Synthesis and purification of thermally sensitive oligomer-enzyme conjugates of poly(N-isopropylacrylamide)-trypsin

Using chain-transfer polymerization, we have synthesized oligomers of poly(N-isopropylacrylamide) [poly(NIPAAm)] with one carboxyl group at the end of each oligomer chain. The lower critical solution temperature (LCST) of the oligomers is very close to that of homo-poly(NIPAAm) lacking the end carboxyl group. The carboxyl groups were activated in methylene chloride using N,N'-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS). A conjugate of trypsin with the preactivated oligomer has been prepared. We studied the effect of oligomer to enzyme (O/E) ratio in the feed on the O/E ratio of the conjugate (the average number of oligomer chains conjugated to one trypsin molecule), assuming that only the primary amino groups of lysine residues and the amino terminal of trypsin would react. The O/E ratio of the conjugate was estimated by determination of the remaining primary amine groups on the trypsin molecule. More than 95% of the conjugate can be recovered by thermally induced precipitation.