Purification and characterization of human serum biotinidase.
Biotinidase has been purified from human serum to a specific activity of 1900 units/mg protein by a five-step procedure. After ammonium sulfate precipitation (33-55% cut) it was purified by DEAE-Sephacel, hydroxylapatite, octyl-Sepharose CL-4B, and Sephadex G-100 chromatography. The purified enzyme showed a single silver staining band with polyacrylamide gel electrophoresis under denaturing and non-denaturing conditions. Biotinidase is a glycoprotein. The sialic acid residues in the molecule are not required for enzyme activity. The Mr of human serum biotinidase estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Ferguson plot) and by sedimentation analysis was 68,000. Human serum biotinidase showed maximum activity in the pH range 6.0 to 7.5 with N-(d-biotinyl) p-aminobenzoate as substrate. However, with biocytin as substrate, the maximal activity of the enzyme was in the pH range 4.5 to 6.0. Using structural analogs of the substrate we have shown that biotinidase is not a general proteolytic enzyme and has specific structural requirements in the substrate for hydrolysis.
D000581:Amidohydrolases / Q000097:blood / Q000302:isolation & purification*; D000596:Amino Acids / Q000032:analysis; D043523:Biotinidase; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D013379:Substrate Specificity; D013696:Temperature
D000596:Amino Acids; D000581:Amidohydrolases; D043523:Biotinidase