title: Effects of polyethylene glycol on bovine intestine alkaline phosphatase activity and stability.
abstract: In this study, we evaluated the effects of polyethylene glycol (PEG) on bovine intestine alkaline phosphatase (BIALP) activity and stability. In the hydrolysis of p-nitrophenylphosphate (pNPP) at pH 9.8 at 20 °C, the k(cat)/K(m) values of BIALP plus 5-15% w/v free PEG with molecular masses of 1, 2, 6, and 20 kDa (PEG1000, PEG2000, PEG6000, and PEG20000 respectively) were 120-140%, 180-300%, 130-170%, and 110-140% respectively of that of BIALP without free PEG (1.8 µM(-1) s(-1)), indicating that activation by PEG2000 was the highest. Unmodified BIALP plus 5% PEG2000 and BIALP pegylated with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine exhibited 1.3-fold higher activity on average than that of BIALP without free PEG under various conditions, including pH 7.0-10.0 and 20-65 °C. The temperatures reducing initial activity by 50% in 30-min incubation of unmodified BIALP plus 5% PEG2000 and pegylated BIALP were 51 and 47 °C respectively, similar to that of BIALP without free PEG (49 °C). These results indicate that the addition of PEG2000 and pegylation increase BIALP activity without affecting its stability, suggesting that they can be used in enzyme immunoassay with BIALP to increase sensitivity and rapidity.
authors: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
authors: Satoshi
authors: S
authors: Sekiguchi
authors: Kiyoshi
authors: K
authors: Yasukawa
authors: Kuniyo
authors: K
authors: Inouye
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry*; D000818:Animals; D002417:Cattle; D004795:Enzyme Stability; D007422:Intestines / Q000201:enzymology*; D011092:Polyethylene Glycols / Q000737:chemistry*
chemical_list: D011092:Polyethylene Glycols; D000469:Alkaline Phosphatase