摘要
本文详细地叙述了提取、分离、提纯豌豆球蛋白及其亚基的方法:用硼酸钠缓冲液提取豌豆球蛋白,用85%饱和度硫酸铵沉淀除去其它蛋白质杂质,再用凝胶柱进一步提纯;提纯的豌豆球蛋白通过DE_(52)-纤维素阴离子树脂柱(8M尿素作为变性分离试剂)和S_(200)凝胶柱(70%甲酸作为变性分离试剂)则可分离出豌豆球蛋白的各个亚基;试验结果还表明:在pH8.5时,33,000的亚基携带少量负电荷;而12,000的亚基携带大量负电荷。
The purification of vicilin andits subunits from pea seeds is descri-bed in detail.Pea seed meal wasextracted with borate buffer and theextract was made 85% saturation with(NH_4)_2 SO_4.The material precipi-tated was collected by centrifugationand discarded;the supernatant,whichcontained vicilin,was dialysed andlyophilised and then the vicilin waspurified by gel filtration.The vici-lin subunits were separated and puri-fied by ion-exchange chromatographyon DEAE-cellulose (in 8M urea asdenaturant) and by gel-filtration onsephacryl S-200 (in 70 % formic acidas denaturant).Analysis by gel elec-trophoresis showed that these me-thods gave preparation of purified vi-cilin and its separated componentsubunits.The results also showedthat at pH 8.5 the subunit of Mr33,000 has the least negative chargeand the 12,000 Mr subunit has themost negative charge.ACKNOWLEDGEMENTS We thank Mr.Dave Bown for in-valuable technical advice and assis-tance.We also thank Drs.R.R.D.Croy,Dr.M.Evans and Dr.J.N.Yarwood for their suggestions andhelp.This work was supported byfinancial aid from Department of Bot-any,University of Durham.U.K.
