The interplay between copper(II), human serum albumin, fatty acids, and carbonylating agent interferes with Cys 34 thiol reactivity and copper binding.

Cys34 thiol group of human serum albumin (HSA) represents major plasma antioxidant. Its reactivity is influenced by multiple factors. The influence of fatty acids (FA; saturated, mono, and poly unsaturated acids from fish oil) binding to HSA, on copper(II) binding affinity and Cys34 thiol group accessibility/reactivity, in the presence of carbonylation agent (methylglyoxal, MG) was examined.

Quantification of total content of non-esterified fatty acids bound to human serum albumin.

Non-esterified fatty acids bound to the human serum albumin (HSA) contribute to several HSAs properties of special concern in pathologies, for instance to the reactivity of the free HSA-Cys34 thiol group (important antioxidative thiol pool in plasma), and to the affinity for binding of molecules and ions (for example cobalt as a prominent biomarker in heart ischemia). Therefore, the method for determination of FAs bound to HSA was developed. FAs were released from HSA (previously isolated from serum by ammonium sulfate precipitation) using acidic copper(II) sulfate in phosphoric acid, extracted by n-heptane-chloroform (4:1, v/v) mixture, spotted on TL silica-gel and then developed with n-heptane-chloroform-acetic acid (5:3:0.

Binding of enterolactone and enterodiol to human serum albumin: increase of cysteine-34 thiol group reactivity.

The interaction of polyphenolic molecules with human serum albumin (HSA) could lead to changes in the reactivity of the HSA Cys34 thiol group (HSA-SH). The influences of enterolactone (EL) and enterodiol (ED) binding on HSA-SH reactivity in fatty acid (FA)-free HSA, and in HSA with bound stearic acid (S) in S/HSA molar ratios of 1:1 and 4:1, were investigated by the determination of the pseudo first order rate constants (k') for the thiol reaction with 5,5'-dithiobis-(2-nitrobenzoic acid). The binding affinities and binding sites of EL and ED were also determined, using fluorescence measurements of the intrinsic fluorescence of Trp214 and diazepam (binding site marker).

HSA carbonylation with methylglyoxal and the binding/release of copper(II) ions.

The potential of carbonylation with methylglyoxal to alter HSA's binding affinity for copper(II) ions and its influence on the release of copper(II) ions from copper-HSA complexes were studied. The affinity of HSA to coordinate copper(II) decreased upon carbonylation of the Cys34-SH group. Carbonylation of copper-HSA complexes caused a decrease in Cys34-SH content, conformational changes and the release of copper(II) ions.

Fatty acids binding to human serum albumin: Changes of reactivity and glycation level of Cysteine-34 free thiol group with methylglyoxal.

Fatty acids (FAs) binding to human serum albumin (HSA) could lead to the changes of Cys-34 thiol group accessibility and reactivity, i.e. its scavenger capacity and antioxidant property.

The efficiency of compounds with α-amino-β-mercapto-ethane group in protection of human serum albumin carbonylation and cross-linking with methylglyoxal.

α-Oxoaldehydes, which are produced in higher quantities in diabetes, uremia, oxidative stress, inflammation and aging, react with the amino, guanidine and thiol groups of proteins and cause the formation of advanced glycated end-products and protein cross-linking. To prevent these reactions, the efficiency of low molecular mass thiols with an α-amino-β-mercapto-ethane group (Cys, penicillamine and N-acetylcysteine (NAcCys, with a blocked amino group)) as scavengers of methylglyoxal, compared with glutathione (GSH) and the biguanidine derivative metformin, was investigated. The time courses of the reactions of the aforementioned compounds with methylglyoxal were assayed.

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.

Improving the reliability of human serum albumin-thiol group determination.

The thiol (Cys34) content of human serum albumin (HSA-SH) decreases during oxidative and carbonyl stress and, therefore, could represent a useful parameter in clinical practice. Nevertheless, the reliability of HSA-thiol determination with Ellman's method depends on the purity of isolated HSA. Determination of total serum thiols (mmol/L) and HSA-SH content (mmol -SH/mmol HSA) after HSA isolation from diabetic patient and control sera by a two-step precipitation with ammonium sulfate (AS), as well as HSA-SH contribution (%) to total serum thiols, was assessed.