【摘要】 为了探讨竹红菌甲素对红细胞膜蛋白的光敏交联机理,我们使用了一些专一性及非专一性的基团修饰剂来修饰膜蛋白,试图说明膜蛋白的光敏交联究竟是由膜蛋白的巯基光氧化所引起的,还是膜蛋白的氨基酸与其侧链氨基之间的交联所引起的。我们分别采用N-乙基顺丁烯二酰抱亚胺(NEM)修饰膜蛋白的巯基,用N-溴代琥珀酰亚胺(NBS)来修饰色氨酸残基,用乙氧甲酸酐(DEP)修饰组氨酸残基,及用琥珀酸酐(SA)修饰氨基。测定了红细胞膜修饰前后及有竹红菌甲素存在时,光照前后的膜蛋白巯基及膜氨基的变化,膜蛋白内源性荧光的变化,以及对膜蛋白形成交联的影响。结果表明:NEM、DEP和NBS修饰的膜, 在有甲素存在时,光照对巯基影响很小,而对SA修饰的膜有明显的光敏作用。甲素对膜蛋白氨基的影响小于巯基,仅降低含量20%。甲素光照能引起NEM和SA修饰的膜内源荧光下降。甲素对NEM处理的膜仍能引起交联,但SA处理过的膜能抗交联,说明氨基在膜蛋白光敏交联中起着重要的作用。更多还原

【Abstract】 The purpose of the present work isto elucidate the mechanism of hypocrel-lin A-sensitized cross-linking of membrane proteins. In our experiments,some modifica-tion reagents of specified and nonspecifiedgroups were used for membrane proteinmodification.Experiments were designedto elucidate whether photosensitized cross-linking of erythrocyte membrane proteinsis caused by photooxjdation of SH groupsin membrane proteins or by reaction be-tween photooxidized amino acid residuesand amino groups.N-ethylmaleimide（NEM）was used in SH group modifica-tion.Tryptophan and histidine modifica-tions were carried out using N-bromosuc-cinimide（NBS）and diethyl pyrocarbonate（DEP）.The NH₂ group modification ofmembrane was accomplished with the aidof succinic anhydride（SA）.Erythrocytemembranes before and after modificationwere irridiated in the presence of hypo-crellin A.The changes of SH groups andNH₂ groups in membrane after irradiationwere observed,The changes of endogenousfluorescence of membrane proteins andphotodynamic effects on cross-linkingformation of membrane proteins wereexamined. The results show that modified membrane proteins with NEM,DEP andNBS were illuminated in the presence ofhypocrellin A,which caused no markedchange in SH groups content,but causedsignificant decrease in SH groups content,if membrane proteins treated with SA（Fig.5）. Hypocrellin A-sensitized illuminationmade diminution of NH₂ groups in mem-brane proteins,but the change ofNH₂ group population was only 20%and not so large as in the case of SHgroups（Fig.6）. When modified membrane proteinwith NEM and SA were irradiated inthe presence of hypocrellin A,a decreasein endogenous fluorescence intensity ofprotein was observed（Fig.8）. The results by SDS-polyacrylamidegel electrophoresis analysis are shown inFig.9.Experiments were performed with ??NEM and SA treated erythrocyte mem-branes.It can be seen that NEM modifiedmembrane proteins in the presence of hy-pocrellin A had no effect on light-induced cross-linking,but SA treated mem-branes were almost completely protectedagainst cross-linking.This phenomenonindicates that NH₂ groups play an impor-tant role in the photosensitized cross-lin-king of membrane proteins.更多还原