A Preliminary Study On The Biological Characters Of Antioxidant Enzymes Cross-linked Polyhemoglobin

Antioxidant enzymes cross-linked polyhemoglobin(PolyHb-SOD-CAT)is a kind of red blood cell substitutes.To establish a method to prepare PolyHb-SOD-CAT and to analyse the biological characters,we isolated Hb from red blood cell and purified Hb using Anion Exchange Chromatography.Glutaraldehyde was used to crosslink hemogloibin and antioxidant enzymes.PolyHb-SOD-CAT(300kD)was prepared with hollow-fiber ultrafiltration,and was concentrated,frozen in-20℃.The diameter was detected using COULTER N4 Plus particle size analyzer. Drabkins method was used to measure the change of MetHb%of the product preserved in different time and temperature;change of neutrophil phagocytosis of the cells and activation of T lymphocytes and platelet incubated with products was estimated using flow cytometry;aggregation test was made to measure the function of serum accelerin incubated with products;rate of elimination of the products infused in the mouse was detected using Western Blot.Our results reveal that characters of the product were as follow:MetHb%6.61±1.42%,particle size 29nm;products preserved in different time and temperature were autoxidation stable,and PolyHb-SOD-CAT appeared to be more stable than PolyHb during the incubation(37℃8h);no significant influence on the function of neutrophil phagocytosis and serum accelerin aggregation was measured;T lymphocytes and platelet stayed inactivated during the incubation;and the product could still be detected 48h after infused into the mouse.ELISA tests showed weak or no antigenicity.Besides,to investigate the antioxitant character of PolyHb-SOD-CAT,We examined the effect of PolyHb-SOD-CAT on human umbilical vein endothelial cells subjected to hydrogen peroxide(H₂O₂).The level of glutathione(GSH)and Ca²⁺in cells were measured using the chemical colorimetry.Location of NF-kB was detected using the imrnunohistochemistry technique.Expression of the endothelial nitric oxide synthase(eNOS)mRNA and heme oxygenase-1(HO-1)mRNA were assessed by RT-PCR.Cellular death was evaluated by flow cytometry.Our results reveal that in H₂O₂ group,content of GSH in cells was 0.0064±0.0039 mmol/L,content of Ca²⁺in cells was 690.05±145.11 nmol/ml,and incubation with H₂O₂ alone produced greater activation of NF-kB,,greater expression of eNOS and HO-1 mRNA and increases in cellular death compared with control.However,comparing with H₂O₂ alone,content of GSH in cells was 0.0214±0.0098 mmol/L,content of Ca²⁺in cells was 172.43±24.09 nmol/ml in PolyHb-SOD-CAT+H₂O₂ group,most NF-kB was still in cytoplasm, expression of eNOS and HO-1 mRNA and cellular death decreased.PolyHb-SOD-CAT inhibited H₂O₂-induced oxidative damage to vascular endothelial cells.The above results showed that PolyHb-SOD-CAT could be a kind of potential safe and effective red blood cell substitutes with better antioxidant stability,and could alleviate ischemia-reperfusion injury.