Development of a simple and rapid determination method of <alpha_1> -acid glycoprotein in plasma.

一种阳离子荧光auramine O （AO）与人<alpha_1> -酸性糖蛋白（<alpha_1> -AG）结合后，荧光明显增强。用荧光光谱法和平衡分析法研究了AO与蛋白质的相互作用。AO通过单一位点与蛋白质结合，解离常数为24 m。已知与蛋白质结合的各种基本药物，如氯丙嗪、丙咪嗪、去西帕明、奎宁、心得安和利多卡因，竞争性地抑制AO与蛋白质的结合。抑菌实验得到的这些基本药物的解离常数与其他方法（平衡透析、蛋白质本征荧光猝灭、差值分光光度法）和文献中得到的解离常数相当。结果表明，AO可能是一种有效的荧光探针，可与< α _1> -AG上的单个基本药物结合位点结合。此外，建立了AO荧光法测定血清中< α _1> -AG的简便方法，并与常规放射免疫扩散法进行比较，验证了该方法的有效性。在酸性条件下，deae -纤维素处理的人血清几乎完全去除< α _1> -AG (< α _1> -AG)，白蛋白和< β > -脂蛋白浓度变化不大，而磺胺水杨酸处理的人血清除< α _1> -AG外几乎全部去除。这些结果表明，用磺胺水杨酸和DEAE纤维素处理血清可用于评估< α _1> -AG对基础药物血清结合的贡献。

A cationic fluorescent auramine O (AO) exhibited an intense increase in fluorescence after bindingto human <alpha_1> -acid glycoprotein ( <alpha_1> -AG). The interaction between AO and the protein was studied by fluorescence spectroscopy and by equilibruim dyalysis. AO binds to the protein via a single site with adissociation constant of 24 M. Various basic drugs such as chlorpromazine, imipramine, desipramine, quinidine, propranolol and lidocaine, which are known to bind to the protein, competitively inhibited the AO binding to the protein. The dissociation constants of these basic drugs ontained from such inhibitory experiments were comparable to those obtained with other methods (equilibrium dialysis, quenching of protein intrinsic fluorescence, and the difference spectrophotometric method) and from the literature. It is concluded that AO may be a useful fluorescence probe that binds to a single basic drug binding site on <alpha_1> -AG. In addition, a simple fluorometric method for the determination of <alpha_1> -AG in serum was developed using AO, and the validity of this method was confirmed by comparing it with the conventional radial immunodiffusion method.Treatment of human serum with DEAE-cellulose in acid conditions almost completely removed <alpha_1> -acid glycoprotein ( <alpha_1> -AG) with little change in the concentration of albumin and <beta> -lipoprotein,while treatment with sulphosalicylic acid removed almost all the proteins except <alpha_1> -AG. These results suggest that treatment of serum with sulphosalicylic acid and DEAE cellulose is useful inassessing the contribution of <alpha_1> -AG to the serum binding of basic drugs.