Studies on the Site-specific PEGylation Induced Interferences Instigated in Uricase Quantification Using the Bradford Method

Abstract

Uricase from Bacillus fastidiosus was site-specifically PEGylated using methoxypolyethyleneglycol-maleimide (mPEG-mal) of different molecular weights (750 Da, 5 kDa, 10 kDa) via Thiol PEGylation strategy. The obtained monoPEGylated uricase conjugates were characterized using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and the molecular weight of single subunit of the conjugate was found to be 42.6, 48.1 and 56.3 kDa with respect to different molecular weights of m-PEG-mal. The influence of PEGylation on the quantification of uricase using protein quantification techniques like Bradford assay, RP-HPLC detection and Dumbroff method has been evaluated. A gradual decline in the absorbance value was observed with the advancement of the PEGylation reaction, indicating an interferences in the protein quantification due to PEGylation. The extent of interference highly dependence on mPEG-mal concentration and its chain length. The present study indicates that the quantification of PEGylation induced interferences caused in protein measured ought to be prudently measured at every discrete step of the PEGylation process.