents to oxygen, or as a dehydrogenase (XDH) that utilizes NAD or oxygen as the final electron acceptor [3], [6], [7], [8], [9]. Both forms contain an internal electron transport system that is capable of producing ROS [7], [10], [11]. The physiological substrates, xanthine and hypoxanthine, bind oxidized enzyme and donate two electrons into the molybdenum cofactor reducing it from MoVI to MoIV. Substrates are hydroxylated by H2O at the molybdenum site as the electrons travel via two iron-sulfide residues to flavine–adenine dinucleotide (FAD). Reduced FAD can be divalently reoxidized by oxygen to produce hydrogen peroxide, or univalently reoxidized in two steps to generate two equivalents of superoxide radical (reviewed in [3], [12], [13], [14]). Superoxide in particular has been identified as the probable reactive oxygen species that contributes to cardiac dysfunction in failing myocardium [1], [2], reviewed [3], [4], [5].
Several studies in tissue culture, isolated hearts, laboratory animals and in cardiac patients have indicated that the XOR inhibitors, allopurinol and oxypurinol, are useful in preventing the formation of superoxide and improving cardiac function [1], [2], reviewed in [3], [4], [5]. However, allopurinol by itself cannot prevent the generation of superoxide by XOR [15]. It is an efficient alternative substrate of XOR that must first be converted to oxypurinol, the actual inhibitor [16], [17], [18], [19], [20], [21], [22]. In contrast to allopurinol, which binds to the oxidized MoVI, oxypurinol binds to reduced MoIV. The reduced XOR–oxypurinol complex then slowly rearranges into a tightly bound inhibitory complex. Consequently, during the course of the reaction (with and without the physiological substrates) the rate of product formation prematurely decelerates as the enzyme becomes strongly inhibited by oxypurinol.
Although the inhibition appears to be irreversible, oxypurinol can slowly dissociate from XOR. Oxypurinol is referred to as a pseudo irreversible inhibitor that “inactivates” the enzyme.
In 1970 [18], allopurinol was shown to reduce cytochrome C while being oxidized by XO. A contemporary study demonstrated that the reduction of cytochrome C by xanthine and XO was mediated by superoxide [11]. Because allopurinol may be generating superoxide, we have studied the reaction of allopurinol with XO and confirmed that allopurinol does produce superoxide during its conversion to oxypurinol. We also examined the variables that determine the quantity of superoxide formed during the reaction.
Section snippets
Materials
Xanthine, allopurinol, uric acid, 8-methyl xanthine, EDTA disodium salt and horse heart Cytochrome C type III were purchased from Sigma (St. Louis, MO). Oxypurinol was obtained from Sigma (St. Louis, MO) and from DSM Pharmaceuticals (Greenville, NC). Analytical grade phosphate buffer, acetonitrile, tetrabutylammonium hydrogen sulfate and trichloroacetic acid were purchased from Fisher. Amicon ultrafree centrifugal filter devices 5000 NMWL were from Millipore Corporation (Bedford, MA). Catalase
Superoxide production by xanthine and allopurinol
The production of superoxide from XO was monitored spectrophotometrically by following the reduction of cytochrome C in reactions at pH 6.8 and 37 °C. The data in Fig. 1 shows that during the complete conversion of 20 μM xanthine to uric acid (confirmed by the direct spectral assay), approximately 5 μM superoxide was formed. This represents approximately 13% of the total electron flux.1
Discussion
The XO-catalyzed conversion of allopurinol to oxypurinol clearly generates superoxide during the reaction. Furthermore, the complete conversion of allopurinol to oxypurinol or xanthine to uric acid produced similar amounts of superoxide. Ironically, allopurinol is one of the inhibitors of XO commonly studied to prevent its production of superoxide, which is deleterious to inflamed tissue [1], [2] (reviewed in [3], [4], [5]). We found that the amount of superoxide produced depended on the
Acknowledgements
We gratefully acknowledge Dr. Russ Hille for providing the xanthine oxidase used in these studies and for helpful discussion and Dr. Alan Ezrin for contributions and guidance.
References (33)
- et al.
Xanthine oxidase activities: evidence for two catalytically different types
Arch Biochem Biophys
(1978) - et al.
The regulation of rat liver xanthine oxidase. Conversion in vitro of the enzyme activity from dehydrogenase (type D) to oxidase (type O)
J Biol Chem
(1969) - et al.
Purification and properties of the NAD+-dependent (type D) and O2-dependent (type O) forms of rat liver xanthine dehydrogenase
Arch Biochem Biophys
(1976) - et al.
The reaction of reduced xanthine dehydrogenase with molecular oxygen. Reaction kinetics and measurement of superoxide radical
J Biol Chem
(1997) - et al.
Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein)
J Biol Chem
(1969) Molybdenum-containing hydroxylases
Arch Biochem Biophys
(2005)Oxypurinol as an inhibitor of xanthine oxidase-catalyzed production of superoxide radical
Biochem Pharmacol
(1988)- et al.
On the mechanism of inactivation of xanthine oxidase by allopurinol and other pyrazolo[3,4-d]pyrimidines
J Biol Chem
(1970) - et al.
Stoichiometric inhibition of reduced xanthine oxidase by hydroxypyrazolo [3,4-d]pyrimidines
J Biol Chem
(1970) - et al.
4-Hydroxypyrazolo (3,4-d)pyrimidine as a substrate for xanthine oxidase: loss of conventional substrate activity with catalytic cycling of the enzyme
Biochem Biophys Res Commun
(1970)
Inhibition of urate production by allopurinol
Biochem Pharmacol
Human and bovine xanthine oxidases. Inhibition studies with oxipurinol
Biochem Pharmacol
The reductive half-reaction of xanthine oxidase. The involvement of prototropic equilibria in the course of the catalytic sequence
J Biol Chem
Studies on milk xanthine oxidase. Some spectral and kinetic properties
J Biol Chem
The microestimation of succinate and the extinction coefficient of cytochrome C
Biochim Biophys Acta
Quantitative aspects of the production of superoxide anion radical by milk xanthine oxidase
J Biol Chem
Cited by (63)
Febuxostat attenuates vascular calcification induced by vitamin D3 plus nicotine in rats
2021, European Journal of Pharmaceutical SciencesCitation Excerpt :Febuxostat (FEB) is a potent selective inhibitor of xanthine oxidase (XO) enzyme (Frampton, 2015). The XO system is a powerful generator of reactive oxygen species (ROS) in the vascular system (George and Struthers, 2009, Galbusera et al., 2006). In view of the central contribution of XO-induced oxidative stress to the initiation and progression of VC (Byon et al., 2008), it is conceivable that specific inhibition of XO in the vascular millieu by FEB may help in attenuating VC.
Protective effect of curcumin on chloroform as by-product of water chlorination induced cardiotoxicity
2014, Biomedicine and Preventive NutritionDesign of an electrochemical biosensing system for xanthine detection and a study on binding interaction of ketoconazole with xanthine oxidase
2014, Colloids and Surfaces A: Physicochemical and Engineering AspectsBiocompatible ligands modulate nanozyme activity of CeO<inf>2</inf> nanoparticles
2023, New Journal of Chemistry