Biotinidase

Biotinidase hydrolyzes biocytin (ε-N-biotinyllysine) to the vitamin biotin and lysine. Biotinidase does not release biotin that is covalently bound to intact holocarboxylases. Carboxylases must be proteolytically degraded to biocytin or small biotinyl peptides before hydrolysis can occur. Biotinidase in blood probably originates from the liver. Biotinidase in serum is sialylated, but in rat liver from which blood is removed by perfusion with physiological saline, biotinidase is asialylated. Results of subcellular fractionation of rat hepatocytes suggest that the enzyme is enriched in the microsomal fraction and, to a lesser extent, in the lysosomal fraction. The simplest and most commonly used method for measuring biotinidase activity uses N-(d-biotinyl) 4-aminobenzoate (BPABA) as substrate. Hydrolysis of BPABA results in the liberation of biotin and 4-aminobenzoate (PABA). Biotinidase is assayed by measuring the hydrolysis of BPABA. The PABA released is diazotized, coupled to a naphthol reagent, and determined by its absorbance at 546 nm. The estimates of the molecular weight of biotinidase determined by gel filtration chromatography in different laboratories vary greatly.