BIOCYTIN-SPECIFIC 110-KDA BIOTINIDASE FROM HUMAN SERUM

Biotinidase purification from human serum was performed under new prot ocol. With HPLC biotinidase assay instead of colorimetric method and u sing non-ionic surfactant, 110-kDa biotinidase was discovered and co-p urified in addition to the previously identified 76-kDa biotinidase. T his 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 e stimated as 59 kDa. Total amino acid analysis indicated 30% higher abs olute amounts of amino acids in 110-kDa enzyme. The following differen ces were observed from kinetic study: the 110-kDa enzyme showed a 10-f old 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 biocyti n concentration in human serum is extremely low. The pathogenesis of a n inborn error of the metabolism such as a variant form of biotinidase deficiency, which presented an atypical clinical course, might be rel ated to these isoenzymes in terms of their different roles in the body .