Stability and conformational change of methoxypolyethylene glycol modification for native and unfolded trypsin

The effect of succinimidyl carbonates activated methoxypolyethylene glycol (mPEG-SC) on the catalytic properties and conformation of native trypsin and dynamic high-pressure microfluidisation (DHPM) induced unfolded trypsin was studied. The thermal stability of unfolded trypsin was enhanced more significantly than that of native trypsin between 45 and 70 degrees C. The autolysis analysis indicated that modified unfolded trypsin was markedly more resistant to autolysis compared to modified native trypsin between 40 and 180 min. Upon mPEG-SC conjugation, the K-m value of the enzyme decreased by about 2-fold, and the catalytic efficiency (K-cat/K-m) increased by about 3-4-fold. Moreover, the increased thermal stability of unfolded trypsin might be due to the lower surface hydrophobicity and the higher hydrogen bond formation after mPEG-SC modification, which was reflected in the decrease of UV absorbance, the quenching and blue shift of fluorescence spectra, as well as the increase of beta-sheet content. (C) 2013 Elsevier Ltd. All rights reserved.