Characterization of a uricase from Bacillus fastidious A.T.C.C. 26904 and its application to serum uric acid assay by a patented kinetic uricase method.

An intracellular uricase from Bacillus fastidious A.T.C.C. 26904 was characterized and evaluated for serum uric acid assay by a patented kinetic uricase method. The active uricase was 151 kDa by gel filtration through Sephadex G-200. Both SDS/PAGE and matrix-assisted laser-desorption ionization-time-of-flight MS resolved a single polypeptide with a molecular mass of approx. 36.0 kDa. The N-terminal sequence was AERTMFYGKGDV. The optimum pH for this uricase ranged from 9.0 to 10.5. At pH 9.2, the Km (Michaelis-Menten constant) was 204+/-14 micromol/l (n=8) and the Ki (inhibition constant) for xanthine was 41+/-7 micromol/l (n=5). By analysing the data monitored within 5 min at 0.03 unit/ml uricase, this kinetic uricase method gave linear response to uric acid in reaction solution from 1.3 to 60 micromol/l. Aside from other common errors, 30 micromol/l xanthine in the reaction solution caused no error in this kinetic uricase method, while it caused negative error in the indirect equilibrium method by peroxidase-coupled assay of H2O2. Uric acid in clinical sera by this kinetic uricase method (Ck) closely and positively correlated with that from the indirect equilibrium method (Ce) (Ck = 0.008+1.081 x Ce, r>0.990, n=99). However, Bland-Altman analysis suggested inconsistency between Ck and Ce. These results indicated that this kinetic uricase method using this uricase was reliable for serum uric acid assay with enhanced resistance to xanthine besides other common errors.