Document Type : Research Paper
Authors
- Zhilla Zarei 1
- Nabi Khaliliaqdam 2
- Shahriar Saeidian 1
- Bahaaldin Rashidzadeh 3
- Mohammad Ghadermarzi 1
1 Department of Biology, Payame Noor University, Tehran, Iran
2 Department of Agriculture, Payame Noor University, Tehran, Iran
3 Department of Chemistry, Payame Noor University, Tehran, Iran
Abstract
Background and objectives: Plants possess efficient antioxidant defense systems that neutralize and eliminate toxic levels of reactive oxygen species (ROS). These systems include both enzymatic and non-enzymatic components. Ascorbate peroxidase (APX) is a key enzyme in this defense system, playing a crucial role in regulating the levels of toxic ROS in various intracellular compartments. The expression of APX is modulated during certain developmental stages as well as under biotic and abiotic stress conditions, highlighting the importance of APX activity in controlling hydrogen peroxide (H₂O₂) levels within cells.
Methodology: In this study, an enzyme extract was prepared from black mulberry (Morus nigra L.), and its kinetic properties were evaluated in the presence of different salts. The activity of ascorbate peroxidase (APX) was determined by measuring the oxidation of the ascorbate substrate at a wavelength of 290 nm, using an extinction coefficient of 18.2 mM⁻¹ cm⁻¹. The optimum temperature for enzyme activity was determined by measuring APX activity at temperatures ranging from 25 to 80 °C in 50 mM Tris–HCl buffer (pH 7.6) at 5 °C intervals. To evaluate thermal stability, the enzyme solution was incubated in a water bath at temperatures between 25 and 80 °C for 30 minutes. The reaction mixture was then maintained at room temperature for 10 minutes, after which APX activity was measured. To determine the most appropriate response of ascorbate peroxidase to the different levels of the studied treatments, linear and non-linear regression analyses were performed due to the limited number of treatments.
Results: The electrophoretic profile of ascorbate peroxidase in blackberry leaf extract revealed two isoenzymes at pH 6.5 and 8 (APX-LI and APX-LII), while one isoenzyme (APX-F) was detected in the fruit extract at pH 6.5. The substrates ascorbate, pyrogallol, and guaiacol increased peroxidase activity at low concentrations; however, at higher concentrations, they exhibited inhibitory effects on enzyme activity, likely due to substrate inhibition mechanisms that help regulate oxidative stress. Nonlinear regression analysis showed that the activities of APX-F and APX-LII followed the Gompertz exponential model, whereas the activity of APX-LI followed a monophasic incremental exponential function. The nonlinear regression analysis of the effect of sodium chloride, based on a planar exponential function with a decreasing phase, indicated that APX-LI is more sensitive to sodium chloride in the environment than APX-LII. In the presence of ferrous sulfate, the slope of APX-LII activity was greater than that of APX-LI, suggesting that APX-LII is more sensitive to ferrous sulfate. The maximum activity of APX-LII was observed at a concentration of 0.138 mM iron chloride. In the presence of zinc sulfate, APX-LII reached 50% of its maximum activity later than the other two enzymes, indicating that this isoenzyme is less sensitive to zinc sulfate in the environment. The optimum temperatures for maximum enzyme activity were 35 °C for APX-F, 30 °C for APX-LI, and 45 °C for APX-LII.
Conclusion: High concentrations of metal ions reduce the plant’s ability to cope with oxidative stress. Based on the results of the present study, the effects of zinc ions (zinc sulfate), iron chloride, iron sulfate, and sodium chloride on peroxidase activity were evaluated. The results indicated that sodium chloride at low concentrations had little effect on peroxidase activity; however, as sodium chloride concentration increased, structural changes occurred, leading to peroxidation. These structural alterations were associated with a significant reduction in enzyme activity, approaching nearly zero in all three isozymes. Zinc ions inhibit peroxidase activity by binding to the enzyme's active site, inducing unfavorable structural changes that interfere with substrate binding and catalytic activity. Nonlinear regression analysis of the activities of the three enzymes (APX-LI, APX-LII, and APX-F) at different temperature levels, based on a two-piece function, showed that the slope of the increasing phase of enzyme activity in the first part of the APX-LI model was greater than that of APX-LII and APX-F. This finding indicates that APX-LI is more sensitive to changes in ambient temperature in blackberries.
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