人类胎盘碱性磷酸酶的分离纯化

ISOLATION AND PURIFICATION OF HUMAN PLACENTAL ALKALINE PHOSPHATASE

①目的分离纯化人类胎盘碱性磷酸酶（ＰＬＡＰ）。②方法应用正丁醇抽提、丙酮沉淀、热处理、ＤＥＡＥ－纤维素和ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－５０柱层析，从人类胎盘中提纯ＰＬＡＰ；聚丙烯酰胺凝胶电泳（ＰＡＧＥ）和高压液相层析（ＨＰＬＣ）鉴定其纯度；十二烷基硫酸钠－聚丙烯酰胺凝胶电泳测其亚基分子量。③结果ＰＬＡＰ的纯化倍数为１６１倍，比活力为１３８ｍｍｏｌ·ｓ－１／ｇ；纯化的ＰＬＡＰ经ＰＡＧＥ鉴定为单一区带，ＨＰＬＣ分析为单一对称峰；测得酶的亚基分子量为６４．６９ｋｕ．④结论纯化的ＰＬＡＰ已达电泳纯和层析纯。

Objective To establish methods for isolation and purification of human alkaline phosphatase(PLAP). Methods PLAP was extracted by n butyl alcohol at 4℃,precipitated by cold acetone, heated for 40min at 65℃ and purified through chromatography on DEAE cellulose column and DEAE SephadexA 50 column. The purified PLAP was identified by polyacrylamide gel electrophoresis(PAGE) and high performance liquid chromatography(HPLC).A subunit molecular weight was determined by sodium dodecyl sulfate polyacrylamide gel eletrophoresis. Results PLAP was purified 161 folds by this procedure, the specific activity of PLAP was 138mmol·s -1 /g. Purified PLAP showed a single band after PAGE and a single peak after HPLC. A subunit molecular weight of PLAP was 64 69ku. Conclusion Purified PLAP in this study had reached the pruity of electrophoresis and chromatography and could be used in the study of its conformation and properties.