Xanthine Dehydrogenase from Leaves of Leguminous Plants: Purification, Characterization and Properties of the Enzyme

Abstract

Summary A high level of xanthine oxidoreductase purification was achieved from leaves of ten leguminous plants. Under native conditions the enzyme purified from Vicia faba, Lens esculenta, Cicer arietinum, Glycine max. and Phaseolus vulgaris appeared electrophoretically homogeneous while that from Lupinus albus, Trifolium repens and pratense, Vicia sativa, and Vigna unguiculata was accompained by one or two minor contaminating proteins. Xanthine oxidizing enzymes isolated from leaves of leguminous plants did not react with molecular oxygen at a significant rate, indicating that all of them are xanthine dehyrogenases (XDH) (EC 1.1.1.204, formerly EC 1.2.1.37.). Under (SDS)-denatured conditions one main subunit of about 130 kDa was revealed in Vicia faba and Lens esculenta XDHs while in Glycine max. and Phaseolus vulgaris XDHs there were 130 and 150 kDa subunits both occurring at about the same molar ratio. The visible absorption spectrum of the pure protein was between 312 and 390 nm, related to the molybdopterin component of the enzyme with a lower absorption from 420 to 510 nm mainly due to flavin chromophores and a negligible absorption close to 550 nm reflecting the absorption of the iron-sulphur centres. The fluorescence excitation spectrum showed peaks at 274 and 368 nm, similar to pterin and xanthopterin. Fluorescence emission spectrum was characterized by two maxima at about 400 and 460 nm, typical of flavin chromophores. Superoxide dismutase and catalase had a slight stimulatory effect on enzyme activity with NAD+ as acceptor. By contrast, catalase strongly enhanced the activity when artificial acceptors such as phenazine methosulphate and methylene blue were used. The effect of inhibitors, particularly p-hydroxy-mercuribenzoate and salicylhydroxamic acid, agree with those reported for other xanthine oxidases and dehydrogenases. The affinity for reducing substrates was rather high with Km values of 8-17 μmol/L while a wide Km range from 8-19 (PMS) to 106-267 μmol/L (methylene blue) was determined for oxidizing substrates. All XDHs were inhibited under anaerobiosis and by incubating the enzyme with the physiological electron donors xanthine and hypoxanthine, but these effects were reversed by a prolonged reaction time. Serological studies with polyclonal rabbit antisera against XDHs from Vicia faba, Lens esculenta and Phaseolus vulgaris indicated that all of the enzymes purified are more or less strictly related serologically.