Surface plasmon resonance imaging studies of protein-carbohydrate interactions
Surface plasmon resonance imaging studies of protein-carbohydrate interactions
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DOI：
10.1021/ja034165u
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发表时间：
2003-05-21
影响因子：
15
通讯作者：
Corn, RM
中科院分区：
文献类型：
--
作者：
Smith, EA;Thomas, WD;Corn, RM
利用表面等离子体共振(SPR)成像技术研究了碳水化合物与蛋白质之间的相互作用。利用形成表面二硫键的固定化方案将硫醇修饰的碳水化合物固定在金膜上，并制备碳水化合物阵列。利用偏振调制傅里叶变换红外反射吸收光谱表征了碳水化合物的吸附步骤，并利用聚二甲基硅氧烷微通道将探针化合物固定在金膜上的不同位置。用SPR成像监测糖结合蛋白刀豆蛋白A(ConA)和黄花素与单糖、甘露糖和半乳糖组成的阵列的结合。利用SPR成像测量完成了以下工作：(I)建立ConA和Jacalin在碳水化合物表面相互作用的吸附等温线，(Ii)监测蛋白质与呈现不同组成的固定化碳水化合物表面的结合，(Iii)测量ConA和Jacalin分别向甘露糖和半乳糖方向的溶液平衡解离常数。吸附系数分别为2.2+/-0.8×10(7)M-1和5.6+/-1.7×10(6)M-1。结果表明，半乳糖与茉莉碱相互作用的溶液平衡解离常数为16+/-5um，刀豆素与甘露糖相互作用的溶液平衡解离常数为200+/-50um。
Carbohydrate arrays fabricated on gold films were used to study carbohydrate-protein interactions with surface plasmon resonance (SPR) imaging. An immobilization scheme consisting of the formation of a surface disulfide bond was used to attach thiol-modified carbohydrates onto gold films and to fabricate carbohydrate arrays. The carbohydrate attachment steps were characterized using polarization modulation Fourier transform infrared reflection absorption spectroscopy; and poly(dimethylsiloxane) microchannels were used to immobilize probe compounds at discrete locations on a gold film. The binding of the carbohydrate-binding proteins concanavalin A (ConA) and jacalin to arrays composed of the monosaccharides mannose and galactose was monitored with SPR imaging. SPR imaging measurements were employed to accomplish the following: (i) construct adsorption isotherms for the interactions of ConA and jacalin to the carbohydrate surfaces, (ii) monitor protein binding to surfaces presenting different compositions of the immobilized carbohydrates, and (iii) measure the solution equilibrium dissociation constants for ConA and jacalin toward mannose and galactose, respectively. Adsorption coefficients (KADS) of 2.2 +/- 0.8 x 10(7) M-1 and 5.6 +/- 1.7 x 10(6) M-1 were obtained for jacalin adsorbing to a galactose surface and ConA adsorbing to a mannose surface, respectively. The solution equilibrium dissociation (K-D) constant for the interaction of jacalin and galactose was found to be 16 +/- 5 muM, and for ConA and mannose was found to be 200 +/- 50 muM.