Study of the kinetic characteristics of d-amino acid oxidase using 2,2′-azinodi(3-ethylbenzothiazoline-6-sulphonic acid) as oxygen acceptor
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1994
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A simple kinetic method for assay of d-amino acid oxidase (d-AAO) catalytic activity using 2,2′-azinodi(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) as chromogen is described. Hydrogen peroxide produced in the reaction catalysed by d-AAO stoichiometrically oxidezes ABTS in a peroxidase-catalysed reaction. The absorbance of the chromogen measured at 410 nm is directly proportional to d-AAO activity. The estimated values of the Michaelis-Menten constants (Km) for d-alanine, d-proline, d-valine, d-2-aminobutyrate and d-leucine, determined in 100 mM pyrophosphate buffer (pH 8.8) at 30°C, were 2.60, 3.40, 4.60, 1.98 and 0.14 mM, respectively. The pH optima for the d-AAO activity was found to be between 8.6 and 8.8. The optimum temperature was about 30°C and the activation energy 8.89 kJ mol-1. The Km values for peroxidase, as an auxiliary enzyme in the indicator reaction, were 19.0, 20.8, 17.3 and 27.2 U 1-1 for d-alanine, d-proline, d-2-aminobutyrate and d-leucine, respectively, as subst...rate. The Km value for ABTS was 1.16 mM. The inhibition by sodium benzoate was found to be competitive with an inhibition constant, Ki, of 2.7 × 10-5 M. In all experiments, 50.5 μM of free flavine adenine dinucleptide was added to the reaction mixture to avoid dissociation of the holoenzyme.
Кључне речи:
Amino acid oxidase / Catalytic activity / Enzymatic methods / Kinetic methodsИзвор:
Analytica Chimica Acta, 1994, 285, 3, 369-375Институција/група
PharmacyTY - JOUR AU - Ignjatović, Svetlana AU - Majkić-Singh, Nada PY - 1994 UR - https://farfar.pharmacy.bg.ac.rs/handle/123456789/115 AB - A simple kinetic method for assay of d-amino acid oxidase (d-AAO) catalytic activity using 2,2′-azinodi(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) as chromogen is described. Hydrogen peroxide produced in the reaction catalysed by d-AAO stoichiometrically oxidezes ABTS in a peroxidase-catalysed reaction. The absorbance of the chromogen measured at 410 nm is directly proportional to d-AAO activity. The estimated values of the Michaelis-Menten constants (Km) for d-alanine, d-proline, d-valine, d-2-aminobutyrate and d-leucine, determined in 100 mM pyrophosphate buffer (pH 8.8) at 30°C, were 2.60, 3.40, 4.60, 1.98 and 0.14 mM, respectively. The pH optima for the d-AAO activity was found to be between 8.6 and 8.8. The optimum temperature was about 30°C and the activation energy 8.89 kJ mol-1. The Km values for peroxidase, as an auxiliary enzyme in the indicator reaction, were 19.0, 20.8, 17.3 and 27.2 U 1-1 for d-alanine, d-proline, d-2-aminobutyrate and d-leucine, respectively, as substrate. The Km value for ABTS was 1.16 mM. The inhibition by sodium benzoate was found to be competitive with an inhibition constant, Ki, of 2.7 × 10-5 M. In all experiments, 50.5 μM of free flavine adenine dinucleptide was added to the reaction mixture to avoid dissociation of the holoenzyme. T2 - Analytica Chimica Acta T1 - Study of the kinetic characteristics of d-amino acid oxidase using 2,2′-azinodi(3-ethylbenzothiazoline-6-sulphonic acid) as oxygen acceptor VL - 285 IS - 3 SP - 369 EP - 375 DO - 10.1016/0003-2670(94)80078-2 ER -
@article{ author = "Ignjatović, Svetlana and Majkić-Singh, Nada", year = "1994", abstract = "A simple kinetic method for assay of d-amino acid oxidase (d-AAO) catalytic activity using 2,2′-azinodi(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) as chromogen is described. Hydrogen peroxide produced in the reaction catalysed by d-AAO stoichiometrically oxidezes ABTS in a peroxidase-catalysed reaction. The absorbance of the chromogen measured at 410 nm is directly proportional to d-AAO activity. The estimated values of the Michaelis-Menten constants (Km) for d-alanine, d-proline, d-valine, d-2-aminobutyrate and d-leucine, determined in 100 mM pyrophosphate buffer (pH 8.8) at 30°C, were 2.60, 3.40, 4.60, 1.98 and 0.14 mM, respectively. The pH optima for the d-AAO activity was found to be between 8.6 and 8.8. The optimum temperature was about 30°C and the activation energy 8.89 kJ mol-1. The Km values for peroxidase, as an auxiliary enzyme in the indicator reaction, were 19.0, 20.8, 17.3 and 27.2 U 1-1 for d-alanine, d-proline, d-2-aminobutyrate and d-leucine, respectively, as substrate. The Km value for ABTS was 1.16 mM. The inhibition by sodium benzoate was found to be competitive with an inhibition constant, Ki, of 2.7 × 10-5 M. In all experiments, 50.5 μM of free flavine adenine dinucleptide was added to the reaction mixture to avoid dissociation of the holoenzyme.", journal = "Analytica Chimica Acta", title = "Study of the kinetic characteristics of d-amino acid oxidase using 2,2′-azinodi(3-ethylbenzothiazoline-6-sulphonic acid) as oxygen acceptor", volume = "285", number = "3", pages = "369-375", doi = "10.1016/0003-2670(94)80078-2" }
Ignjatović, S.,& Majkić-Singh, N.. (1994). Study of the kinetic characteristics of d-amino acid oxidase using 2,2′-azinodi(3-ethylbenzothiazoline-6-sulphonic acid) as oxygen acceptor. in Analytica Chimica Acta, 285(3), 369-375. https://doi.org/10.1016/0003-2670(94)80078-2
Ignjatović S, Majkić-Singh N. Study of the kinetic characteristics of d-amino acid oxidase using 2,2′-azinodi(3-ethylbenzothiazoline-6-sulphonic acid) as oxygen acceptor. in Analytica Chimica Acta. 1994;285(3):369-375. doi:10.1016/0003-2670(94)80078-2 .
Ignjatović, Svetlana, Majkić-Singh, Nada, "Study of the kinetic characteristics of d-amino acid oxidase using 2,2′-azinodi(3-ethylbenzothiazoline-6-sulphonic acid) as oxygen acceptor" in Analytica Chimica Acta, 285, no. 3 (1994):369-375, https://doi.org/10.1016/0003-2670(94)80078-2 . .