人尿胰蛋白酶抑制剂与1-苯胺基-8-萘磺酸的相互作用研究

用荧光光谱、等温滴定微量热(ITC)及分子模建3种方法研究了人尿胰蛋白酶抑制剂(UTI)与1-苯胺基-8-萘磺酸(ANS)的相互作用.结果表明:在UTI上有4个以静电相互作用为主要作用力的特异性的ANS结合位点,分别命名为位点Ⅰ,Ⅱ,Ⅲ和Ⅳ;其中位点Ⅰ位于UTI上结构域Ⅱ的第98位色氨酸(Trp98)附近,位点Ⅱ位于结构域Ⅰ和结构域Ⅱ的相互作用区,位点Ⅲ与位点Ⅳ位于结构域Ⅰ;这4个特异性结合位点所处的区域疏水性较强.ITC实验测得另外5个非特异性结合位点,其主要相互作用力是以ANS的磺酸基与UTI分子表面的带正电的基团间形成的盐键,表明在中性缓冲液中,有5个带正电的氨基酸残基暴露在UTI分子表面.

Interaction between Urinary Trypsin Inhibitor and 1-Anilino-8-Naphthalisene Sulfonate

The interaction between Urinary Trypsin Inhibitor(UTI) and 1-anilino-8-naphthalisene Sulfonate(ANS) was investigated by fluorescence spectrum,Isothermal Titration Calorimetry(ITC) and molecular model.The results of the three experiments identically revealed the presence of four specific binding sites on UTI for ANS,and the interaction between UTI and ANS in the four specific binding sites were driven mainly by electrostatic interaction.The four binding sites were named as site Ⅰ,Ⅱ,Ⅲ and Ⅳ respectively.Site Ⅰ was located in domain Ⅱ and near to Trp98;site Ⅱ were located in the interaction region of the two domains;site Ⅲ and site Ⅳ were located in domain Ⅰ.The results indicated there were four hydrophobic patches in UTI.But the data of ITC experiments showed the presence of other five nonspecific binding sites.The interaction between UTI and ANS in the five nonspecific binding sites were driven mainly by the formation of salt band between the sulfonates of ANS and positive residues on the surface of UTI,which indicated there were five positive residues in the surface of UTI molecular in the neutral buffer.