Chuang Victor Tuan Giam, Otagiri Masaki
Faculty of Pharmaceutical Sciences, Kumamoto University, Japan.
Pharm Res. 2002 Oct;19(10):1458-64. doi: 10.1023/a:1020496314081.
This study was undertaken to investigate how fatty acids cause the allosteric binding of drugs to human serum albumin (HSA). The influence of fatty acids on the binding of ketoprofen (KP), an NSAID, to HSA was examined by using a photoaffinity labeling technique.
Ultrafiltration was performed to quantitate the concentration of free KP. HSA, photolabeled with KP in the presence of myristate (MYR), octanoate, and diazepam, was cleaved with cyanogen bromide, separated by Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequently analyzed by autoradiography.
The addition of MYR at molar ratios from 4 to 5, but not from 1 to 2, causes substantial increases in unbound KP for KP:HSA ratios of 0.5 and 1. The addition of two or more moles of MYR, octanoate, and diazepam per mole of HSA caused a pronounced decrease in the labeling of the 11.6- and 13.5-kDa peptides. However, only MYR showed an increase in labeling of the 20 kDa and, especially, the 9.4-kDa peptides. At MYR:HSA ratios in excess of 3, a decrease in the extent of labeling of the 9.4-kDa peptide was observed.
Long-chain fatty acids regulate the binding properties of HSA in a complex manner, in which a simultaneous competitive and allosteric mechanism operates and which mainly involves domain I.
本研究旨在探讨脂肪酸如何导致药物与人血清白蛋白（HSA）的变构结合。通过光亲和标记技术研究了脂肪酸对非甾体抗炎药酮洛芬（KP）与HSA结合的影响。
采用超滤法定量游离KP的浓度。在肉豆蔻酸（MYR）、辛酸和地西泮存在下用KP进行光标记的HSA，用溴化氰裂解，通过Tricine十二烷基硫酸钠聚丙烯酰胺凝胶电泳分离，随后进行放射自显影分析。
对于KP:HSA比值为0.5和1的情况，以4至5的摩尔比添加MYR，但不是1至2的摩尔比，会导致未结合的KP大幅增加。每摩尔HSA添加两摩尔或更多摩尔的MYR、辛酸和地西泮会导致11.6 kDa和13.5 kDa肽段的标记显著减少。然而，只有MYR显示20 kDa尤其是9.4 kDa肽段的标记增加。当MYR:HSA比值超过3时，观察到9.4 kDa肽段的标记程度降低。
长链脂肪酸以复杂的方式调节HSA的结合特性，其中同时存在竞争和变构机制，且主要涉及结构域I。
