Carboxylated poly(N-isopropylacrylamide) (pNIPAM) was prepared in ethyl alcohol at 70°C by radical polymerization using 4,4′-azobis(N,N′-cyanopentanoic acid) (V-501) as the initiator. Trypsin was conjugated to carboxylated pNIPAM using carbodiimide and the pNIPAM-trypsin conjugate was subsequently fractionated by gel filtration. The lower critical solution temperature (LCST) of the pNIPAM-trypsin conjugates changed with the molecular weight of the coupled pNIPAM. The enzymatic activity of the conjugate and its temperature dependence was affected by both the molecular weight and the amount of the coupled pNIPAM. The relative activity of the conjugates decreased above the LCST, and one of the reasons for this could be a decrease in the diffusion of the substrate upon shrinkage of the coupled pNIPAM above the LCST. The enzymatic activity changed reversibly with temperature when the conjugates contained a certain amount of pNIPAM. In addition, the conjugates were more resistant against heat treatment than native trypsin. Circular dichroism measurements indicated that the tertiary structure of the conjugates was different from that of native trypsin.
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