Preparation, Conformational Structure, and Proteolytic Activity of Papain Covalently Conjugated to Poly(ethylene glycol)-Tethered Lipid Bilayer Membranes

Abstract

Conjugation of enzymes to lipid membranes is a key approach to reconstitute fascinating features of cell organelles and to deduce the nature of membrane-involved biological events. In this work, papain was covalently conjugated via a cross-linker to phospholipid vesicles (liposomes) tethered with poly(ethylene glycol) (PEG) at 25 °C and pH = 7.0, followed by chromatographic purification. The size of the PEG moiety and the type of cross-linker were optimized to obtain PEG-tethered liposome-conjugated papain (liposome-PEG-papain). Slight conformational changes of the membrane-conjugated papain in both its secondary and tertiary structures were revealed using circular dichroism and intrinsic fluorescence measurements. Notably, heat treatment of a liposome-PEG-papain dispersion at 77 or 84 °C caused permeabilization of the lipid membranes to 5(6)-carboxyfluorescein. Furthermore, liposome-PEG-papain exhibited the digestion activity of casein at 37 °C and pH = 7.6. The structural flexibility of liposomes as enzyme carriers may provide the opportunity to functionalize the membrane-conjugated biomacromolecules.