化学
显色的
磷酸盐
水解
基质(水族馆)
亲核细胞
贪婪
劈理(地质)
共轭体系
组合化学
立体化学
色谱法
催化作用
生物化学
抗原
有机化学
海洋学
聚合物
生物
遗传学
地质学
岩土工程
断裂(地质)
工程类
作者
Qinjian Zhao,Robert D. Sitrin
标识
DOI:10.1006/abio.2001.5175
摘要
Aluminum-containing adjuvants are widely used in a variety of vaccine products, such as recombinant proteins, virus-like particles, conjugated polysaccharides, and recently DNA vaccines. Aluminum-containing adjuvants are also known to have a high affinity to inorganic phosphate and its mono- or diesters. Since phosphate groups are present in many antigens as well as the natural physiological environment, a better understanding of the interactions between phosphate and phospho-containing species could help in the design of improved vaccines. This report describes a convenient and novel continuous procedure to measure the avidity denoted by the new term "phosphophilicity" of phosphate and phosphate esters to the surface of aluminum-containing adjuvants. The assay measures the rate of hydrolysis of a fluorogenic substrate-6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP)-with a microplate reader. This method was based on the fundamental bioorganic phenomenon that when a tight binding event occurs, the effective concentration of nucleophile(s) will be significantly increased in the proximity of the P atom for a nucleophilic reaction (i.e., the cleavage of the P&bond;O bond) to take place. A very good leaving group (pK(a) of DiFMU approximately 4.7) in the phosphate monoester substrate makes the assay highly sensitive. Top reading of the nascent fluorescence makes the assay very convenient with no need to separate the particulate adjuvants from the reaction mixtures. The results from this assay are consistent with catalysis of the chromogenic phosphate mono- or diesters.
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