Polyethylene Glycol Monomethyl Oxygen Radicals - Aldehyde (mpeg - Ald) Chemical Modification Research Of Superoxide Dismutase (sod)

ObjectiveSuperoxide dismutase (SOD) can protect the organism through scavenging superoxide anion. It was widely used for anti-radiation agent, anti-aging agent, anti-inflammatory agent, and so on. More and more important position was held in medical value. However, because the half-life of SOD elimination (usually intravenous only6-15min) was very short in vivo, this restricted in its clinical effectiveness and practicality. Modified SOD by chemical methods to increased its stability and its half-life in vivo become a hot realm of research.Methods:The modified SOD process by monomethoxypoly(ethylene glycol)-aldehyde (mPEG-ALD, mw5,000) was established in my master’s degree paper. Firstly in order to explore if it is possible for Aldehyde group to modify amino group, small moleculer compounds such as para hydroxy benzaldehyde and L-Lysine was subjected to coupling as a model material instead of expensive SOD to get a optimal modified condition. After the pre-experiment succeeded, we use raPEG-ALD to modify SOD. Then according to the established conditions, modified SOD was prepared by the same procedure, and was analyzed by SDS-PAGE gel electrophoresis.The L-Lysine derivatives was prepared by coupling reation with para hydroxy benzaldehyde at room temperature in PH5.3PBS buffer for4hrs to form shiff base, then by reduction reaction with sodium cyanoborohydride for4hrs to form acid amid construction which is a stable and site-specific modified construction. The structure of the derivative(L-lysine) modified by para hydroxy benzaldehyde was additionally confirmed by IR analysis besides above mentioned analysis. Then suitable material ratio and coupling temperature were optimized. The optimal material ratio is para hydroxy benzaldehyde:sodium cyanoborohydride: L-Lysine (5:2:1). And the optimal reaction temperature is room temperature. Then L-lysine derivative is analyzed by SDS-PAGE gel eletrophoresis. The result also proves that amino group can be modified by Aldehyde group. And we establ ish an optimal buffer PH is10.7.ResuIts:It’s proved to be possible that the Aldehyde group can modify the amino group. So we use macromolecule mPEG-ALD to modify active protein SOD. Material ratio is mPEG-ALD:sodium cyanoborohydride:SOD(6:200:1). Filting the derivatives through a bag filter(MW10000). Then use SDS-PAGE gel eletrophoresis to analyze the product. The result proves that mPEG-ALD can modify active protein (i.e. SOD) successfully.Conclusion:From the results above, we conclude that mPEG-ALD can modify active protein with high specific regioselectivity.But which specific site is modified is still not clear and need further research.