PEGylation Of Glutathione Peroxidase 1 And Characterization

Reactive oxygen（ROS）is the intermediate product of cell metabolism.With appropriate concentration,ROS takes part in the regulation of various important signal transductions and the functions of cell.Under normal physiological conditions,the production and elimination of ROS keep dynamic balance to support physiological function of cells.However,the balance will be broken after coming to adverse environments.The abnormal accumulation of ROS within cells will bring damage to DNA,protein and lipid,leading to many health-threating diseases,such as:cardiovascular disease,diabetes,ischemia-reperfusion injury,aging or even cancer.Glutathione peroxidase（GPx）is an important antioxidase,scavenging various peroxides,including H₂O₂,and fighting aging and pathological changes caused by reactive oxygen species（ROS）.Among them,GPx1 is the first discovered selenium-containing antioxidant enzyme and can efficiently catalyze the reduction of H₂O₂.GPx1 can also take part in the signal pathway of insulin and influence occurrence and development of diabetes.However,due to the limited source and poor stability,GPx1 is difficult to be used in clinic for various disease therapies.To obtain long-term and stable protein drugs,chemical modification and genetic engineering method were usually used.PEG,as a FDA approved material used for research and production of drugs and cosmetics,is a biocompatible biomaterial.PEGylation technique is often used for protein modification to lower immunogenicity and prolong the half-life and stability.This method has been successfully applied in many protein drugs and peptide drugs,and some of them have already been commercialized.In current study,we chose 5 kD-mPEG-SC to modify the amino groups of GPx1.First of all,we determined the optimal condition of this reaction by controlling the molar ratio of GPx1 to mPEG-SC,the reaction time,the pH of the reaction system and the reaction temperature.The product was characterized by SDS-PAGE,MALDI TOF and 1H-NMR.And we obtained the final product by dialysis and molecular sieve.The changes of particle size and Zeta potential before and after modification were evaluated by Mastersizer Micro.We studied the enzyme activity and catalytic kinetics of the modified product and compared the stability of GPx1 and SC-mPEG-GPx1.We obtained the optimum reaction conditions,that is,the pH is 8.0,the molar ratio is 1:60 at 20℃for 20 min.SDS-PAGE showed that mPEG-SC was successfully modified onto GPx1.As expected,the molecular weight of SC-mPEG-GPx1 was 29154D determined by MALDI TOF.The size distribution and Zeta potential of SC-mPEG-gpx1 was evaluated by Dynamic Light SCattering（DLS）.As the results indicated SC-mPEG-GPx1 is smaller than GPx1 and we speculated it was easier to transport in body.In this study,we were mostly concerned about the activity of SC-mPEG-GPx1 after PEGylation.In order to exclude the effect of free mPEG-SC on activity determination,free mPEG-SC was used as a control.As the result shown that mPEG-SC have no effect on the catalytic activity.The activity of SC-mPEG-GPx1 is 202.16 U/mg.Kinetic analysis of SC-mPEG-GPx1 revealed it’s a typical ping-pong mechanism.In addition,we explored the optimal catalysis condition of SC-mPEG-GPx1.When the temperature is 37℃and the pH is 9,the SC-mPEG-GPx1 had the maximum activity.For the stability study,we kept GPx1 and SC-mPEG-GPx1 at 4℃for several days.As the results indicated the stability of SC-mPEG-GPx1 was much higher than that of GPx1 and about 20%activity was reserved after 115 days’incubation.In summary,we successfully modified GPx1 with mPEG-SC,and found that SC-mPEG-GPx1 has higher catalytic activity,which will advance the application of glutathione peroxidase 1 in clinic in near future.