| SOD1 is one of the most important antioxidant enzymes in the body,which maintains the homeostasis of O2·-and H2O2.SOD 1 locates within the whole cell,including membrane gaps of mitochondria and nucleus,which can protect the body from oxidative damage by eliminating redundant O2·-.Meanwhile,SOD 1 is also one of the key proteins in regulating intracellular redox signaling.In addition,as a protein drug,SOD1 is widely applied in antioxidant,anti-inflammatory,anti-cancer,anti-aging areas.However,as is known to all,macromolecule such as SOD1 can not penetrate cell-membrane automatically.Intravenous SOD1 is mostly adherent to the outer membrane so that it is not able to enter cells and makes a difference.Chemical modification is widely used in improving protein delivery efficiency.The chemical modification of protein refers to the modified groups react with the specific amino acid side chains of natural proteins to realize a series of functions such as positioning,modification,functionalization and fixing,etc.Via chemical modification,the stability,physical and chemical properties,function and delivering efiency of SOD 1 are significantly improved.We mainly carried out the work in following areas:1.Three fluorescent SOD1s were obtained by chemical modification.Nonspecificly modified products SOD1-FITC,SOD1-RhB and N-terminl specificly modified product SOD1-pRhB were used in the following studies including spectral propertie,structure analysis,enzymatic activity and anti-enzymatic hydrolysis ability,etc.N-terminl specific modification does not need harsh reaction conditions and products are with high purity.Via this method,we obtained the N-terminl specificially modified SOD1-pRhB.Compared with wild type SOD1,SOD1-pRhB had little change in structure and enzymatic activity.2.The fluorescence microscope,confocal laser scanning microscope and flow cytometry were used to research the transmembrane ability of modified SOD1.Experimental results confirmed that SOD1-pRhB could automatically penetrate the cell-membrane of multiple mammalian cells without any other carriers.This chemical modification strategy could be used in a variety of proteins modification and delivery.3.Transmembrane mechanism was studied according to the structure characteristics of SOD1 and pRhB.We confirmed that SOD1-pRhB entered cells via active transport which is energy-dependent and do not disturb cell-memberane.The Relationship between modification and transmembrane ability of SOD1-pRhB was analyzed.The cationic segment(pRhB)of SOD1-pRhB played a great role in transmembrane.Combined with rseults of inhibitors experiments,intracellular distribution of SOD1-pRhB and danamic procedure of internalization and excretion,the correlation between SOD 1-pRhB delivery and cationic compound RhB was confirmed.4.The research of ethanediamine and Methylmaleic anhydride modified SOD1 was also carried out.By means of these modification,we obtained two different charged SOD1-(NH2)n and SOD1-(COOH)n.SOD1-(NH2)n was a supercharged protein,could electrostaticly interact with negative macromolecules such as DNA.This capacity of SOD1-(NH2)n made it possible to be used in gene dilivery.5.Combined with the two modified strategies of the nonspecific amino modification and N-terminl specific modification,bifunctional(NH2)n-SOD1-pRhB was obtained.(NH2)n-SOD1-pRhB proformed higher transmembrane ability compared with SOD1-pRhB.Meanwhile it could interact with DNA with higher afinity.These results were crucial to make use of SOD1 as a gene delivery carrier. |
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