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 temperatur e (LCST) of the oligomers is very close to that of homo-poly(NIPAAm) l acking the end carboxyl group. The carboxyl groups were activated in m ethylene chloride using N,N'-dicyclohexylcarbodiimide (DCC) and N-hydr oxysuccinimide (NHS). A conjugate of trypsin with the preactivated oli gomer 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), assumin g 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 es timated by determination of the remaining primary amine groups on the trypsin molecule. More than 95% of the conjugate can be recovered by t hermally induced precipitation.