岩豆凝集素分子修饰与圆二色性的关系研究

Molecular Modification and Circular Dichroism of Millettia dielsiana Harms.Lectin

天然态岩豆凝集素（ＭＤＬ）远紫外圆二色性（ＣＤ）谱显示２１６ｎｍ处单一负峰，是一种高β－折叠构象凝集素；近紫外ＣＤ谱呈现２８２ｎｍ处负峰和２６０～２７５ｎｍ及２９５ｎｍ处的负肩，经Ｎ－乙基顺丁烯二酰亚胺（ＮＥＭＩ）和对氯汞苯甲酸（ＰＣＭＢ）修饰ＭＤＬ的巯基，其近紫外ＣＤ谱未发生变化，远紫外ＣＤ谱仅发生细微变化，ＭＤＬ凝集活性保持不变；ＰＣＭＢ过量时，ＣＤ谱呈现典型的无规卷曲谱形，ＭＤＬ完全丧失凝集活性，去除ＰＣＭＢ后，活性又全部恢复．二硫苏糖醇（ＤＤＴ）修饰ＭＤＬ的二硫键并用碘乙酸（ＩＣＨ２ＣＯＯＨ）保护巯基，ＭＤＬ远紫外ＣＤ谱２１６ｎｍ处的负峰红移至２２５ｎｍ，且显著减小；同时，近紫外ＣＤ谱２８２ｎｍ处负峰几乎消失，两负肩分别保持完整，分子中α－螺旋降低，无规卷曲增加较多，ＭＤＬ凝集活性未发生变化．用Ｎ－溴代丁二酰亚胺（ＮＢＳ）修饰ＭＤＬ分子中的色氨酸，导致２１６ｎｍ负峰蓝移至２０８ｎｍ且变小，分子中无规卷曲和α－螺旋增加，β折叠减少，近紫外ＣＤ谱２９５ｎｍ负肩消失，２８２ｎｍ负峰红移至２８７ｎｍ，ＭＤＬ凝集活性完全丧失．

To explain the roles of thiol groups (—SH),disulfide bonds (—S—S—)and tryptophan in structure and function of a legume lectin from Millettia dielsiana Harms(abbrev.MDL),the conformational change of MDL was studied by circular dichroism (CD).Native MDL contained about 16 2% α helix and 46 3% β sheet,its CD spectra exhibited a negative peak at 216 nm,near ultraviolet (UV)CD spectra showed a negative band at 282 nm and two negative shoulders centered at 260～275 nm and 295 nm.Treated MDL with N ethylmaleimide and p chloromercuribenzoate only resulted in minor change of CD spectra.Modification of MDL by dithiothreitol and iodoacetic acid,caused the band red shift from 216 nm to 225 nm and the extremum moves upward.the negative peak at 282 nm disappeaed,while the two shoulders intact,since there was no tyrosine in MDL,so the peak at 282 nm was contributed by —S—S—.The content of helix and sheet decreased,while MDL kept all activity.So the carbohydrate binding site was located at subunit and the domain was consisted of monomer,not monomer monomer interaction.The second and tertiary structure elements in MDL exhibited less interaction at the monomer monomer interface region,since MDL was a dimeric glycoprotein composed of identical subunit.Treated by N bromosuccinimide,MDL lost activity completely,the band at 216 nm blue shift to 208 nm with decreased extremum;the negative shoulder at 295 nm disappeared and β sheet decreased.So this shoulder was contributed by tryptophan,and tryptophans were all on the surface of MDL and crucial for the conformation and activity of MDL.Some tryptophan may be located or around the carbohydrate binding site.