The composition of milk xanthine oxidase

Abstract

The composition of milk xanthine oxidase has been reinvestigated. When the enzyme is prepared by methods that include a selective denaturation step in the presence of sodium salicylate the product is obtained very conveniently and in high yield, and is homogeneous in the ultracentrifuge and in recycling gel filtration. It has specific activity higher than previously reported preparations of the enzyme and its composition approximates closely to 2mol of FAD, 2g-atoms of Mo and 8g-atoms of Fe/mol of protein (molecular weight about 275000). In contrast, when purely conventional preparative methods are used the product is also homogeneous by the above criteria but has a lower specific activity and is generally comparable to the crystallized enzyme described previously. Such samples also contain 2mol of FAD/mol of protein but they have lower contents of Mo (e.g. 1.2g-atom/mol). Amino acid compositions for the two types of preparation are indistinguishable. These results confirm the previous conclusion that conventional methods give mixtures of xanthine oxidase with an inactive modification of the enzyme now termed ;de-molybdo-xanthine oxidase', and show that salicylate can selectively denature the latter. The origin of de-molybdo-xanthine oxidase was investigated. FAD/Mo ratios show that it is present not only in enzyme purified by conventional methods but also in ;milk microsomes' (Bailie & Morton, 1958) and in enzyme samples prepared without proteolytic digestion. We conclude that it is secreted by cows together with the active enzyme and we discuss its occurrence in the preparations of other workers. Studies on the milks of individual cows show that nutritional rather than genetic factors determine the relative amounts of xanthine oxidase and de-molybdo-xanthine oxidase. A second inactive modification of the enzyme, now termed ;inactivated xanthine oxidase', causes variability in activity relative to E(450) or to Mo content and formation of it decreases these ratios during storage of enzyme samples including samples free from demolybdo-xanthine oxidase. We conclude that even the best purified xanthine oxidase samples described here and by other workers are contaminated by significant amounts of the inactivated form. This may complicate the interpretation of changes in the enzyme taking place during the slow phase of reduction by substrates. Attempts to remove iron from the enzyme by published methods were not successful.

Purification of highly active milk xanthine oxidase by affinity chromatography on sepharose 4B/folate gel

“…The precipitated enzyme was dissolved in a minimal volume of 0.1 M pyrophosphate (pH 8.5) containing 0.2 mM EDTA and was passed through a Sephadex G-25 column equilibrated with the same buffer. The AFR 25°C value of the pooled enzyme was 195, as shown in table 1, a value which is very close to the maximum values reported in [2,4,6]. On the other hand, the AFR 25°C value of the pooled inactive fractions was 38.1.…”

“…Two forms of non-functional enzyme were subsequently indicated: one form containing a full complement of the oxidation-reduction groups;and another form lacking molybdenum [2,4,5]. However, final proof of the correctness of Morell's hypothesis came with the resolution of the active and inactive forms of the enzyme by affinity chromatography [6].…”

“…It is well known that an inactive form of xanthine oxidase, the desulfo-form, is present in milk xanthine oxidase preparations, in addition to an inactive form lacking molybdenum [1][2][3][4]. The existence of the desulfo-form in the enzyme preparations is considered to be caused by spontaneous release of sulfur during storage or purification.…”

The (2R)‐hydroxycarboxylate‐viologen‐oxidoreductase from Proteus vulgaris is a molybdenum‐containing iron‐sulphur protein

“…This was described for other molybdoenzymes, such as the quinoline-4-carboxylic acid oxidoreductase from Agrobacterium sp. 1 B (Bauder and Lingens, 1992) and the xanthine oxidases from human (Abadeh et al, 1992) and cow milk (Hart et al, 1970). The isolation of about 0.7 mol pterin-6-carboxylic acid after permanganate oxidarion of the HVOR confirmed the assumption that it contains 1 mol molybdenum bound to a pterin-containing cofactor.…”

Purification of 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans. An iron-sulfur protein

“…Protein [20], non-heme iron [21], molybdenum [22], labile sulfur [23] and total flavin [24, 251 were determined by described procedures. Iron as well as Se, Co, Mg, Ca, Cu, Zn, Mn, Mo, Ni and V were also analysed by plasma emission spectroscopy (Plasma Scan 71 0, Labtest, D-4030 Ratingen).…”