Biocytin-specific 110-kDa biotinidase from human serum

Area of Science:

• Biochemistry
• Enzymology
• Human Physiology

Background:

• Biotinidase is crucial for biotin metabolism, recycling the vitamin from dietary sources.
• Previous studies identified a 76-kDa biotinidase in human serum.
• The existence and role of other biotinidase isoenzymes remain largely unexplored.

Purpose of the Study:

• To develop a novel protocol for biotinidase purification from human serum.
• To identify and characterize novel biotinidase isoenzymes.
• To investigate the kinetic properties and potential physiological roles of identified isoenzymes.

Main Methods:

• High-performance liquid chromatography (HPLC) biotinidase assay.
• Purification using non-ionic surfactant.
• Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and N-glycanase treatment for protein core estimation.
• Total amino acid analysis.
• Enzyme kinetics studies (Km, kcat/Km).

Main Results:

• A novel 110-kDa biotinidase isoenzyme was co-purified alongside the 76-kDa form, accounting for 5% of total activity.
• The protein core of the 110-kDa enzyme was estimated at 72 kDa (vs. 59 kDa for 76-kDa), with higher amino acid content.
• The 110-kDa isoenzyme demonstrated a 10-fold lower Km and a 9-fold higher kcat/Km for biocytin compared to the 76-kDa enzyme.
• The 110-kDa enzyme is proposed as the physiological biocytin hydrolase (biocytinase) due to its efficiency at low biocytin concentrations.

Conclusions:

• Human serum contains at least two distinct biotinidase isoenzymes with differing kinetic properties.
• The newly identified 110-kDa biotinidase is likely the primary enzyme responsible for biocytin hydrolysis in vivo.
• Differences in isoenzyme function may contribute to the varied clinical presentations of biotinidase deficiency.