Dextrin–trypsin and ST-HPMA–trypsin conjugates: Enzyme activity, autolysis and thermal stability
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- 作者：Benjaporn Treetharnmathurot ; Lucile Dieudonné ; Elaine L. Ferguson ; Dirk Schmaljohann ; Ruth Duncan ; Ruedeekorn Wiwattanapatapee
- 关键词：Dextrin ; Semi-telechelic poly[N-(2-hydroxypropyl)methacrylamide] ; Polymer– ; protein conjugates ; Thermal stability ; Trypsin ; Polymer therapeutics
- 刊名：International Journal of Pharmaceutics
- 出版年：2009
- 出版时间：21 May 2009
- 年：2009
- 卷：373
- 期：1-2
- 页码：68-76
- 全文大小：1220 K
文摘
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 8000 g/mol, dextrin I; or 61,000 g/mol, dextrin II), and a ST-HPMA–COOH (Mw 10,100 g/mol). The conjugates had a trypsin content of 54, 17 and 3 wt % respectively with <5 % free protein. When amidase activity (KM, Vmax and Kcat) was determined by using N-benzoyl-l-arginine p-nitroanilide (BAPNA) as substrate, trypsin KM values were not altered by conjugation, but the Vmax was 6–7-fold lower, and the substrate turnover rate (Kcat) decreased by 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 °C, and also exhibited improved thermal stability in the autolysis assays at 40 °C.