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Title: Effects of amines and aminoalcohols on bovine intestine alkaline phosphatase activity
Authors: Sekiguchi, Satoshi
Hashida, Yasuhiko
Yasukawa, Kiyoshi  kyouindb  KAKEN_id
Inouye, Kuniyo
Author's alias: 井上, 國世
Keywords: Alkaline phosphatase
Amine
Aminoalcohol
Bovine intestine alkaline phosphatase
Enzyme immunoassay
Issue Date: Jul-2011
Publisher: Elsevier Inc.
Journal title: Enzyme and Microbial Technology
Volume: 49
Issue: 2
Start page: 171
End page: 176
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 Kcat 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 Kcat values increased with increasing concentrations of diethanolamine, triethanolamine, and N-methylethanolamine and reached 1240 ± 60, 1450 ± 30, and 2250 ± 80 s[−1], respectively, at 1.0 M. On the other hand, the Kcat values at 0.05-1.0 M 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 Km values increased with increasing concentrations of diethanolamine and N-methylethanolamine, but not triethanolamine: the Km value at 1.0 M diethanolamine (0.83 ± 0.15 mM) was 12-fold higher than that at 0.05 M (0.07 ± 0.01 mM), and that at 1.0 M N-methylethanolamine (2.53 ± 0.20 mM) was 14-fold higher than that at 0.2 M (0.18 ± 0.02 mM), while that at 1.0 M triethanolamine (0.31 ± 0.01 mM) was similar as that at 0.2 M (0.25 ± 0.01 mM), suggesting that the mechanisms of BIALP activation are different between the aminoalcohols.
Rights: © 2011 Elsevier Inc.
This is not the published version. Please cite only the published version. この論文は出版社版でありません。引用の際には出版社版をご確認ご利用ください。
URI: http://hdl.handle.net/2433/143675
DOI(Published Version): 10.1016/j.enzmictec.2011.04.019
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