Characterization of n 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 K m (Michaelis-Menten constant) was 204 ± 14 μmol/l (n = 8) and the K i (inhibition constant) for xanthine was 41 ± 7 μmol/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 μmol/l. Aside from other common errors, 30 μmol/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 H 2 O 2 . Uric acid in clinical sera by this kinetic uricase method (C k ) closely and positively correlated with that from the indirect equilibrium method (C e ) (C k = 0.008 + 1.081 x C e , r > 0.990, n = 99). However, Bland-Altman analysis suggested inconsistency between C k and C e . 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.