Thermodynamic binding parameters of individual epitopes of multivalent carbohydrates to concanavalin a as determined by "reverse" isothermal titration microcalorimetry
Thermodynamic binding parameters of individual epitopes of multivalent carbohydrates to concanavalin a as determined by "reverse" isothermal titration microcalorimetry
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DOI：
10.1021/bi015829k
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发表时间：
2002-01-29
期刊：
影响因子：
2.9
通讯作者：
Brewer, CF
中科院分区：
文献类型：
--
作者：
Dam, TK;Roy, R;Brewer, CF
前一篇论文[Dam，T. K.，罗伊河，Page，D.，和Brewer，C. F.（2002）Biochemistry 41，1351-1358]证明，具有末端3，6-二-O-（α-D-吡喃甘露糖基）-α-D-吡喃甘露糖苷残基的二价、三价和四价碳水化合物类似物与凝集素伴刀豆球蛋白A（ConA）结合的等温滴定微量热法（ITC）数据的Hill图显示，在类似物与凝集素结合后，负协同性增加。本研究演示了“反向”ITC实验，其中凝集素滴定到二价和三价类似物的溶液中。结果提供了直接确定的热力学的ConA的两个多价类似物的个人表位的结合。从反向ITC得到的n值（每个碳水化合物分子的结合位点数）证明了二价和三价类似物上的两个功能性结合表位，证实了先前用两种碳水化合物得到的“正常”ITC结果[Dam，T. K.，罗伊河，达斯，S。K.，Oscarson，S.，和Brewer，C. F.等人（2000）J. Biol. Client. 275，14223-14230]。反向ITC测量显示，ConA对二价类似物的第一表位的微观亲和常数比其第二表位大18倍，ConA结合三价类似物的第一表位的微观亲和常数比其第二表位大53倍。数据还表明，二价和三价类似物上的两个表位的结合的微观熵基本上相同，并且表位的微观K-a值的差异是由于它们的结合值的不同微观熵。这些发现与前一篇论文中这些类似物与ConA结合的负Hill系数增加一致。
The preceding paper [Dam, T. K., Roy, R., Page, D., and Brewer, C. F. (2002) Biochemistry 41, 1351-1358] demonstrated that Hill plots of isothermal titration microcalorimetry (ITC) data for the binding of di-, tri-, and tetravalent carbohydrate analogues possessing terminal 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside residues to the lectin concanavalin A (ConA) show increasing negative cooperativity upon binding of the analogues to the lectin. The present study demonstrates "reverse" ITC experiments in which the lectin is titrated into solutions of di- and trivalent analogues. The results provide direct determinations of the thermodynamics of binding of ConA to the individual epitopes of the two multivalent analogues. The n values (number of binding sites per carbohydrate molecule) derived from reverse ITC demonstrate two functional binding epitopes on both the di- and trivalent analogues, confirming previous "normal" ITC results with the two carbohydrates [Dam, T. K., Roy, R., Das, S. K., Oscarson, S., and Brewer, C. F. (2000) J. Biol. Client. 275, 14223-14230]. The reverse ITC measurements show an 18-fold greater microscopic affinity constant of ConA for the first epitope of the divalent analogue versus its second epitope and a 53-fold greater microscopic affinity constant of ConA binding to the first epitope of the trivalent analogue versus its second epitope. The data also demonstrate that the microscopic enthalpies of binding of the two epitopes on the di- and trivalent analogues are essentially the same and that differences in the microscopic K-a values of the epitopes are due to their different microscopic entropies of binding values. These findings are consistent with the increasing negative Hill coefficients of these analogues binding to ConA in the previous paper.