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.