Studies On Superoxide Dismutase Modified With Monomethoxy-Polyethylene Glycol

The Superoxide dismutase (SOD) is one kind of metal enzyme,distributes in organism extremely broadly, is studies at present up to theknown 2,000 many kinds of enzymes in reports most enzymes. As a result ofthe metal subunits difference, it may divide into four kinds, respectively isCu/Zn-SOD, Mn-SOD, Fe-SOD and Ni-SOD. SOD is one kind of importantoxygen free radical elimination medicinal preparation, can catalyze the (O2-. )disproportionation responded, moreover its existence is possible related withorganism senile, the tumor , autoimmunity disease and the radiation shieldand so on, therefore it has very great medical value and the widespreadapplication prospect. But the natural SOD unstability, has immunogenicity,the function is relatively unitary, but has limited its application. Modified canstrengthen the enzyme stability, reduces immunogenicity. The reports userthe sucrose, the dextran, the albumin, the gelatin, the starch, thepolyethylene glycol (PEG) and so on modified SOD . This article uses thesingle methoxy polyethylene glycol (mPEG) to ox blood Cu/Zn-SODmodified, and to compare the SOD nature and modified.We use three different methods to activate the single methoxypolyethylene glycol(MPEG), the three methods respectively are: N-Hydroxysuccinimide activation method (NHS), N,N'-Carbonyldiimidazoleactivation methods and p-Toluenesulfonyl chloride activation method. Afterthe activation obtained three kind of activation derivatives: SS -MPEG, N,N'-Carbonyldiimidazole-MPEG, Tosyl -MPEG. After the impurity, hascarried on an activation determination to three kinds of MPEG activationderivative. (1) because under the alkalinity condition , the N-Hydroxysuccinimide has the characteristic absorption in 260nm, with thestandard N-Hydroxysuccinimide manufacture standard curve, determinesSS-MPEG OD260 then to obtain SS-SOD activation, (2) N,N'-Carbonyl-diimidazole-MPEG may use the element analytic method, determines N thepercentage can get the activation grade, (3) Tosyl -SOD has the characteristicin 261nm place to absorb the peak, according to the above may determine itsactivation grade.With the three kinds of activation derivatives which obtains to themodified SOD, according to mole of ratio 20: 1 in the pH9.2 sodiumtetraborate responded 1 hour, the product in the pH7.3 phosphoric acid buffersolution dialysis 2 hours, move the surplus sodium tetraborate, lays asidethe polyethylene glycol outside the dialysis bag to cause the productconcentration to the small volume. Takes a sample to Sephadex -the G75column, collects the first peak, after the freeze drying obtains the whitemPEG -SOD.Uses the TNBS method to determine mPEG – SOD activication grade,under the pH8.5 boric acid buffer system, determines OD420 take the glycineas the standard amino acid, the manufacture standard curve. Around natureand modified SOD OD420 change around, then extracts nature and modifiedprotein how amino acid change, by the two differences, extracts each theMPEGnumber of molecular protein.Around uses the pyrogallic acid to decorate the SOD active changesituation from the oxo-process determination, the enzyme vigor unitdefinition is: 25 ℃, in the 325nm wave length place, each minutesuppression pyrogallic acid reaches 50% from the oxidized speed to need theenzyme quantity definition is an enzyme vigor unit.the polyacrylamide gelatin electrophoresis the gelatin density is 7%, 1%carrier amphoteric electrolyte result (the pH gradient is 3.5-10). This enzymein and so on the electric focusing electrophoresis presents a zymoprotein belt,its isoelectric point is 4.1.SOD stability trial: studied mPEG -SOD in different pH, under thetemperature condition stability, as well as tolerance to trypsin and pepsin.The result indicated, stability of the mPEG -SOD has the remarkableenhancement in whatever pH, the temperature or trypsin and the pepsintolerance compared to the natural enzyme.