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2011/09/06

  • available-technologies
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Bovine intestine alkaline phosphatase (BIALP) is widely used as a signaling enzyme in sensitive assays such as enzyme immunoassay (EIA). The sensitivity and rapidity of the assay increase with increasing the activity of the enzyme. Therefore, its activation and stabilization are of great interest. The assay of BIALP has been done conventionally in 0.05-0.5 M borate buffer at pH 9.8, 25℃ with the molecular activity (kcat) value of 120-130 s-1 in the hydrolysis of p-nitrophenyl phosphate (pNPP). We evaluated the effects of various aminoalcohols and amines on BIALP activity. The kcat values in the presence of ethanolamines were much higher than that observed in borate buffer, and the values increased with increasing their concentrations. In the presence of 1 M diethanolamine, triethanolamine, and N-methylethanolamine, the values were 1200, 1500, and 2300 s-1, respectively(Fig. 1).

fig1
Fig. 1, Dependence of the enzyme reaction rate (vo) on the pNPP concentration in the BIALP-catalyzed hydrolysis of pNPP in the presence of aminoalcohols at pH 9.8, at 20℃. The initial enzyme concentration ([E]o) is 40-400 pM. The vo/[E]o is plotted against pNPP concentrations. Solid line represents the best fit of the Michaelis-Menten equation using the nonlinear least-squares methods. Symbols for the buffers: 1.0 M diethanolamine, ◯; 1.0 M ethanolamine, △; 1.0 M triethanolamine, □; 1.0 M N-methylethanolamine, ◇; and 0.5 M borate-NaOH; .

On the other hand, ethanolamine, ethylamine, methylamine, and dimethylamine did not show such remarkable activating effects. It is noted that the Michaelis constant (Km) values under the conditions examined are much lower than the solubility of pNPP, and thus the ethanolamines should be suitable as components of the BIALP reaction buffer in EIA.

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