Treetharnmathurot Benjaporn, Dieudonné Lucile, Ferguson Elaine L, Schmaljohann Dirk, Duncan Ruth, Wiwattanapatapee Ruedeekorn
Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand.
Int J Pharm. 2009 May 21;373(1-2):68-76. doi: 10.1016/j.ijpharm.2009.02.008. Epub 2009 Feb 21.
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.
最近，我们使用单甲氧基聚（乙二醇）（mPEG）-胰蛋白酶偶联物证明，PEG分子量（1100 - 5000 g/mol）和连接子化学性质都会影响蛋白质的自溶速率和热稳定性。这些重要因素常常被忽视，但它们可以指导早期选择最佳聚合物/化学性质，以合成适合后期制剂开发的先导聚合物治疗剂。由于我们目前正在开发糊精和半遥爪聚[N-（2-羟丙基）甲基丙烯酰胺]（ST-HPMA）-蛋白质偶联物作为新型治疗剂，本研究的目的是以胰蛋白酶偶联物为模型，研究聚合物对活性、自溶及其热稳定性的影响，并与mPEG偶联物获得的数据进行比较。首先使用琥珀酰化糊精（分子量约8000 g/mol，糊精I；或约61,000 g/mol，糊精II）和ST-HPMA-COOH（分子量约10,100 g/mol）合成胰蛋白酶偶联物。这些偶联物的胰蛋白酶含量分别约为54%、17%和3%，游离蛋白<5%。当以N-苯甲酰-L-精氨酸对硝基苯胺（BAPNA）为底物测定酰胺酶活性（K(M)、V(max)和K(cat)）时，胰蛋白酶的K(M)值在偶联后未改变，但V(max)约低6 - 7倍，底物周转率（K(cat)）降低约5 - 7倍。在30至70摄氏度之间的所有温度下，糊精II-胰蛋白酶偶联物比其他偶联物和天然胰蛋白酶更稳定，并且在40摄氏度的自溶试验中也表现出更好的热稳定性。
