Uricase, an enzyme used for the treatment of hyperuricemia, is conjugated with polysialic acid (PSA) of average molecular weight of 10 kDa by reductive amination in presence of NaCNBH3 in order to improve its pharmacological properties. Polysialylation with 50-,100-,150- and 200-fold molar excess of PSA increased the percentage substitution of the free amino groups on enzyme surface (46, 66, 78 and 80 % respectively). The SDS-PAGE is used to visualize the conjugates with increased molecular weight and it retained almost 65 % of their initial specific activity after conjugation. The stability studies at physiological condition reveals improved stability and activity than the native enzyme. The apparent KM of the enzyme has increased slightly from 4.18 × 10-5 M to 5.46 × 10-5 M suggesting that the affinity of the substrate to the enzyme has not been altered to a higher extent. The conjugates, when probed against anti-uricase antibodies generated in rabbit, showed a clean decline in the affinity by 35 % and also have retained double the catalytic activity than that of the native enzyme after exposure to antiserum. The results suggest that uricase-PSA conjugates can be used as an alternative to the conventional synthetic polymer-enzyme conjugates.
|Number of pages||8|
|Journal||International Journal of Peptide Research and Therapeutics|
|Publication status||Published - 01-01-2014|
All Science Journal Classification (ASJC) codes
- Molecular Medicine
- Drug Discovery
- Analytical Chemistry