title: HPLC separation of human serum albumin isoforms based on their isoelectric points.
abstract: 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.
authors: Laboratorio de Enzimología, Facultad de Ciencias, Universidad de la República, Montevideo, 11400, Uruguay.|Laboratorio de Fisicoquímica Biológica, Facultad de Ciencias, Universidad de la República, Montevideo, 11400, Uruguay.|Center for Free Radical and Biomedical Research, Universidad de la República, Montevideo, 11800, Uruguay.
authors: Lucía
authors: L
authors: Turell
authors: Center for Free Radical and Biomedical Research, Universidad de la República, Montevideo, 11800, Uruguay.|Unidad de Cristalografía de Proteínas, Institut Pasteur de Montevideo, Montevideo, 11400, Uruguay.
authors: Horacio
authors: H
authors: Botti
authors: Center for Free Radical and Biomedical Research, Universidad de la República, Montevideo, 11800, Uruguay.|Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo, 11800, Uruguay.
authors: Lucía
authors: L
authors: Bonilla
authors: Laboratorio de Enzimología, Facultad de Ciencias, Universidad de la República, Montevideo, 11400, Uruguay.|Center for Free Radical and Biomedical Research, Universidad de la República, Montevideo, 11800, Uruguay.
authors: María José
authors: MJ
authors: Torres
authors: Department of Pharmacology and Chemical Biology, University of Pittsburgh Medical Center, Pittsburgh, PA, 15213, USA.
authors: Francisco
authors: F
authors: Schopfer
authors: Department of Pharmacology and Chemical Biology, University of Pittsburgh Medical Center, Pittsburgh, PA, 15213, USA.
authors: Bruce A
authors: BA
authors: Freeman
authors: Laboratorio de Biofármacos: Control de Calidad y Desarrollo, Institut Pasteur de Montevideo, Montevideo, 11400, Uruguay.
authors: Larissa
authors: L
authors: Armas
authors: Laboratorio de Biofármacos: Control de Calidad y Desarrollo, Institut Pasteur de Montevideo, Montevideo, 11400, Uruguay.
authors: Alejandro
authors: A
authors: Ricciardi
authors: Laboratorio de Enzimología, Facultad de Ciencias, Universidad de la República, Montevideo, 11400, Uruguay.|Center for Free Radical and Biomedical Research, Universidad de la República, Montevideo, 11800, Uruguay.
authors: Beatriz
authors: B
authors: Alvarez
authors: Center for Free Radical and Biomedical Research, Universidad de la República, Montevideo, 11800, Uruguay.|Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo, 11800, Uruguay.
authors: Rafael
authors: R
authors: Radi
mesh_terms: D000368:Aged; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008297:Male; D008875:Middle Aged; D020033:Protein Isoforms / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D012709:Serum Albumin / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification*
chemical_list: D020033:Protein Isoforms; D012709:Serum Albumin
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