Hasan A, Smith J B, Qin W, Smith D L
H.E.J. Research Institute of Chemistry, University of Karachi, Pakistan.
Exp Eye Res. 1993 Jul;57(1):29-35. doi: 10.1006/exer.1993.1095.
Acetylation of the lysines of bovine lens alpha A-crystallin has been examined after 0-48-hr incubations of whole alpha-crystallins in 100 mM aspirin. The alpha A-crystallins were isolated after the incubation, proteolytically digested into peptides which were separated by reversed phase HPLC and analysed by mass spectrometry. For the reaction conditions used in this investigation, acetylated lysyl residues were the principal products. The extent of acetylation was quantified from the intensities of the peaks in the fast atom bombardment mass spectra of the modified and unmodified peptides. The modified lysine containing peptides demonstrated that all seven lysyl residues of alpha A-crystallin reacted with aspirin; the extent of acetylation at each lysyl residue varied. Plots of the extent of acetylation vs. time were used to calculate rate constants for the reaction at each lysyl residue. The rate constant for the acetylation of Lys 166, the most reactive, was about seven times greater than for Lys 88, the least reactive. These rate constants were used to calculate the yield of predicted products for the reaction of alpha-crystallin with therapeutic concentrations of aspirin. Comparison of the yield of acetylated alpha-crystallin with the yield of carbamylated alpha-crystallin that might occur due to renal failure indicates that aspirin is not likely to be an effective inhibitor of cataract due to carbamylation of lysyl residues.
将整个α-晶体蛋白在100 mM阿司匹林中孵育0 - 48小时后，检测了牛晶状体αA-晶体蛋白赖氨酸的乙酰化情况。孵育后分离出αA-晶体蛋白，将其用蛋白酶消化成肽段，通过反相高效液相色谱分离并进行质谱分析。对于本研究中使用的反应条件，乙酰化的赖氨酰残基是主要产物。从修饰和未修饰肽段的快原子轰击质谱图中峰的强度对乙酰化程度进行定量。含修饰赖氨酸的肽段表明αA-晶体蛋白的所有七个赖氨酰残基都与阿司匹林发生了反应；每个赖氨酰残基的乙酰化程度各不相同。用乙酰化程度与时间的关系图来计算每个赖氨酰残基反应的速率常数。反应活性最高的Lys 166的乙酰化速率常数约为反应活性最低的Lys 88的七倍。这些速率常数用于计算α-晶体蛋白与治疗浓度阿司匹林反应的预测产物产率。将乙酰化α-晶体蛋白的产率与因肾衰竭可能产生的氨甲酰化α-晶体蛋白的产率进行比较，结果表明阿司匹林不太可能因赖氨酰残基的氨甲酰化而成为有效的白内障抑制剂。
