title: 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.
authors.affiliation: Division of Pharmacology, Drug Safety and Metabolism, Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan. haradada@otsukakj.co.jp
authors.forename: Daisuke
authors.initials: D
authors.lastname: Harada
authors.forename: Makoto
authors.initials: M
authors.lastname: Anraku
authors.forename: Hikaru
authors.initials: H
authors.lastname: Fukuda
authors.forename: Shinsaku
authors.initials: S
authors.lastname: Naito
authors.forename: Kumiko
authors.initials: K
authors.lastname: Harada
authors.forename: Ayaka
authors.initials: A
authors.lastname: Suenaga
authors.forename: Masaki
authors.initials: M
authors.lastname: Otagiri
mesh_terms: D000111:Acetylcysteine / Q000032:analysis / Q000493:pharmacokinetics*; D000368:Aged; D004220:Disulfides / Q000032:analysis / Q000493:pharmacokinetics*; D005260:Female; D006801:Humans; D008297:Male; D011108:Polymers / Q000493:pharmacokinetics*; D011485:Protein Binding / Q000502:physiology; D011726:Pyridinium Compounds / Q000493:pharmacokinetics*; D012709:Serum Albumin / Q000032:analysis / Q000493:pharmacokinetics*
chemical_list: D004220:Disulfides; D011108:Polymers; D011726:Pyridinium Compounds; D012709:Serum Albumin; C014677:1-methylpyridinium; D000111:Acetylcysteine