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) ther mal treatment (60 degrees 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 lec tin concanavalin A (overall performance: 1096-fold purification, 42% r ecovery). The purified enzyme has a specific activity of 34 U mg(-1) ( 25 degrees C), and is a homopentamer of M-r approximate to 125,000 (HP LC analysis) showing a single band on SDS-polyacrylamide gel electroph oresis (M-r approximate to 26,000) after staining with silver nitrate. The kinetic constants of the purified enzyme for oxalate are K-m 0.27 mM and k(cat) 22 s(-1) (37 degrees C), whereas at [oxalate] greater t han or equal to 4 mM the 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 acti vated by 1 mM each of Ca2+ (1.7-fold) and Pb2+ (2.6-fold). Irreversibl e inactivation studies with denatured (70 degrees C) and native (37 de grees C) enzyme using the sulfhydryl-attacking reagent 5,5-dithiobis(2 -nitrobenzoic) acid (1.4 mM), in the presence and absence of SDS, resp ectively, have shown that denatured OXO (4% SDS, 10 min, 100 degrees C ) exhibited 10 HS groups per molecule, whereas native OXO displayed on e accessible HS group per molecule after approximately 15 min incubati on and, over the same period, maintained its catalytic activity to 90% . Furthermore, native OXO treated with beta-mercaptoethanol (1 mM) los t 83% of its catalytic activity within 5 min. These findings indicate that some cysteines may preserve the catalytic activity of OXO by main taining the integrity of its tertiary structure via disulfide bond for mation. (C) 1997 Academic Press.