The nature of catalytic activities of milk xanthine oxidase

Abstract

The widely accepted view that xanthine oxidase is a flavoprotein whose flavine prosthetic group is alternately reduced by the substrate and oxidized by the hydrogen acceptor has not been firmly established experimentally. It rests almost entirely on the observations of Ball (1939a) and Corran, Dewan, Gordon & Green (1939) that preparations of the enzyme from milk contain flavin-adenine dinucleotide (FAD). A number of investigators (Ball, 1939a; Corran et al. 1939; Horecker & Heppel, 1949; Kalckar, Kjeldgaard & Klenow, 1950) have found that the FAD in xanthine oxidase preparations is reduced when xanthine or hypoxanthine is added under anaerobic conditions, but the reduction is incomplete and, in the only study in which the rate was measured, the authors (Corran et al. 1939) concluded that the reduction was too slow to account for the catalytic activity of the enzyme. Ball (1939a, b) suggested that xanthine oxidase contained a second prosthetic group in addition to FAD. He claimed to have split the enzyme reversibly into its apo-enzyme and prosthetic group moieties by prolonged dialysis. The apo-enzyme was reactivated by the supernatant obtained by heating and centrifuging an enzyme preparation but not by pure FAD. Since the supernatant solution contained FAD, Ball suggested that the active enzyme contained as prosthetic groups both FAD and an additional component present in the heated enzyme preparation. However, he pointed out that it was possible that this additional component would be active on its own. The significance of Ball's findings has been questioned by Kalckar et al. (1950) who found that the reactivation obtained was due to the presence in the heated supernatant of sulphydryl compounds which removed metal ions acquired by the intact enzyme during dialysis. Thus, the reversible splitting of xanthine oxidase into apoenzyme and prosthetic group has not yet been achieved. Corran et al. (1939) believed that the typical reddish tinge of the enzyme was due to a second prosthetic group. This view, however, has since been criticized by Lowry, Bessey & Crawford (1949), who have suggested that since the red tinge disappears entirely when the enzyme is denatured, it may be

Properties of Xanthine Dehydrogenase Variants from Rosy Mutant Strains of Drosophila melanogaster and their Relevance to the enzyme's Structure and Mechanism

“…Another possible inactive contaminant is the demolybdo enzyme (Bray, 1975). Measurement of the extent of bleaching by xanthine of the ultraviolet-visible spectrum of samples of the purified enzyme may be used (Morell, 1952) to estimate the proportion of the molecules that are in the functional form. For the Drosophila xanthine dehydroganase variants, these measurements (see Materials and Methods) were technically difficult because of the small amounts available, because of the difficulty of concentrating the purified enzymes and because of the instability of [G101 1EIxanthine dehydrogenase.…”

Expression of Drosophila melanogaster xanthine dehydrogenase in Aspergillus nidulans and some properties of the recombinant enzyme

“…However, a more direct way to measure the functionality of XDH is to determine the extent to which the UV\visible spectrum of the enzyme is bleached immediately on addition of xanthine. This provides a measure of the proportion of the enzyme molecules in the functional form [33][34][35]. Whereas precision of the measurements of bleaching at 450 nm was limited by the amounts of enzyme available, and the experimental conditions may not have been optimal, our data (Table 2) suggest, in contrast to the activity data, that our preparation of recombinant enzyme contains only some 40 % functional enzyme.…”

Nature and Function of Metalloflavoproteins

“…Highly active preparations have been obtained in a number of laboratories ( [14][15][16][17][18]. The starting material for these various procedures is either buttermilk or cream.…”

“…This reduction of the bound flavin appears to proceed in two steps: an initial rapid but incomplete phase (15), followed by a slow and complete reduction of all the FAD (17). Morrell has explained this phenomenon by assuming two forms of the enzyme (variable from preparation to preparation), one with catalytically active FAD which is rapidly reduced; the other is catalytically inert but possesses a prosthetic group still slowly reducible by the FAD of the active species (17). On treatment and aging, the first variety would be transformed into the second.…”

“…(15,18,21) though it is apparently incapable of bleaching the FAD (17). The latter phenomenon is probably referable to the very low redox potential of the enzyme (18).…”