Xanthine Accumulation during Hypoxanthine Oxidation by Milk Xanthine Oxidase

Abstract

Accumulation of xanthine as well as uric acid, was found as a result of hypoxanthine oxidation by xanthine : oxygen oxidoreductase from milk. The ratio xanthineluric acid increases with hypoxanthine concentration, and decreases with the enzyme concentration and the reaction time. The methodical consequences of the xanthine accumulation during hypoxanthine oxidation are discussed. The possibility of regulation of the xanthineluric acid ratio in the cell by changes of enzyme and/or hypoxanthine levels is suggested.Hypoxanthine oxidation (hydroxylation) in living cells is believed to be a two-step reaction :Hypoxanthine --, Xanthine -Uric Acid catalysed by one enzyme, xanthine oxidase.suggested that a pool of xanthine may arise in vivo during hypoxanthine oxidation. Transient accumulation of xanthine by the milk enzyme in vitro was reported by Bergmann and Dikstein [2]. Xanthine is also accumulated by the action of xanthine dehydrogenase from chicken [3] and from snail, Helix pomatiu [4], on hypoxanthine. It seems that this enzyme may play some role in the regulation of the xanthineluric acid ratio in living organisms. The present study was undertaken with the aim of obtaining some information about the pools of these two purines arising in the course of hypoxanthine oxidation.The model enzyme, xanthine : oxygen oxidoreductase from milk, was used for this purpose. I I1 MATERIALS AND METHODS ReagentsXanthine oxidase (milk) preparations were purchased from P-L Biochemicals, Inc. (Milwaukee, Vis., U.S.A.) and Koch-Light Laboratories Ltd (Colnbrook, Bucks, England) ; they were used without further purification. The ratios of absorbance a t 280 and 450 nm for these preparations were found to be 11.6 and 9.0, respectively; the preparations were stored a t 4 "C and were diluted 1 :lo0 with 0.03 M Tris-RC1 buffer pH 8.0, before use. Highly purified milk xanthine oxidase (batch 31 S, A::? = 29, Enzyme. Xanthine oxidase or xanthine : oxygen oxidoreductase (EC 1.2.3.2).act./A,,, = 159) was the kind gift of Dr R.

Substrate Orientation and Catalytic Specificity in the Action of Xanthine Oxidase

“…We attribute this to the much greater extent to which xanthine competes with hypoxanthine at pH 7.0 relative to pH 8.5 (K m xanthine is 13 times smaller than K m hypoxanthine at pH 7.0, but the K m values are comparable at pH 8.5). These results place on a more quantitative basis the earlier results of Jezewska (32), who also observed significant accumulation of xanthine in the course of enzyme action on hypoxanthine.…”

Hypoxanthine-based enzymatic sensor for determination of pork meat freshness

“…This percentage was similar at all the temperatures tested (data not shown) within of 8.48% (CV). The study of Hx oxidation is complicated, due to the oxidative degradation of both, Hx and X occurs simultaneously (Escribano, García-Cánovas, & García-Carmona, 1988) although the accumulation of X during the enzymatic reaction is a reliable proof that the catalytic affinity of the XO is bigger for Hx than for X as already observed (Jezewska, 1973).…”

“…For this reason it is necessary to dilute the meat sample appropriately to fit the linear detection range of the enzyme sensor and to obtain a good quantification of Hx. High concentrations of Hx can inhibit the enzyme by excess of substrate or product or by competition between the Hx and X by the active sites of the enzyme (Jezewska, 1973). Readings greater than 1 mg/L of consumed oxygen or enzyme rate of 0.064 mg/L * s À1 during the enzymatic reaction suggest an excess of Hx.…”

A new multi-wavelength model-based method for determination of enzyme kinetic parameters

“…Therefore, R is defi ned as a function of nonlinear parameters only. Since 1973, several different methods have been developed and are available for the fi tting of any number of parameters to a given measurement (Jezewska 1973;Lopez et al 2002). According to Maeder and Zuberbühler (1990), these methods can be classifi ed into two groups: (a) direct methods, where the sum of squares, ssq, of R is minimized, and (b) the Newton-Gauss-Levenberg/Marquardt (NGL/ M) method, where the residuals, R, are used to guide the iterative process toward the minimum.…”