Effects of amines and aminoalcohols on bovine intestine alkaline phosphatase activity

Abstract

Bovine intestine alkaline phosphatase (BIALP) is widely used as a signaling enzyme in sensitive assays such as enzyme immunoassay (EIA). In this study, we evaluated the effects of various aminoalcohols and amines on the activity of BIALP in the hydrolysis of p-nitrophenyl phosphate (pNPP) at pH 9.8, at 20 °C. The k(cat) values at 0.05 M diethanolamine, 0.1 M triethanolamine, and 0.2 M N-methylethanolamine were 190±10, 840±30, and 500±10 s(-1), respectively. The k(cat) values increased with increasing concentrations of diethanolamine, triethanolamine, and N-methylethanolamine and reached 1240±60, 1450±30, and 2250±80 s(-1), respectively, at 1.0M. On the other hand, the k(cat) values at 0.05-1.0M ethanolamine, ethylamine, methylamine, and dimethylamine were in the range of 100-600 s(-1). These results indicate that diethanolamine, triethanolamine and N-methylethanolamine highly activate BIALP and might be suitable as a dilution buffer of BIALP in EIA. Interestingly, the K(m) values increased with increasing concentrations of diethanolamine and N-methylethanolamine, but not triethanolamine: the K(m) value at 1.0M diethanolamine (0.83±0.15 mM) was 12-fold higher than that at 0.05M (0.07±0.01 mM), and that at 1.0M N-methylethanolamine (2.53±0.20 mM) was 14-fold higher than that at 0.2M (0.18±0.02 mM), while that at 1.0M triethanolamine (0.31±0.01 mM) was similar as that at 0.2M (0.25±0.01 mM), suggesting that the mechanisms of BIALP activation are different between the aminoalcohols.

The heat released during catalytic turnover enhances the diffusion of an enzyme

“…1). Values are in agreement with the ones from the supplier (Sigma) and found in the literature 17–20 . ΔH is the enthalpy of the chemical reaction measured in bulk; values are reported in the literature for catalase 30 , urease 1 , triose phosphate isomerase 22 and measured by isothermal titration calorimetry for alkaline phosphatase (see Extended Data Fig.…”

“…1). All enzymes followed Michaelis–Menten kinetics with k cat and K M values in agreement with those previously reported in the literature 17–20 (Table 1). …”

Stabilization of Bovine Intestine Alkaline Phosphatase by Sugars

“…BIALP-catalyzed hydrolysis of pNPP was performed as described previously. 21,22) Briefly, the reaction was initiated by mixing 2,000 mL of 0.3-6.0 mM pNPP in 1.0 M diethanolamine-HCl, 1.0 mM MgCl 2 , and 20 mM ZnCl 2 at pH 9.8 with 1,000 mL of 120 pM BIALP thermally treated in the presence of sugars (90 mM sucrose, 90 mM trehalose, 90 mM maltose, 180 mM glucose, or 180 mM fructose) or 120 or 240 pM BIALP thermally treated in the absence of y To whom correspondence should be addressed. Tel: +81-75-753-6266; E-mail: inouye@kais.kyoto-u.ac.jp Abbreviations: ALP, alkaline phosphatase; BIALP, bovine intestine alkaline phosphatase; EIA, enzyme immunoassay; pNPP, p-nitrophenyl phosphate sugars.…”

“…[9][10][11][12][13][14][15][16][17][18][19][20] It is now widely used to stabilize not only enzymes but also various biomaterials. We have found that amines, 21) aminoalcohols, 21) and polyethylene glycol 22) activate BIALP, suggesting that they can be used as activators of BIALP in EIA. In this study, we examined the effects of three disaccharides (sucrose, trehalose, and maltose) and two monosaccharides (glucose and fructose) on BIALP activity and stability.…”

Effects of Polyethylene Glycol on Bovine Intestine Alkaline Phosphatase Activity and Stability

“…We have found that amines and aminoalcohols activate BIALP. 13) Polyethylene glycol (PEG) is a hydrophilic, biocompatible polymer. The covalent attachment of PEG to proteins (pegylation) is effective for advanced clinical uses.…”