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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

Corrales Escobosa, Alma Rosa, Wrobel, Katarzyna, Yanez Barrientos, Eunice, Jaramillo Ortiz, Sarahi, Ramirez Segovia, Alejandra Sarahi, Wrobel, Kazimierz
Analytical and bioanalytical chemistry 2015 v.407 no.4 pp. 1149-1157
binding sites, copper, gel chromatography, glucose, glycation, homeostasis, human serum albumin, nitrogen, quantitative analysis, reversed-phase liquid chromatography, tandem mass spectrometry
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.