Kinetic Studies of Covalent Binding between N-acetyl-L-cysteine and Human Serum Albumin Through a Mixed-disulfide Using an N-methylpyridinium Polymer-based Column

Summary: The binding properties of the disulfide covalent bond between N-acetyl-L-cysteine (NAC) and human serum albumin (HSA) were investigated. HSA, purified from either healthy subjects or renal failure patients, was incubated with NAC in buffer and analyzed by 4VP-EG-Me column chromatography, which can distinguish between the redox states of the only free thiol of HSA. Although intact HSA was found to consist of mainly three sub-types, marcaptoalbumin (HMA), cysteine-bound nonmercaptoalbumin (HNA Cys ) and a further oxidized form (HNA oxy ), the formation of a new type of nonmercaptoalbumin (HNA NAC ) was confirmed after incubation with NAC. Interestingly, NAC rapidly dissociated Cys from HNA Cys and NAC itself bound very slowly to HSA. These findings suggest that the interaction between NAC and HSA proceeds in a 2-step processes. The first-order binding and dissociation rate constants of NAC to healthy HSA (k on,NAC ) and Cys from healthy HNA Cys (k off,Cys ) were approximately 0.0032and 1.3 (h −1 ), respectively. On the other hand, HSA from renal failure patients showed decreased HMA and increased HNA Cys . The k on,NAC and k off,Cys were 0.0094 and 0.45 (h −1 ), respectively, suggesting that the pathological state may affect the binding properties of HSA and NAC.