不同生理时期梅花鹿血液GSH-Px含量测定及其纯化

研究不同生理时期梅花鹿全血中GSH-Px含量变化及其纯化技术,为开发鹿血抗衰老资源奠定理论基础.用DINB法测定梅花鹿的溶血液、血细胞内容物、血浆和血细胞细胞膜中GSH-Px含量并计算全血GSH-Px含量；利用10％～100％饱和度的(NH4)2SO4盐析和柱层析技术纯化GSH-Px;用SDS-聚丙烯酰胺凝胶电泳测定其亚基相对分子质量.试验结果表明:全血GSH-Px含量以生茸期最高为(1 973.07士25.43)U·mL-1,与配种期的(1 727.74士12.46)U·mL-1差异极显著(P＜0.01),与生茸前期的(1 961.83士16.54)U·mL-1差异不显著(P＞0.05)；GSH-Px的初始比活力为24.9 U·mg-1,经纯化后得到GSH-Px 0.37 mg· mL-1,最终比活力1 347.7U·mg-1,纯化倍数为54.1倍,回收率25.00％；鹿血GSH-Px亚基的相对分子质量为17.2 ku.结果提示生茸期鹿血的GSH-Px含量最高,开发利用价值最大,GSH-Px含量与生理特性相关；鹿血GSH-Px盐析条件为40％～80％饱和度的(NH4)2SO4,此时所得GSH-Px纯化倍数较高.

Measurement and Purification of GSH-Px in Different Physiological Period of Sika Deer Blood

The contents of GSH-Px in the whole blood of sika deer within different physiological period and the technique for purification GSH-Px,were studied for developing anti-aging resources of deer blood and laid a theoretical basis.The contents of GSH-Px in the blood cells,plasma,dissolved blood and cell membrane were detected by using DTNB method and the contents of GSH-Px in the whole blood were calculated.A 10%-100% of saturation(NH4)2SO4 salting-out and column chromatography technology were used to purify the GSH-Px and the SDS-PAGE was used to measure its subunit molecular weight.The results showed that the highest contents of GSH-Px appeared during the time of antler developing period((1 973.07±25.43) U·mL-1),which was significantly higher(P<0.01) than that in the service period((1 727.74±12.46) U·mL-1).However,no significant difference(P>0.05) was found between the value in antler developing period and the pre-antler period((1 961.83±16.54) U·mL-1).GSH-Px initial specific activity was 24.9 U·mg-1 and 0.37 mg·mL-1 purified GSH-Px was achieved.Finally specific activity was 1 347.7 U·mg-1 and 54.1 times purification rate and 25% recovery rate were achieved.The relative molecular weight of subunit for GSH-Px was 17.2 kDa.The results demonstrated that there are some certainly correlation between the change of GSH-Px content and the physiological period of the deer.The saturation of 40%-80%(NH4)2SO4 is the best condition for GSH-Px purification.