Kinetic analysis of covalent binding between N-acetyl-L-cysteine and albumin through the formation of mixed disulfides in human and rat serum in vitro
Kinetic analysis of covalent binding between N-acetyl-L-cysteine and albumin through the formation of mixed disulfides in human and rat serum in vitro
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DOI：
10.1023/a:1020749211745
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发表时间：
2002-11-01
影响因子：
3.7
通讯作者：
Otagiri, M
中科院分区：
文献类型：
--
作者：
Harada, D;Naito, S;Otagiri, M
目的。通过体外实验对N-乙酰-L-半胱氨酸与白蛋白的共价结合进行了动力学评价。将C-14-NAC与人或大鼠血清孵育后，用阴离子交换高效液相色谱法进行分析。白蛋白结合的C-14-NAC通过测量白蛋白部分中的放射性来定量。紫外色谱图和/或放射色谱图表明C-14-NAC与人或大鼠白蛋白之间存在稳定的共价键。通过分析该蛋白在血清中结合的时间依赖性，得到了该蛋白的一级结合和解离速率常数(k(On)和k(Off))。血清在二氧化碳培养箱中处理，以避免氧化干扰，并分别测定初始速率。人和大鼠血清的Kon值分别为0.33(h(-1))和0.48(h(-1))。需要L-半胱氨酸引发与白蛋白结合的C-14-NAC的解离。加入适量的L半胱氨酸后，人和大鼠血清的Koff值分别为0.30~1.0h(-1)和0.54~1.4h(-1)。大鼠血清的k(ON)和K(OFF)值与NAC在体内的血浆蛋白结合动力学一致，表明该方法是一种可靠的体外蛋白结合评价方法。人血清和大鼠血清之间的蛋白质结合动力学没有物种差异。
Purpose. Covalent binding between N-acetyl-L-cysteine (NAC) and albumin was evaluated kinetically by conducting in vitro experiments.Methods. After C-14-NAC was incubated with human or rat serum, the solution was analyzed by anion-exchange HPLC. The albumin-bound C-14-NAC was quantified by measuring the radioactivity in the albumin fraction.Results. Ultraviolet chromatograms and/or radiochromatograms indicated the presence of a stable covalent bond between C-14-NAC and either human or rat albumin. By analyzing the time dependence of this protein binding in serum, the first-order binding and dissociation rate constants (k(on) and k(off)) were obtained. The serum was treated in a CO2 incubator to avoid oxidative interference, and the initial rates were determined separately. The kon values obtained were 0.33 (h(-1)) and 0.48 (h(-1)) for human and rat serum, respectively. L-Cysteine was required to initiate the dissociation of C-14-NAC bound to albumin. Following the addition of appropriate amounts of L-cysteine, the koff values were determined to be 0.30-1.0h(-1) and 0.54-1.4h(-1) for human and rat serum, respectively.Conclusions. The k(on) and k(off) values obtained for rat serum were in good agreement with the in vivo plasma protein binding kinetics of NAC in rats, indicating the reliability of this in vitro method for evaluating protein binding. No species differences in protein binding kinetics were found between human and rat serum.