Biocytin-specific 110-kDa biotinidase from human serum

Abstract

Biotinidase purification from human serum was performed under new protocol. With HPLC biotinidase assay instead of colorimetric method and using non-ionic surfactant, 110-kDa biotinidase was discovered and co-purified in addition to the previously identified 76-kDa biotinidase. This newly identified enzyme accounted for 5% of the total biotinidase activity. Protein core of 110 kDa was estimated as 72 kDa by use of N-glycanase and SDS-PAGE analysis, while that of the 76-kDa enzyme was estimated as 59 kDa. Total amino acid analysis indicated 30% higher absolute amounts of amino acids in 110-kDa enzyme. The following differences were observed from kinetic study: the 110-kDa enzyme showed a 10-fold lower K m value and a 9-fold higher kcat/K m value for biocytin than those of 76-kDa enzyme. Thus, 110-kDa enzyme is more likely to be the physiological biocytin hydrolase (biocytinase), since the biocytin concentration in human serum is extremely low. The pathogenesis of an inborn error of the metabolism such as a variant form of biotinidase deficiency, which presented an atypical clinical course, might be related to these isoenzymes in terms of their different roles in the body.