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 approximately 8000 g/mol, dextrin I; or approximately 61,000g/mol, dextrin II), and a ST-HPMA-COOH (Mw approximately 10,100g/mol). The conjugates had a trypsin content of approximately 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 approximately 6-7-fold lower, and the substrate turnover rate (K(cat)) decreased by approximately 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.