Catalytic antibodies. Circular dichroism and UV-vis studies of antibody-metalloporphyrin interactions
Catalytic antibodies. Circular dichroism and UV-vis studies of antibody-metalloporphyrin interactions
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DOI：
10.1021/ic00052a019
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发表时间：
1992-12
影响因子：
4.6
通讯作者：
E. Keinan;E. Benory;S. Sinha;A. Sinha-Bagchi;D. Eren;Z. Eshhar;B. Green
中科院分区：
文献类型：
--
作者：
E. Keinan;E. Benory;S. Sinha;A. Sinha-Bagchi;D. Eren;Z. Eshhar;B. Green
利用圆二色性和紫外可见性测量研究了几种水溶性金属卟啉与单克隆抗体（mab）之间的相互作用，这些单克隆抗体是针对结构相关的二羟基锡（IV）卟啉而引发的。先前研究的一些单克隆抗体-金属卟啉复合物被证明具有类似血红蛋白的活性，例如苯乙烯被碘索苯催化环氧化。单克隆抗体-金属卟啉络合物的形成通常伴随着显著的深色移和卟啉Soret波段吸收最大值的低/高色变。在同一区域（350-450 nm），可以检测到络合物形成时的诱导CD光谱。它们遵循比尔定律，表现出等吸行为，并且作为抗体卟啉比率的函数，棉花效应的一般形状没有显着变化。信号强度（椭圆度）随着抗体与金属卟啉比值的增加而增加，达到饱和络合的上限，均表明特异性的1:1结合现象。观察到信号形状和信号强度对特异金属和抗体的强烈依赖，表明诱导CD可能是两个看似相同的MAB克隆明确分化的有力工具。在强烈诱导CD信号的情况下，可以使用滴定曲线获得单克隆抗体-金属卟啉解离常数，其中椭圆度作为抗体：卟啉比率的函数进行测量，并以Scatchard图表示。金属卟啉-单抗配合物的UV-vis吸光度和诱导CD谱在6 - 11的较宽pH范围内保持不变，表明这些配合物具有显著的稳定性，反映了半抗原羧酸基与抗体结合位点之间的静电相互作用的主导作用。
Circular dichroism and UV-vis measurements were used to study the interaction between several water-soluble metalloporphyrins and monoclonal antibodies (MABs) that were elicited against a structurally related dihydroxytin(IV) porphyrin, la. Some of the MAB-metalloporphyrin complexes studied were previously shown to mimic hemoprotein-like activity, such as catalytic epoxidation of styrene by iodosobenzene. MAB-metalloporphyrin complex formation is usually accompanied by significant bathochromic shift and hypo-/hyperchromicity changes of the absorption maxima in the porphyrin Soret band region. Induced CD spectra in the same region (350-450 nm) are detected upon complex formation. They follow Beer’s law, exhibit isosbestic behavior, and show no significant change in the general shape of the Cotton effect as a function of the antibody:porphyrin ratio. Signal intensity (ellipticity) increases with increasing antibody:metalloporphyrin ratio, reaching an upper limit that represents saturation complexation, all indicating a specific 1:l binding phenomenon. Strong dependence of both signal shape and signal intensity upon the specific metal and antibody were observed, suggesting that induced CD may serve as a powerful tool for the unequivocal differentiation of two seemingly identical MAB clones. In cases of intense induced CD signals, it is possible to obtain MAB-metalloporphyrin dissociation constants using a titration curve where ellipticity is measured as a function of the antibody:porphyrin ratio and presented as a Scatchard plot. Both the UV-vis absorbance and induced CD spectrum of the metalloporphyrin-MAB complex remain unchanged over a broad pH range between 6 and 11, indicating remarkable stability of these complexes and reflecting the dominant role of electrostatic interactions between the hapten carboxylate groups and the antibody combining site.