Articles
THE DISTINCTIVE FEATURES OF ASCORBATE PEROXIDASE IN DORMANT CROCUS SATIVUS L. CORM
Article number
650_12
Pages
119 – 125
Language
English
Abstract
The presence of ascorbate peroxidase activity was investigated in an extract prepared from dormant Crocus sativus L. corm.
The enzymatic activity was measured by following spectrophotometrically the oxidation of sodium ascorbate at 290 nm, in the presence of hydrogen peroxide, in citrate-phosphate buffer, upon addition of the extract.
After correction for ascorbate auto-oxidation, the extract exhibited ascorbate peroxidase activity with an optimum pH of 8.0. At that pH, Km was 0.15 ± 0.02 mM for ascorbate, and 0.05 ± 0.005 mM (three times less) for hydrogen peroxide.
Apparent Vmax, calculated per mg protein in the extract, was 54 ± 6 μM.min-1 for ascorbate and 205 ± 15 μM.min-1 (four times more) for hydrogen peroxide.
The catalytic efficiency (per mg protein in the extract) was 0.36 ± 0.08
min-1 for ascorbate and 4.1 ± 0.6 min-1 for hydrogen peroxide, thus eleven times that for ascorbate.
The pseudo-first order rate constant for ascorbate as the varying substrate was 0.0089 ± 0.0006 min-1 and it was 0.5 ± 0.06 min-1 (fifty five times higher) for hydrogen peroxide as the varying substrate.
Thus, ascorbate peroxidase activity was detected in dormant C. sativus L. corm extract.
The enzyme obeyed simple Michaelis-Menten kinetics with either hydrogen peroxide or ascorbate.
But, surprisingly, in contrast to other ascorbate peroxidases as well as to other hemoproteins, it was insensitive to up to 100 mM cyanide, 100 mM azide or 100 mM aminotriazole.
This may correspond to a specific requirement of metabolic control activity of this enzyme in dormant corm.
The enzymatic activity was measured by following spectrophotometrically the oxidation of sodium ascorbate at 290 nm, in the presence of hydrogen peroxide, in citrate-phosphate buffer, upon addition of the extract.
After correction for ascorbate auto-oxidation, the extract exhibited ascorbate peroxidase activity with an optimum pH of 8.0. At that pH, Km was 0.15 ± 0.02 mM for ascorbate, and 0.05 ± 0.005 mM (three times less) for hydrogen peroxide.
Apparent Vmax, calculated per mg protein in the extract, was 54 ± 6 μM.min-1 for ascorbate and 205 ± 15 μM.min-1 (four times more) for hydrogen peroxide.
The catalytic efficiency (per mg protein in the extract) was 0.36 ± 0.08
min-1 for ascorbate and 4.1 ± 0.6 min-1 for hydrogen peroxide, thus eleven times that for ascorbate.
The pseudo-first order rate constant for ascorbate as the varying substrate was 0.0089 ± 0.0006 min-1 and it was 0.5 ± 0.06 min-1 (fifty five times higher) for hydrogen peroxide as the varying substrate.
Thus, ascorbate peroxidase activity was detected in dormant C. sativus L. corm extract.
The enzyme obeyed simple Michaelis-Menten kinetics with either hydrogen peroxide or ascorbate.
But, surprisingly, in contrast to other ascorbate peroxidases as well as to other hemoproteins, it was insensitive to up to 100 mM cyanide, 100 mM azide or 100 mM aminotriazole.
This may correspond to a specific requirement of metabolic control activity of this enzyme in dormant corm.
Authors
L. Ghamsari, E. Keyhani
Keywords
aminotriazole, ascorbate, azide, cyanide, inhibition, kinetics, oxidative stress
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