Effect of ageing of human serum albumin in vitro on surface hydrophobicity and binding sites of metronidazole

Abstract

The fluorescence characteristic of the “alkaline-ageing” process was performed. The quenching of the aged form of human serum albumin (AHSA) fluorescence by acrylamide (Ac) was smaller than that of native HSA, in contrast to the negatively charged anion iodide quencher. The comparison of quenching of fluorescence probes ANS and DNSA bound to aged and native forms of HSA allows for the conclusion that “alkaline-ageing process” causes an increase of hydrophobicity within the binding site located in subdomain IIA. This conclusion was confirmed by the F coefficients calculated for the emission fluorescence spectra of A- and N-forms of HSA excited at 295 nm and 275 nm which show that the increase of hydrophobicity is more significant within tyrosyl than within tryptophanyl residues. The binding constants metronidazole–HSA as well as the number of the class of binding sites were determined by the use of the Scatchard and Klotz-plot analysis. Ageing of HSA causes an increase of the quenching constant determined from the Stern–Volmer equation for λ ex 275 nm. However ageing does not affect the K Q value for λ ex 295 nm. The influence of ageing of human serum albumin on its surface hydrophobicity was also studied with the use of 8-anilino-1-naphthalenesulfonic acid (ANS) as the fluorescence probe. At the ANS fluorescence excitation wavelength λ ex 360 nm the change in surface hydrophobicity is not observed for both N- and A-forms of HSA. The increase of surface hydrophobicity of the A-form in comparison with that of the native form at λ ex 295 nm indicates that within subdomain IIA an alteration of HSA conformation takes place.