Preparation Of Long-term Uricase The (uricase-peg40kd Conjugates) And The Basic Nature Of The Study

Objective: To obtain uricase-PEG (PEG-40,000) and compare native uricase with uricase-PEG in properties.Methods: Uricase of molecular weight 60,000 was obtained from crudeuricase by Superdex 75 chromatography, The uricase was modified by molecular weight 40,000 of PEG. Effects of pH, temperature, PEG content and uricase content and reaction time etc. in changes on modification reactionof uricase were analyzed by SDS-PAGE in non-rduceing conditions. Uricase-PEG was isolated by molecular sieve chromatography on Superdex 75 column. Purity and apparent molecular weight of Uficase-PEG were reviewed by SDS-PAGE and gradient gel PAGE. Modification degree for Uricase-PEG was determined by TNBS method. Comparisons of kinetic properties of uricase-PEG and native uricase were analyzed by common technology of enzymologyResults: As a result, the optimal reaction conditions of uricase-PEGylation were established as pH8.5 with 0.2mmol/L boric acid buffer, 25℃or room temperature), PEG∶NHS=1∶20 (w/w), reaction time(30min) and stop reaction reagent (Glycin) or none, uricase-PEG. complex was successfully isolated by Superdex75 chromatography, Result of SDS-PAGE showed apparent molecular weight of uricase-PEG was 100,000 and 140,000zed. by. Modification degree of uricase with PEG was assayed with 30% by TNBS method. The optimal pH and pH stability of uricase-PEG complex were the same as uricase's and not changed. The optimal temperature of uricase-PEG complex was 45℃and higher than uricase. Thermo stability of uricase-PEG was higher than uricase.Conclusion: It was shown that Uricase-PEG was more stable than native SOD in some properties.