Human serum albumin (HSA) is the most abundant protein in plasma. Cys34, the only free Cys residue, is the predominant plasma thiol and a relevant sacrificial antioxidant. Both in vivo circulating HSA and pharmaceutical preparations are heterogeneous with respect to the oxidation state of Cys34. In this work, we developed an external pH gradient chromatofocusing procedure that allows the analysis of the oxidation status of HSA in human plasma and biopharmaceutical products based on the different apparent isoelectric points and chemical properties of the redox isoforms. Specifically, reduced-mercury blocked HSA (HSA-SHg(+)), HSA with Cys34 oxidized to sulfenic acid (HSA-SOH) and HSA oxidized to sulfinate anion (HSA-SO2(-)) can be separated with resolutions of 1.4 and 3.1 (first and last pair) and hence quantified and purified. In addition, an N-terminally degraded isoform (HSA3-585) in different redox states can be resolved as well. Confirmation of the identity of the chromatofocusing isolated isoforms was achieved by high resolution whole protein MS. It is proposed that the chromatofocusing procedure can be used to produce more exact and complete descriptions of the redox status of HSA in vivo and in vitro. Finally, the scalability capabilities of the chromatofocusing procedure allow for the preparation of highly pure standards of several redox isoforms of HSA.
Keywords: 5,5′-dithiobis-(2-nitrobenzoate); AEHIC; Chromatofocusing; DEAE; DTNB; EPGC; GSH; HMA; HNA1 and 2; HSA; HSA lacking the amino terminal dipeptide Asp1-Ala2; HSA with Cys34 oxidized to sulfenic acid; HSA with Cys34 oxidized to sulfinate anion; HSA with Cys34 oxidized to sulfonate anion; HSA(3–585); HSA–SH; HSA–SH with Cys34 blocked by a Hg(II) ion; HSA–SHg(+); HSA–SO(2)(−); HSA–SO(3)(−); HSA–SOH; HSA–SSC; HSA–SSG; HSA–SSNAC; HSA–SSR; HSA–SX; Human serum albumin; N-acetylcysteine; NAC; Plasma; Posttranslational modifications; Redox isoforms; SH/HSA; anion exchange-hydrophobic interaction chromatography; diethylaminoethyl; external pH gradient chromatofocusing; human mercaptalbumin; human nonmercaptalbumin 1 and 2, according to the AEHIC fractions reported by Era et al. [1]; human serum albumin; isoelectric point; mixed disulfide of HSA with N-acetylcysteine; mixed disulfide of HSA with cysteine; mixed disulfide of HSA with glutathione; mixed disulfides of HSA and low molecular weight thiols; mol of thiol per mol of albumin; pI; reduced glutathione; reduced native HSA; spontaneous decay product of HSA–SOH.
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