PEG-HRP於有機溶劑中反應之研究

Abstract

Pegylated horseradish peroxidase (PEG-HRP) was prepared by reacting HRP with monomethoxypoly(ethylene glycol), activated by 4-nitrophenyl chloroformate. The optimal conversion, close to 100 %, was observed in 0.1M sodium borate buffer pH9.0 with a [HRP]/[PEG] ratio of 5 x 10-3 at 25℃ for 3 hrs. The optimal activity of PEG-HRP in aqueous solution was observed at pH9.0及50℃. PEG-HRP is soluble in hydrophobic solvents such as benzene and toluene and was thus used for the polymerization of 2-naphthol in acetonitrile. The molecular weight of the polymers, exhibiting fluorescence properties, was found to increase with acetonitrile concentration.

本研究利用4-nitrophenyl chloroformate活化mPEG5000，以此活化mPEG進行辣根過氧化氫酶(Horseradish Peroxidase,HRP)化學修飾，探討最適化修飾條件及修飾化反應對HRP於水相、有機相中性質的影響，並利用修飾後HRP於有機溶劑中進行2-奈酚(2-naphthol)聚合反應。辣根過氧化氫酶最適化修飾條件為，0.1M sodium borate buffer pH9.0 ，HRP 3.5mg/ml，mPEG5000 100mg/ml，25℃反應3小時，修飾反應轉化率由SDS-PAGE分析可達100%。修飾後HRP於水相活性較未修飾HRP高，最適反應pH值與溫度分別為pH9.0及50℃，熱穩定性則沒有因修飾反應而增加。修飾後HRP可溶於Benzene、Toluene等疏水溶劑，在含50%(v/v)以上DMF或ACN與 potassium phosphate buffer混合液系統中，仍有70%以上的殘餘活性。修飾後HRP於ACN/potassium phosphate buffer混合液系統中進行2-奈酚聚合反應，產物重量平均分子量隨著ACN溶劑比例上升而增加，ACN 90%時平均分子量可達1600，螢光分析中顯示產物具有螢光反應。