Dextrin-trypsin and ST-HPMA-trypsin conjugates: Enzyme activity, autolysis and thermal stability

Using monomethoxy poly(ethylene glycol) (mPEG)-trypsin conjugates we recently showed that both PEG molecular weight (1100-5000 g/mol) and linker chemistry affect the rate of protein autolysis and thermal stability. These important factors are often overlooked but they can guide the early choice of optimal polymer/chemistry for synthesis of a lead polymer therapeutic suitable for later formulation development. As we are currently developing dextrin- and semi-telechelic poly[N-(2-hydroxypropyl)methacrylamide] (ST-HPMA)-protein conjugates as new therapeutics, the aim of this study was to examine the effect of polymer on activity, autolysis and its thermal stability using trypsin conjugates as a model and compare to the data obtained for mPEG conjugates. Trypsin conjugates were first synthesized using succinoylated dextrin (Mw similar to 8000 g/mol, dextrin 1; or similar to 61,000 g/mol, dextrin II), and a ST-HPMA-COOH (Mw similar to 10,100 g/mol). The conjugates had a trypsin content of similar to 54,17 and 3 wt% respectively with <5% free protein. When amidase activity (K-M, V-max and K-cat) was determined by using N-benzoyl-L-arginine p-nitroanilide (BAPNA) as substrate, trypsin K-M values were not altered by conjugation, but the V-max was similar to 6-7-fold lower, and the substrate turnover rate (K-cat) decreased by similar to 5-7-fold. The dextrin II-trypsin conjugate was more stable than the other conjugates and native trypsin at all temperatures between 30 and 70 degrees C, and also exhibited improved thermal stability in the autolysis assays at 40 degrees C. (C) 2009 Elsevier B.V. All rights reserved.