title: Effect of different glycation agents on Cu(II) binding to human serum albumin, studied by liquid chromatography, nitrogen microwave-plasma atomic-emission spectrometry, inductively-coupled-plasma mass spectrometry, and high-resolution molecular-mass spectrometry.
abstract: The ability of human serum albumin to capture unbound copper under different clinical conditions is an important variable potentially affecting homeostasis of this element. Here, we propose a simple procedure based on size-exclusion chromatography with on-line UV and nitrogen microwave-plasma atomic-emission spectrometry (MP-AES) for quantitative evaluation of Cu(II) binding to HSA upon its glycation in vitro. The Cu-to-protein molar ratio for non-glycated albumin was 0.98 ± 0.09; for HSA modified with glyoxal (GO), methylglyoxal (MGO), oxoacetic acid (GA), and glucose (Glc), the ratios were 1.30 ± 0.22, 0.72 ± 0.14, 0.50 ± 0.06, and 0.95 ± 0.12, respectively. The results were confirmed by using ICP-MS as an alternative detection system. A reduced ability of glycated protein to coordinate Cu(II) was associated with alteration of the N-terminal metal-binding site during incubation with MGO and GA. In contrast, glycation with GO seemed to generate new binding sites as a result of tertiary structural changes in HSA. Capillary reversed-phase liquid chromatography with electrospray-ionization quadrupole-time-of-flight tandem mass spectrometry enabled detection and identification of Cu(II) coordinated to the N-terminal metal-binding site (Cu(II)-DAHK) in all tryptic digests analyzed. This is the first report confirming Cu(II)-DAHK species in HSA by means of high-resolution tandem mass spectrometry, and the first report on the use of MP-AES in combination with chromatographic separation.
authors.affiliation: Chemistry Department, Division of Natural and Exact Sciences, University of Guanajuato, L. de Retana 5, 36000, Guanajuato, Mexico.
authors.forename: Alma Rosa
authors.initials: AR
authors.lastname: Corrales Escobosa
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Wrobel
authors.forename: Eunice
authors.initials: E
authors.lastname: Yanez Barrientos
authors.forename: Sarahi
authors.initials: S
authors.lastname: Jaramillo Ortiz
authors.forename: Alejandra Sarahi
authors.initials: AS
authors.lastname: Ramirez Segovia
authors.forename: Kazimierz
authors.initials: K
authors.lastname: Wrobel
mesh_terms: D001665:Binding Sites; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D019327:Copper Sulfate / Q000032:analysis* / Q000737:chemistry; D005947:Glucose / Q000737:chemistry*; D006031:Glycosylation; D006037:Glyoxal / Q000737:chemistry*; D006038:Glyoxylates / Q000737:chemistry*; D006801:Humans; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011765:Pyruvaldehyde / Q000737:chemistry; D012709:Serum Albumin / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D013054:Spectrophotometry, Atomic; D053719:Tandem Mass Spectrometry
chemical_list: D006038:Glyoxylates; D012709:Serum Albumin; D006037:Glyoxal; D011765:Pyruvaldehyde; D005947:Glucose; C031150:glyoxylic acid; D019327:Copper Sulfate