Studies On The Interaction Of SO₂ And SO₂ Derivatives With Protein By Fluorescence Spectrophtometry

Sulfur dioxide (SO₂) is one of the most important air pollutants all over the world, which exists as low concentrations in the atmosphere and high concentrations in some industrial areas. SO₂ has been paid more and more attention to its toxic effects. Recently few data are available on the interaction and molecular mechanism between SO₂ and protein in vivo/ vitro, although some biochemical changes, genetic toxicity, oxidative stress and DNA damage have been detected.In order to explore the interaction mechanism of SO₂ and SO₂ derivatives with protein, the spectrum feature of bovine serum albumin (BSA) with BSA-SO₂ derivative system was observed by using fluorescence spectrum assay and ultraviolet absorbance spectrum assay. Then the effects of protein oxidative damage and DNA damage induced by SO₂ derivatives in different tissues of mice were studied. It will provide based data for the protein oxidative damage, nucleic acid damage, the occurrence of diseases, development, the prevention, control and clinical diagnosis.1. Studies on the interaction of SO₂ and SO₂ derivatives with BSA by Fluorescence SpectrophtometryIt was found that the two results are hardly different. The results suggest that SO₂ and SO₂ derivatives can quench the fluorescence of BSA in stimulanting human physiological conditions. The relative dynamics and thermodynamics constants were gained through the equations of Stern-Volmer and Lmeweaver-Burk at different temperatures. According to these constants, it was concluded that the interaction reaction between SO₂ (or SO₂ derivatives) and BSA is exothermal; the binding reaction was mainly hydrophobic and electrostatic forces; the quenching mechanism belonged to static quenching. The binding sites between them are 0.8042 and 0.9345; the binding constants are 1.15×10⁴ L·mol^-1 and 2.257×10³ L·mol^-1, respectly. It showed that the conjugation reaction between SO₂ and BSA is weaker, only about a binding site. The result is the same as the lower quenching rate of BSA added by SO₂. Three-dimensional fluorescence spectra showed that after SO₂ was added, it happend to the microenvironment of BSA changing, an significant blue shift of the fluorescence peak and the Stokes shift decreasing, but the shape of fluorescence peak had no significant change. This study suggested that SO₂ and SO₂ derivative have a similar role in the interaction of BSA. The study showed indirectly that when SO₂ was inhaled into the body, it would immediately transform to vivo derivatives (sulfite and bisulfite).2. Protein oxidative damage and DNA-protein crosslinks in tissues from mice induced by SO₂ derivative(1) The measurement results of protein carbonyl (PCO) content in different tissues of miceThe protein carbonyl content in different tissues of mice was measured by using spectrophotometric DNPH assay to reflect the degree of protein oxidative damage. The result shows that SO₂ derivatives could lead to protein oxidative damage in liver, lung, heart, spleen and stomach tissues, and it implied that SO₂ derivatives could lead to PCO in a dose-dependent manner. The PCO contents of liver tissue in mice increase (P<0.01); the PCO contents of lung and heart tissue increase (P<0.05) in group of 0.025 g·kg^-1 bw. Although there is an increase in spleen and stomach, it has no significant difference in statistic. At 0.1, 0.4 g·kg^-1 bw, the protein carbonyl contents in liver, lung, heart, spleen increase significantly (P<0.001), while the protein carbonyl contents in stomach also rise(P<0.01).(2) The measurement results of DNA-protein crosslinks (DPC) coefficients in different histiocytes of miceThe DNA-protein crosslinks (DPC) coefficients was measured by using KCl-SDS assay. It will be used to estimate the degree of damage of nucleic acid induced by SO₂ derivatives. The result indicated that SO₂ derivatives caused the damage of DNA in liver, lung, heart, spleen and stomach tissues of mice. It was showed that the degrees of DPC levels in different tissues increased as the concentration of SO₂ derivatives rise. It presents an obvious concentration-dependent manner. All of the related correlation coefficients are higher than 0.9. Compared with the control, the DPC coefficients in liver have highly significant increase (P<0.001); the DPC coefficients in lung, stomach increase significantly (P<0.01) in group of 0.025 g·kg^-1 bw. Although there is an increase in heart and spleen, it has no significant difference in statistic. When the injection doses are 0.1 and 0.4 g·kg^-1 bw, the DPC coefficients in liver, lung and stomach have very significant increase (P<0.001); the DPC coefficients of heart and spleen have highly significant increase (P<0.01).(3) ConclusionIn all, injection of SO₂ derivatives could cause the formation of PCO and DPC in liver, lung, heart, spleen and stomach tissues. The increases of PCO and DPC contents share the same regulation in a concentration (SO₂ derivatives)-dependent manner. It indicated that SO₂ derivatives cloud lead to protein oxidative damage and nucleic acid damage. The results support the topic that SO₂ is a systemic toxicant.