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

Abstract

  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 0.0032 and 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.