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.

Effect of PEG molecular weight and linking chemistry on the biological activity and thermal stability of PEGylated trypsin.

PEGylated proteins are routinely used as therapeutics, but systematic studies of the effect of PEG molecular weight and linking chemistry on the biological activity and particularly the thermal stability of the conjugated protein are rarely made. Here, activated monomethoxypolyethylene glycol (mPEG)s (Mw 1100, 2000 and 5000 g/mol) were prepared using succinic anhydride (SA), cyanuric chloride (CC) or tosyl chloride (TC) and used to synthesise a library of trypsin conjugates. The enzyme activity (KM, Vmax and Kcat) of native trypsin and the mPEG-modified trypsin conjugates was compared using N-benzoyl-l-arginine p-nitroanilide (BAPNA) as a substrate, and their thermal stability determined using both BAPNA and N-alpha-benzoyl-l-arginine ethyl ester hydrochloride (BAEE) as substrates to measure amidase and esterase activity respectively. The effect of conjugate chemistry on trypsin autolysis was also examined at 40 degrees C. PEG-trypsin conjugates containing the higher molecular weight of mPEG (5000 g/mol) were more stable than free trypsin, and the conjugate containing CC-mPEG 5000 g/mol had the best thermal stability.