PRESSURE-INDUCED DISSOCIATION OF FLUORESCEIN FROM THE ANTIFLUORESCEIN SINGLE-CHAIN ANTIBODY 4-4-20
PRESSURE-INDUCED DISSOCIATION OF FLUORESCEIN FROM THE ANTIFLUORESCEIN SINGLE-CHAIN ANTIBODY 4-4-20
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DOI：
10.1021/bi00092a001
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发表时间：
1993-10-19
期刊：
影响因子：
2.9
通讯作者：
VOSS, EW
中科院分区：
文献类型：
--
作者：
COELHOSAMPAIO, T;VOSS, EW
使用流体静压从高亲和力抗Fl单链抗体4-4-20（SCA 4-4-20）解离荧光素（Fl）。通过测量（1）Fl吸收峰的位移，（2）Fl荧光强度的恢复，其在SCA结合时淬灭，或（3）Fl荧光偏振的降低来监测Fl解离。在两种不同的Fl：SCA 4-4-20摩尔比：1：1下研究了压力效应，在该压力下，Fl荧光猝灭约为1.5 μ mol/L。在常压下淬火率为35%;在1：5时淬火率可达95-97%。在这两种情况下，压力诱导的解离有利于蛋白质和配体的伴随稀释。基于压力下Fl荧光偏振的测量计算每个压力下的解离常数（K（D））。K（D）和离解的DELTAG对压力的依赖性允许计算离解过程中涉及的标准体积变化（DELTAV）的大小。根据该研究，Fl-SCA复合物的解离Δ V为约50 mL/mol，其对应于比Fl从完整IgG mAb 4-4-20解离所发现的值高10倍的值[Herron，J.N.，Kranz，D. M.，Jameson，D. M.，& Voss，E. W.，Jr.（1986）Biochemistry 25，4602-4609]。这种差异是解释在一个较高的整体灵活性的unliganded SCA和一个较不稳定的结合位点SCA相对于单克隆抗体。该解释基于以下发现：Fl配体稳定SCA构象，Fl解离不由流体静压诱导的SCA结构中的主要构象变化决定，并且当与SCA配体时Fl比与mAb结合时更容易接近带电的猝灭剂碘化物。最后，Fl从SCA 4-4-20的压力诱导解离的强温度依赖性结合发现Fl半抗原与SCA的缔合在药理学上是不利的事实表明Fl-SCA 4-4-20复合物的形成是熵驱动的过程。
Hydrostatic pressure was used to dissociate fluorescein (Fl) from the high-affinity anti-Fl single-chain antibody 4-4-20 (SCA 4-4-20). Fl dissociation was monitored by measuring (1) the shift in the Fl absorption peak, (2) the recovery in Fl fluorescence intensity, which is quenched upon SCA binding, or (3) the decrease in Fl fluorescence polarization. Pressure effects were studied at two different Fl:SCA 4-4-20 molar ratios: 1:1, at which Fl fluorescence quenching was ca. 35% at atmospheric pressure, and 1:5, at which quenching reached 95-97% under the same conditions. In both cases, pressure-induced dissociation was favored by concomitant dilution of protein and ligand. Dissociation constants (K(D)) at each pressure were calculated on the basis of measurements of Fl fluorescence polarization under pressure. The dependence of K(D), and consequently of DELTAG of dissociation, on pressure permitted calculation of the magnitude of the standard volume change (DELTAV) involved in the dissociation process. According to this study, DELTAV of dissociation for the Fl-SCA complex is -50 mL/mol, which corresponds to a 10-times higher value than that found for dissociation of Fl from the intact IgG mAb 4-4-20 [Herron, J. N., Kranz, D. M., Jameson, D. M., & Voss, E. W., Jr. (1986) Biochemistry 25, 4602-4609]. This difference is explained in terms of a higher overall flexibility of unliganded SCA and of a less stable binding site in SCA relative to mAb. This interpretation was based on the findings that the Fl ligand stabilizes SCA conformation, that Fl dissociation was not determined by primary conformational changes in SCA structure induced by hydrostatic pressure, and that Fl is more accessible to the charged quencher iodide when liganded to SCA than when bound to mAb. Finally, the strong temperature dependence for pressure-induced dissociation of Fl from SCA 4-4-20 combined with the fact that association of the Fl hapten to SCA was found to be enthalpically unfavorable indicates that formation of the Fl-SCA 4-4-20 complex is an entropy-driven process.