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

Abstract

Purpose: Covalent binding between N-acetyl-L-cysteine (NAC) and albumin was evaluated kinetically by conducting in vitro experiments.

Method: After 14C-NAC was incubated with human or rat serum, the solution was analyzed by anion-exchange HPLC. The albumin-bound 14C-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 14C-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 k(on) 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 14C-NAC bound to albumin. Following the addition of appropriate amounts of L-cysteine, the k(off) values were determined to be 0.30-1.0 h(-1) and 0.54-1.4 h(-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.