The study on the interaction between human serum albumin and a new reagent with antitumour activity by spectrophotometric methods

In this work, the binding of 2-hydroxy-3-nitro-9-fluorenone (HNF; a new reagent with antitumour activity) to human serum albumin (HSA) was investigated by fluorescence spectroscopy combined with UV-Vis absorption, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectrophotometric techniques under simulative physiological conditions for the first time. A strong fluorescence quenching reaction of HNF to HSA was observed and the quenching mechanism was suggested as static quenching according to the Stern-Volmer (S-V) equation. The binding constants of HNF with HSA at 300, 3 10 and 320 K were calculated as 6.08 x 10(5), 3.80 x 10(5) and 2.79 x 10(5) M-1, respectively, and corresponding numbers of binding sites (n) were 1.1, 1.0 and 1.0. Experimental results observed showed that the binding of HNF to HSA induced conformational change of HSA. The quantitative analysis data of CD spectra from that of the alpha-helix 60.3% in free HSA to 56.5% in the HNF-HSA complex further confirmed that the secondary structure of the protein was modified by HNF. The thermodynamic parameters, standard enthalpy change (DeltaHdegrees) and the standard entropy change (DeltaSdegrees), were obtained to be -31.10 kJ mol(-1) and 6.87 J mol(-1) K-1, respectively, which indicated that a hydrophobic force played a major role in the interaction of HNF with HSA. All these experimental results and theoretical data clarified that HNF could bind to HSA and be effectively transported and eliminated in body, which could be a useful guideline for further drug design. (C) 2004 Elsevier B.V. All rights reserved.