Chauhan J, Dakshinamurti K
J Biol Chem. 1986 Mar 25;261(9):4268-75.
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.
通过五步程序从人血清中纯化出生物素酶，其比活性达到1900单位/毫克蛋白质。经硫酸铵沉淀（33 - 55%饱和度切割）后，依次通过DEAE - 葡聚糖凝胶、羟基磷灰石、辛基 - 葡聚糖凝胶CL - 4B和葡聚糖凝胶G - 100色谱进行纯化。纯化后的酶在变性和非变性条件下的聚丙烯酰胺凝胶电泳中显示出单一的银染条带。生物素酶是一种糖蛋白。分子中的唾液酸残基对酶活性并非必需。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（弗格森图）和沉降分析估算，人血清生物素酶的相对分子质量为68,000。以N -（d - 生物素基）对氨基苯甲酸为底物时，人血清生物素酶在pH 6.0至7.5范围内表现出最大活性。然而，以生物胞素为底物时，该酶的最大活性处于pH 4.5至6.0范围内。使用底物的结构类似物，我们已表明生物素酶不是一种通用的蛋白水解酶，并且在底物水解方面具有特定的结构要求。
