Bioconjugation of papain with poly(N-vinylpyrrolidone): NMR characterization and study of its enzymatic activity

Abstract

A poly(vinylpyrrolidone) end-functionalized with a carboxylic acid group (PVP–CO2H) was synthesized by reversible addition-fragmentation chain transfer (RAFT)/macromolecular design via the interchange of xanthates (MADIX) polymerization mediated by 4-(O-ethylxanthyl)methyl benzoic acid. The molecular weight of the as-synthesized PVP–CO2H was estimated through UV–vis spectroscopy (Mn(UV–vis) = 7322 g/mol), gel permeation chromatography (GPC) (Mn(GPC) = 8670 g/mol), and 1H NMR, (Mn(NMR) = 8207 g/mol). The values obtained were close with the theoretical molecular weight (Mn(th) = 7925 g/mol). Subsequently, the preformed PVP–CO2H was activated to produce N-succinimidyl poly(vinylpyrrolidone) (PVP–NHS). This precursor was covalently coupled to papain to produce bioconjugate PVP–papain. The functional group modifications in the PVP chain-end were observed by the variations in the chemical shift values by 1H and 13C NMR and FTIR analysis at each step of the synthesis. The molecular weight of the PVP–papain was obtained by SEC–HPLC and suggests that, on average, four or five chains of PVP–CO2H were attached to one papain molecule. Compared with papain, the PVP–papain exhibited significantly improved catalytic activity, pH, and thermal stability. Additionally, the storage studies showed that the catalytic activity of PVP–papain was about 79% versus the native enzyme (29%), and this activity was maintained even when it was stored for 25 days.