Oxalate Oxidase from Barley Roots: Purification to Homogeneity and Study of Some Molecular, Catalytic, and Binding Properties

Oxalate oxidase (OXO) was purified to homogeneity in three steps from roots of barley seedlings. The purification method comprised: (i) thermal treatment (60°C, 10 min), (ii) affinity chromatography on immobilized either Procion turquoise MX-G dye or biomimetic aminoethyl oxamic blue dye, and (iii) affinity chromatography on immobilized lectin concanavalin A (overall performance: 1096-fold purification, 42% recovery). The purified enzyme has a specific activity of 34 U mg−1(25°C), and is a homopentamer ofMr≈125,000 (HPLC analysis) showing a single band on SDS–polyacrylamide gel electrophoresis (Mr≈26,000) after staining with silver nitrate. The kinetic constants of the purified enzyme for oxalate areKm0.27 mmandkcat22 s−1(37°C), whereas at [oxalate] ≥ 4 mmthe enzyme exhibited substrate inhibition. Barley root OXO contains no prosthetic group absorbing at 370 or 450 nm, and riboflavin and FAD have no effect on its activity. The enzyme is activated by 1 mmeach of Ca2+(1.7-fold) and Pb2+(2.6-fold). Irreversible inactivation studies with denatured (70°C) and native (37°C) enzyme using the sulfhydryl-attacking reagent 5,5-dithiobis(2-nitrobenzoic) acid (1.4 mm), in the presence and absence of SDS, respectively, have shown that denatured OXO (4% SDS, 10 min, 100°C) exhibited 10 HS groups per molecule, whereas native OXO displayed one accessible HS group per molecule after approximately 15 min incubation and, over the same period, maintained its catalytic activity to 90%. Furthermore, native OXO treated with β-mercaptoethanol (1 mm) lost 83% of its catalytic activity within 5 min. These findings indicate that some cysteines may preserve the catalytic activity of OXO by maintaining the integrity of its tertiary structure via disulfide bond formation.