The influence of fatty acids on determination of human serum albumin thiol group

During investigation of the changes of the Cys34 thiol group of human serum albumin (HSA) (isolated by affinity chromatography with Cibacron Blue (CB)) in diabetes, we found that the HSA-SH content was higher (11-33%) than the total serum thiol content. The influence of fatty acids (FA) binding to HSA on this discrepancy was investigated in vitro (using fluorescence and CD spectroscopy and GC) and with HSA samples from diabetic (n=20) and control groups (n=17). HSA-bound FA determine the selection of HSA molecules by CB and enhance reactivity and/or accessibility of the SH group. A high content of polyunsaturated FA (35.6%) leads to weaker binding of HSA molecules to CB. Rate constants of DTNB reaction with the SH group of HSA applied to a CB column, bound-HSA and unbound-HSA fractions, were 4.8 x 10(-3), 21.6 x 10(-3), and 11.2 x 10(-3) s(-1), respectively. The HSA-SH group of diabetics is more reactive compared with control individuals (rate constants 20.9 x 10(-3)+/- 4.4 x 10(-3) vs 12.9 x 10(-3)+/- 2.6 x 10(-3) s(-1), P lt 0.05). Recovery values of the SH group obtained after chromatography of HSA with bound stearic acid ranged from 110 to 140%, while those for defatted HSA were from 98.5 to 101.7%. Thus, HSA-bound FA leads to an increase of HSA-SH content and a contribution to total serum thiols, which make the determination of the thiol group unreliable. (C) 2013 Elsevier Inc. All rights reserved