Simple spectroscopic method for titration of binding sites in molecularly imprinted nanogels with hydrolase activity

In this investigation we report the preparation of soluble molecularly imprinted catalytic nanogels with hydrolytic activity. The nanogels were imprinted using a stoichiometric non-covalent approach, employing a phosphate transition state analogue as template and polymerizable tyrosine and arginine units as functional monomers, for catalysis of a carbonate hydrolysis reaction. Full characterization of the rebinding and of the hydrolytic activity was performed, with particular emphasis on a novel titration method developed for the measurement of active site concentrations and the subsequent calculation of accurate catalytic parameters. Considering the features of the template molecule and the functional monomers used, an original method for performing rebinding experiments is described, taking advantage of the change of the visible spectrum evident on binding the sodium salt of the template to the arginine residue present in the nanogel.