岩豆凝集素分子中色氨酸残基的化学修饰及其荧光光谱研究

CHEMICAL MODIFICATION OF TRYPTOPHAN RESIDUES AND STUDIES ON LECTIN FROM MILLETTIA DIELSIANA HARMS EX DIELS

从岩豆 (MillettiadielsianaHarmsexDiels)种子中分离纯化得到的岩豆凝集素 (简称MDL)经链霉蛋白酶水解 ,测得其分子中含有 4个色氨酸 (Trp)残基 .用N 溴代丁二酰亚胺 (NBS)对MDL分子中的色氨酸残基进行化学修饰 ,在无变性剂存在下有 3.6个Trp残基被修饰 ,在变性条件下可修饰 3.8个Trp残基 .修饰后MDL活性均丧失 ,且甘露糖对MDL活性具有保护作用 .表明Trp残基是MDL凝集活性所必需的基团 ,且位于MDL糖结合部位 .同时采用荧光光谱实验发现 ,随着NBS的加入 ,MDL荧光强度不断减弱 ,但发射谱形状和最大发射波长无大的变化 ,提示Trp残基可能位于MDL分子表面的疏水区内 .图 3参

The purified Millettia dielsiana Harms ex Diels Lectin(MDL) was studied by chemical modification of typtophan residues and fluorescence spectroscopy. Result from pronase hydrolysis assay showed that there were four tryptophan(Trp) residues in MDL molecule. Modification of Trp residues with N Bromosuccinimide(NBS) induced obvious changes in the fluorescence emission spectra of MDL and resulted in a complete loss of hemagglutinating activity. There were 3.6 Trp residues modified as demonstrated by modification with NBS in native state and 3.8 Trp residues modified in denaturing condition [ c (urea)=8 mol/L]. The research also found that mannose could prevent MDL from losing its activity after being modified by NBS. It indicated that Trp residues were essential for the activity of MDL and involved at carbohydrate-binding site. Results from fluorescence spectra also implied that Trp residues were located in the hydrophobic pocket of the surface of MDL. Fig 3, Ref 8