The Preparation Of PolyCHb-SOD-CAT And The Study On Its Antioxidant Properties

Background:Increasing worldwide military and civilian needs,the infection risk of blood transfusion and the problems such as the requirement of cross matching before blood transfusion have promoted the development of blood substitutes.Hemoglobin-based oxygen carriers are one of blood substitutes which have attracted much attention.Unlike natural blood,there are many potential advantages for HBOC include convenient transportation and storage which can alleviate the shortage of clinical blood.HBOC also can overcome the risk of viral transmission from blood transfusion and can applie to emergency blood use effectively.However,the main side effects of HBOC such as oxidative stress that limit their application and further development.Our project team have been engaged in the researches of polymerized human cord blood hemoglobin(PolyCHb)for a long period of time and in the previous study the oxidative stress effect induced by polyCHb was found in the rat 70%exchange transfusion experimental model.In order to alleviate the effects of oxidative stress that PolyCHb induced.this project designed a new type of HBOC which inspired from the human body system by using glutaraldehyde as crosslinker to crosslink hemoglobin with antioxidant enzymes,tried to solve the oxidative stress side effect casing by HBOC.Objective:To prepare a polyhemoglobin with antioxidant properties by crosslinking hemoglobin,superoxide dismutase and catalase together.To have a preliminary study on its structure,function,and its antioxidant effects in vitro and vivo.Methods:Using human cord blood to extract hemoglobin,superoxide dismutase(SOD)and catalase(CAT).Using glutaraldehyde as a crosslinker to crosslink hemoglobin with antioxidant enzymes.The molecular weight distribution,the secondary structure of protein and the function of hemoglobin were analyzed through size-exclusion chromatography,circular dichroism(CD)spectra and oxygen dissociation curve respectively.For the conservation experiments in vitro,the anti-oxidability of PolyCHb-SOD-CAT was assessed by detecting the concentrations of methemoglobin,activities of superoxide dismutase(SOD)and catalase(CAT)at 4?and 25?respectively.For the in vivo study,MAP and BPM were measured and the femoral artery blood was taken at 1 hour after intravenous infusion with PolyCHb-SOD-CAT to analyze the arterial blood gas and detect several indexes such as reduced glutathione(GSH)concentration,malondialdehyde(MDA)concentration,8-hydroxy deoxyguanosine glycosides(8-OHDG)concentration,superoxide dismutase(SOD)activity,total antioxidant capacity(T-AOC)in plasma to estimate the leveal of oxidative stress in rats.Results:PolyCHb-SOD-CAT was successfully prepared with human cord blood.The second structure of this product was changed after crosslinking by glutaraldehyde,the oxygen affinity was increased and the cooperative effect was significantly decreased(P<0.01).More than 72.58 percent of the superoxide dismutase(SOD)activity was distributed in product in which the average molecular weight beyond 100 kDa,however more than 60.19 percent of the catalase(CAT)activity was distributed in product in which average molecular weight beyond 350 kDa.In 120 hours of in vitro conservation experiments,MetHb(%)of PolyCHb-SOD-CAT group at 4?increase from 4.97±0.85%to 10.19±1.43%compared with the control group from 9.37±0.77%to 14.92±0.23%;With regard to 25?,MetHb(%)of PolyCHb-SOD-CAT group increases from 4.97±0.11%to 43.58±6.87%compared with control group from 9.07±0.71%to 63.53±4.21%;The superoxide dismutase(SOD)activity of PolyCHb-SOD-CAT was stable both at 4? and 25?,whereas catalase(CAT)activity was stable at 4?and slightly decreased at 25?,from 1363.82±144.88U/gHb to 979.38±99.40 U/gHb.(P<0.05).Infusion of PolyCHb-SOD-CAT can enhance antioxidant enzyme concentration in plasma.In contrast with PolyCHb,PolyCHb-SOD-CAT induced a weaker oxidative stress,produced less MDA(P<0.01)and reduced ROS in plasma effectively(P<0.01).Conclusions:PolyCHb-SOD-CAT with oxygen carrying capacity and antioxidant ability were successfully prepared and showed the antioxidant properties both in vitro and in vivo experiments-inhibiting the self-oxidation of Hb,clearing ROS and reducing the generation of MDA.