野花生豆凝集素半胱氨酸残基的研究

THE STUDY ON CYTEINES IN CROTALARIA MUCRONATA LECTIN

将野花生豆经浸取、硫酸铵分级沉淀、猪胃粘蛋白 Sepharose 4B亲和层析和SephacrylS 2 0 0HR分子筛层析后 ,可得到对人类A型血专一凝集的野花生豆凝集素 (CrotalariamucronataLectin ,CML) .纯化的CML在PAGE和SDS PAGE中均显示单一蛋白带 .在含 8mol/L脲和不含脲的缓冲体系中 ,分别用NEM修饰CML中的半胱氨酸残基 ,经计算被修饰巯基数目分别为 3 1个和 3 2个 ,且修饰后的CML的活性仍不丧失 .通过NR/R单向SDS PAGE和NR/R双向对角线SDS PAGE研究 ,发现CML不含二硫键 ,表明CML中的半胱氨酸残基既与CML的凝集活性无关 。

Crotalaria mucronata lectin (CML) was purified from seeds of Crotalaria mucronata by extraction, fraction with (NH 4) 2SO 4,hog gastric mucin Sepharose 4B affinity chromatography and followed by gel filtration of Sephacryl S 200 HR CML agglutinates type A human red blood cells specially The purifed CML gave one band on acrylamide gel electrophoresis and on SDS acrylamide gel electrophoresis The purified CML modified by N ethymaleimide in the physiological saline containing 8mol/L urea or not has agglutinate activity There are 3 1 sulfhydryl groups in CML modified with 8mol/L urea and 3 2 sulfhydryl groups in CML modified without urea The trials of NR/R single dimensional SDS PAGE and NR/R two dimensional SDS PAGE prove that CML has not disulfide bond The cysteines in CML are no essential for its activity and don't form disulfide bond.