Uniform polymer-protein conjugate by aqueous AGET ATRP using protein as a macroinitiator

In situ aqueous activators generated by electron transfer for atom transfer radical polymerization (AGET ATRP) in air, using an enzyme as a macroinitiator, has been proposed to prepare uniform polymer-protein conjugates with improved stability under adverse conditions. In the first step, an initiator, 2-bromoisobutyryl bromide (BIB), was grafted onto the protein surface by reaction with the amino groups. The second step was in situ AGET ATRP polymerization in air using CuBr(2)/1,1,4,7,7-pentamethyldiethylenetriamine as a catalyst and ascorbic acid as a reducing agent. The effectiveness of this method has been demonstrated using horseradish peroxidase (HRP) as a model protein and acrylamide as the monomer, which yielded HRP-polyacrylamide conjugate with a mean particle size of about 20-30 nm. The grafting of BIB onto HRP and the subsequent polymerization yielding a polyacrylamide chain were confirmed by nuclear magnetic resonance and matrix-assisted laser desorption ionization time-of-flight spectrometry analysis. The size of the conjugate was shown to be a function of monomer loading and reaction time. The HRP conjugates yielded essentially retained the catalytic behavior of HRP in free form, as shown by K(m) and V(max) values, but exhibited significantly enhanced thermal stability against high temperature and trypsin digestion. The use of protein as the macroinitiator prevented the formation of copolymer and thus facilitated purification of the protein conjugate. The uniform size indicates a well-defined composition of protein and polymer, which is essential for applications that request a precise control of the dosage of enzyme activity.