Wen Lili, Chen Yan, Liao Jie, Zheng Xianxian, Yin Zongning
Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of Pharmacy, Sichuan University, Chengdu, Sichuan Province, PR China.
Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of Pharmacy, Sichuan University, Chengdu, Sichuan Province, PR China.
Int J Pharm. 2015 Jan 30;478(2):753-61. doi: 10.1016/j.ijpharm.2014.12.038. Epub 2014 Dec 18.
The purpose of this study was to better understand the preferential binding behavior of arginine to protein as well as the impact of arginine on the conformational and colloidal stability of protein solution. Physical stabilities of model proteins, bovine serum albumin (BSA) and ovalbumin (OVA), were investigated by fluorescence-based and dynamic light scattering techniques in the absence and presence of arginine. We investigated the interactions between arginine and tryptophan or tyrosine residues by conducting solubility and fluorescence studies of two amino acid derivatives, N-acetyl-l-tryptophanamide (NATA) and N-acetyl-l-tyrosinamide (NAYA), in arginine solutions. The result showed that arginine preferentially bond to the aromatic amino acids of proteins mainly through hydrogen bonds and Van der Waals' forces, while the binding constant K of arginine with BSA and OVA at 298K was 41.92 and 5.77L/mol, respectively. The fluorescence quenching, the decreased fluorescence lifetime and the red-shifted ANS peak position revealed that arginine perturbed the local environment of tryptophan and tyrosine residues. We also found the attenuated electrostatic repulsion among BSA and OVA molecules after adding arginine. These findings provided strong evidence that arginine possessed negative effects on tertiary conformational and colloidal stability of BSA and OVA during the preferential binding process.
本研究的目的是更好地理解精氨酸与蛋白质的优先结合行为，以及精氨酸对蛋白质溶液构象和胶体稳定性的影响。在有无精氨酸存在的情况下，通过基于荧光和动态光散射技术研究了模型蛋白牛血清白蛋白（BSA）和卵清蛋白（OVA）的物理稳定性。我们通过对两种氨基酸衍生物N-乙酰-L-色氨酸酰胺（NATA）和N-乙酰-L-酪氨酸酰胺（NAYA）在精氨酸溶液中的溶解度和荧光研究，研究了精氨酸与色氨酸或酪氨酸残基之间的相互作用。结果表明，精氨酸主要通过氢键和范德华力优先与蛋白质的芳香族氨基酸结合，而在298K时精氨酸与BSA和OVA的结合常数K分别为41.92和5.77L/mol。荧光猝灭、荧光寿命缩短和ANS峰位红移表明精氨酸扰乱了色氨酸和酪氨酸残基的局部环境。我们还发现添加精氨酸后BSA和OVA分子之间的静电排斥减弱。这些发现提供了有力证据，表明在优先结合过程中，精氨酸对BSA和OVA的三级构象和胶体稳定性具有负面影响。
