Photosensitized paraquat-induced structural alterations and free radical mediated fragmentation of serum albumin

Abstract

Photosensitization of paraquat with photosynthetically active radiations (PAR) induced substantial production of both the hydroxyl radicals ( ⋅OH) and superoxide anions (O 2 ⋅−) under in vitro conditions. Addition of transition metal ion, Cu (II) enhanced the paraquat-induced ⋅OH radical production by 1.8-fold. Treatment of bovine serum albumin (BSA) with photosensitized paraquat resulted in a dose dependent fragmentation of protein. The quantitative analysis revealed the release of 73 μM acid soluble amino groups and 450 μM carbonyl groups from treated albumin at the highest albumin–paraquat molar ratio of 1:8 in presence of 200 μM Cu (II). The results with the free radical quenchers such as mannitol and superoxide dismutase (SOD) clearly reflected the involvement of ⋅OH radicals in protein fragmentation process. The fluorescence data revealed significantly higher binding of paraquat with serum albumin. The binding constants ( K a) and binding capacity ( n) of albumin for paraquat were determined to be 3.4×10 5 l/mole and 12.9, respectively. Fluorescence emission spectra exhibited significant quenching of the intrinsic fluorescence of albumin upon addition of paraquat at increasing molar ratios. This is attributed to induced conformational changes in protein structure upon paraquat interaction at specific sites on albumin molecule. Most likely, the alkyl group transfers occur from N1 and/or N1′ positions of paraquat to the electron rich sites at critical amino acid residues on treated protein. At higher paraquat concentrations, the albumin–paraquat interaction resulted in adduct formation with concurrent protein alkylation and free radical mediated fragmentation. Thus, on the basis of these results, the paraquat–protein interaction leading to alkylation, structural alterations and/or fragmentation of biological macromolecules has been suggested as an important factor for agrochemical-induced toxicity.