Kaneko K, Chuang V T G, Ito T, Suenaga A, Watanabe H, Maruyama T, Otagiri M
Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan.
Pharmazie. 2012 May;67(5):414-8.
Arylpropionic acid nonsteroidal anti-inflammatory drusg (NSAIDs) primarily bind to subdomain III A (site II) of human serum albumin (HSA). Ketoprofen (KP), an arylpropionic acid that contains a photoreactive benzophenone moiety, was used to photolabel the binding region of site II. LC/Q-TOF mass spectrometry determination revealed that R485 was the amino acid residue that formed covalent adduct with the benzophenone moiety of KP. Point mutation of arginine 485 to alanine showed a slight decrease in the overall binding percentage of KP when compared to that of native HSA. The induced circular dichroism spectral data of KP with both R485A and native albumin confirmed the photolabeling findings. Interestingly, an increase in the extent of [14C]KP covalent adduct formation with the 11.6 kDa peptide derived from subdomain IIB-IIIA was observed for R485A. In contrast, mutation of arginine 410 caused a significant reduction of binding percentage, confirming the importance of this residue in high affinity binding of arylpropionic acid derivatives. This may indicate that while KP's carboxylate interacts electrostatically with arginine 410, the benzophenone moiety may have swung away from helix 6 in the absence of arginine 485. In this study, photolabeling of native and mutants albumins, R485A and R410C with [14C]KP confirmed that R485 involved in the non-electrostatic interaction with the benzophenone moiety of KP, but not vital to hold KP in the binding pocket of subdomain IIIA.
芳基丙酸类非甾体抗炎药（NSAIDs）主要与人血清白蛋白（HSA）的亚结构域IIIA（位点II）结合。酮洛芬（KP）是一种含有光反应性二苯甲酮部分的芳基丙酸，用于对位点II的结合区域进行光标记。液相色谱/四极杆飞行时间质谱测定表明，R485是与KP的二苯甲酮部分形成共价加合物的氨基酸残基。与天然HSA相比，将精氨酸485突变为丙氨酸后，KP的总体结合百分比略有下降。R485A和天然白蛋白与KP的诱导圆二色光谱数据证实了光标记结果。有趣的是，对于R485A，观察到与源自亚结构域IIB-IIIA的11.6 kDa肽形成的[14C]KP共价加合物形成程度增加。相比之下，精氨酸410的突变导致结合百分比显著降低，证实了该残基在芳基丙酸衍生物高亲和力结合中的重要性。这可能表明，虽然KP的羧酸盐与精氨酸410发生静电相互作用，但在不存在精氨酸485的情况下，二苯甲酮部分可能已从螺旋6摆动开。在本研究中，用[14C]KP对天然白蛋白和突变体白蛋白R485A和R410C进行光标记，证实R485参与了与KP二苯甲酮部分的非静电相互作用，但对于将KP保持在亚结构域IIIA的结合口袋中并非至关重要。
