Purification, characterization and clinical application of a membrane bound oxalate oxidase from leaves of Bougainvillea glabra

Abstract

A plastid membrane bound oxalate oxidase from mature leaves of Bougainvillea glabra has been solubilized by taurodeoxycholate (20 mg/mL) and purified upto apparent homogeneity as judged in PAGE, using 0-80% ammonium sulphate precipitation, Sephadex G-100 gel fliteration and DEAE-sephacel ion exchange chromatography. An overall purification of 60.47 fold with 31% yield was achieved. The molecular weight (Mr) of enzyme was 133 kDa with two subunitsof identical Mr (66.5 kDa). The purified enzyme showed optimum activity at pH 5.5 when incubated at 40°C for 4 min. The energy of activation (Ea) was 3.97 kCal/mol. The enzyme showed hyperbolic relationship with its substrate (oxalate) in the range 0.5 mM and 5 Mm. Km for oxalate and Vmax were 1.6 mM and 121 nmoles/H2O2/mL/min. Among the various metal tested onle CuSO4 stimulated the enzyme while NaCl, KCl, HgCl2, and CoCl2 inhibited the enzyme. Among the SH group inhibitors SDS, and#946;-mercaptoethanol, reduced glutathione, sodium dodecyl sulfate and p-chloromercuribenzoate inhibited the enzyme but iodoacetate had practically no effect affect the activity of enzyme. UV and visible spectra of purified enzyme did not show any peak at 370 nm and 450 nm, a characteristic of flavoprotein. Flavins such as riboflavin, FMN and FAD had practically no effect on enzyme indicating that the enzyme is not a flavoprotein. The enzyme gave reddish brown color when heated in orcinol-H2SO4 reagent confirming the glycoprotein nature of Bougainvillea glabra oxalate oxidase. The enzyme consisted of 0.43 mg of reducing sugar per mg of protein. The enzyme was employed for determination of oxalate in plasma and 24 h urine of apparently healthy and urinary stone formers. The enzyme was coupled with ethylene maleic anhydride (EMA) to shift its pH to near physiological pH, encapsulated into liposome and injected into peritoneal cavity of experimental hyperoxaluric rats fed with vitamin B6 deficient where it degraded C14 oxalate.