Radioprotective Effects Of Novel Bifunctional Antioxidant Enzyme

Two bifunctional antioxidant enzymes were developed as promising radioprotectors.DNA fragment of SOD1-R9 was the fusion of human CuZn-SOD(SOD1)and cell permeable peptide R9(RRRRRRRRR)and SOD1-X-R9 were the fusion of human CuZn-SOD(SOD1),cell permeable peptide R9(RRRRRRRRR)or substrate peptide of matrix metalloproteinase X(PLGLAG).Two DNA fragments were synthesized and inserted into the prokaryotic expression vector pGEX-4T-1 with GST tag,respectively.DNA constructs were then transformed into Escherichia coli BL21(DE3)and the highest levels of soluble protein production were obtained when the expression was induced with IPTG(0.2 mmol/L)at 25 ? for 7 h and 11 h,respectively.Two bifunctional antioxidant enzymes GST-SOD1-R9(46 kDa)and GST-SOD1-X-R9(47 kDa)were purified subsequently with ultrasonication of bacterial lysate,ammonium sulfate precipitation,and GST resin column affinity chromatography.They were verified by SDS-PAGE and special enzymatic activity.SOD and GST activities of GST-SOD1-R9 were 2388 and 227 U/mg,respectively and those activities of GST-SOD1-X-R9 were 2954 and 328 U/mg,respectively.Both two proteins lost their antioxidant enzyme activities rapidly when they were heat treating over 50 ? or their pH value were below 5.0.But both of them were stable under physiological condition,and GST-SOD1-X-R9 was more stable than GST-SOD1-R9.In addition,both GST-SOD1-X-R9 and GST-SOD1-R9 were cell permeable and the latter was superior to the former.Furthermore,the protective effect of two bifunctional antioxidant enzymes on 6 Gy whole body irradiated mice was investigated.In this study,a significant deficit in the body weight,the number of peripheral white blood cells,hepatic antioxidant system,and shrunken spleen and thymus were observed in mice of the X-ray irradiation alone group on the 8th day after radiotherapy.Two bifunctional antioxidant enzymes pretreatment were proved to be radioprotective on the whole body irradiated mice not only by significantly enhancing the number of white blood cells but also by significantly enhancing thymus index and spleen index,reducing hepatic MDA level and enhancing hepatic antioxidant enzymes abilities.The high-dosage level of GST-SOD1-R9(1.5 mg/mL)presented comparable radioprotective effect to amifostine while middle-and high-dosage levels of GST-SOD1-X-R9(0.75 mg/mL and 1.5 mg/mL)were superior to GST-SOD1-R9 and amifostine.Especially the middle-dosage levels of GST-SOD1-X-R9,it showed best radioprotective effect.In summary,the bifunctional antioxidant enzyme could be a novel radioprotector and perfect substitution of amifostine for their convenient production technology and remarkable radioprotective ability.