Subramanian N, Subramanian S, Karande A A, Adiga P R
Department of Biochemistry and Centre for Reproductive Biology & Molecular Endocrinology, Indian Institute of Science, Bangalore.
Arch Biochem Biophys. 1997 Aug 15;344(2):281-8. doi: 10.1006/abbi.1997.0196.
An anti-avidin mAb, viz., H12G4, is shown to release bound biotin in a dose-dependent manner from holoavidin and holostreptavidin and inhibit the binding of ligand to the two apoproteins. The release of biotin by this mAb is accompanied by quenching of ligand-induced enhanced fluorescence of the FITC-avidin conjugate. In terms of mechanism of release of bound biotin, we demonstrate that on binding to the Fab fragment of the mAb, the native tetrameric holoavidin undergoes dissociation progressively with time to monomers with no bound biotin associated with the latter. Based on the immunoreactivity associated with defined overlapping fragments of avidin obtained by chemical cleavage, the epitope recognized by mAb H12G4 has been localized to residues 58-96 of the primary sequence. By pepscan method of epitope mapping, this mAb is shown to identify a minimal core sequence of 87RNGK90 in avidin and a corresponding sequence of 84RNAH87 in streptavidin.
一种抗抗生物素蛋白单克隆抗体，即H12G4，已被证明能以剂量依赖的方式从全抗生物素蛋白和全链霉抗生物素蛋白中释放结合的生物素，并抑制配体与这两种脱辅基蛋白的结合。该单克隆抗体释放生物素的过程伴随着FITC-抗生物素蛋白缀合物的配体诱导增强荧光的淬灭。关于结合生物素的释放机制，我们证明，天然四聚体全抗生物素蛋白与单克隆抗体的Fab片段结合后，会随着时间的推移逐渐解离为单体，且后者不与结合的生物素相关联。基于通过化学裂解获得的抗生物素蛋白特定重叠片段的免疫反应性，单克隆抗体H12G4识别的表位已定位到一级序列的58-96位残基。通过表位作图的肽扫描方法，该单克隆抗体被证明能识别抗生物素蛋白中87RNGK90的最小核心序列以及链霉抗生物素蛋白中84RNAH87的相应序列。
