Study of Modification of Drug Binding Site I of Human Serum Albumin by Sodium Hypochlorite by Time Resolved Fluorescent Spectroscopy

—The kinetics of fluorescence decay of the K-35 probe and the tryptophan amino acid residue were measured after the addition of sodium hypochlorite in human albumin serum (HSA). The K-35 fluorescent probe (excitation 405 nm, fluorescence 530 nm) binds to drug binding site I of albumin. During the experiment the molar concentration of albumin in the fraction varied from 0.3 to 20 μM. Oxidation of the fraction with hypochlorite was recorded by the change in the fluorescence of tryptophan also located in binding site I of albumin (excitation 290 nm, fluorescence 350 nm). It was shown that, at serum albumin concentration of about 30 μM, HSA has antioxidant activity which maintains its ability to bind ligands even after exposure to relatively large amounts of hypochlorite (up to 50 oxidizing molecules per protein molecule). HSA retains its ability to bind ligands because the properties of the K-35 probe during oxidation remained almost unchanged and we observed only insignificant changes in binding characteristics. The added hypochlorite is, predominantly, used to oxidize albumin amino acids, including Trp oxidation as observed in the experiment. For 14 sera of donors, we showed significant differences in the fluorescence of oxidized and native tryptophan.