摘要
本文以正常人胎盘为材料,通过匀浆、硫酸铵分级分离、20％正丁醇抽提、Sephadex G-200凝胶过滤及Concanavalin A-Sepharose 4B亲和层析等分离纯化步骤,制备获得了纯化3705倍的酸性β-1,4葡萄糖苷酶,其比活性和获得率分别为277261.33n mol·(mg Prot·h)^(-1)和14.3％。 经agarosn-IEF检验,该纯化的酸性β-1,4葡萄糖苷酶已达蛋白电泳单点纯,PI为5.2。经3～28％梯度PAGE,求知该酶单体分子量为74kD。该纯制酶极不稳定,4℃贮存6天后活性即降低50％以上,与其特异性抗体结合后,活性全部被抑制。
Acid β-1.4-glucosidase(GβA) was purified 3705 fold from human place-nt by homogenization, ammonium sulfate fractionation, n-butanol extraction,sephadex G-200 filtration and concanavlin-A-sepharose 4B affinity chromatography. The specific activity of the yield was 277261.33nmol(mg prot.hr)-1 with 14.3% recovery. Only one protein band could be detected by agarose IEF(PH 3.5-10), indicated that the GβA was purified to homogeneity with PI at 5.2. The subunit MW is 74 kD as showed by 3-28% gradient PAGE. The purified GβA is very unstable, more than 50% of the activity will be lost after storage for 6 days at 4℃. When combining with specific antibody, GβA will be inhibited completely.
关键词
Β-葡萄糖苷酶
纯化
β-Glucosidase, Gaucher Disease, Placenta, Affinity Chromatography
