Document Type : Research Paper
Authors
- shahriar saeidian 1
- Zhilla Zarei 2
- Nabi Khaliliaqdam 3
- Bahaaldin Rashidzadeh 4
- Mohammad Ghadermarzi 2
1 Biochemistry - payame noor university - tehran - Iran
2 MSc biochemistry, Biology Department, Payame Noor University
3 Associate Professor, Agriculture Department, Payame Noor University, Tehran
4 Assistant Professor, Chemistry Department, Payame Noor University, Tehran, Iran
Abstract
Abstract
Background and objectives: Plants have efficient antioxidant systems to neutralize and eliminate toxic levels of reactive oxygen species, which include various enzymatic and non-enzymatic components. Ascorbate peroxidase is a key enzyme in this field that controls the levels of toxic reactive oxygen species in different intracellular compartments. The expression of ascorbate peroxidase is modulated in some developmental stages and under biotic and abiotic stresses, indicating the importance of ascorbate peroxidase activity in controlling H2O2 content in intracellular compartments.
Methodology: In this research, an enzyme extract was prepared from black mulberry with the scientific name Niger Morus and it was measured kinetically in the presence of different salts. The activity of ascorbate peroxidase enzyme was measured according to the amount of oxidation of ascorbate substrate at a wavelength of 290 nm and using the extinction coefficient of 18.2-1mM cm-1. To determine the optimal temperature of enzyme activity, ascorbate peroxidase was measured at 25-80°C in 50 mM Tris-HCL buffer (pH 7-6) with 5°C intervals. To evaluate the thermal stability, the enzyme solution was incubated for 30 minutes in a water bath from 25°C to 80°C, and then the reaction mixture was kept at room temperature for 10 minutes. Finally, ascorbate peroxidase activity was measured. In this research, with the aim of determining the best level of response of ascorbate peroxidase to different levels of the studied treatments, due to the small number of treatments, linear and non-linear regression analysis was used.
Results: The diagram of ascorbate peroxidase in blackberry leaf extract shows two isoenzymes at pH 6.5 and 8 (APX-LI, APX-LII) and one isoenzyme (APX-F) in blackberry fruit extract at pH 6.5. Ascorbate, pyrogallel and guaiacol substrates increase peroxidase activity, although in higher concentrations, they have opposite effects on peroxidase activity by inhibiting the substrate in order to reduce oxidative stress. The nonlinear regression analysis of APX-F and APX-LII followed the Gompertz exponential model and the activity of APX-LI followed the monophasic incremental exponential function. The results of the non-linear regression analysis of the effect of sodium chloride based on the planar exponential function had a decreasing phase and showed that the sensitivity of APX-LI in the environment is higher than that of APX-LII. As a result of ferrous sulfate, the slope of APX-LII activity is higher than that of APX-LI, which indicates that APX-LII is more sensitive to ferrous sulfate. APX-LII has the maximum activity at the concentration of 0.138 mM iron chloride. In the presence of zinc sulfate, APX-LII reaches 50% of its maximum activity much later than the other two enzymes, and this means that this isozyme is less sensitive to the presence of zinc sulfate in the environment. The maximum activity of APX-F was at 35°C, APX-LI was at 30°C and in the case of APX-LII at 45°C.
Conclusion: High concentrations of metal ions lead to reducing the plant's sensitivity in dealing with oxidative stress. Based on the results of the present study, between zinc ions (zinc sulfate), iron chloride, iron sulfate and sodium chloride, it was concluded that sodium chloride in low concentrations does not have much effect on peroxidase activity, but with increasing sodium chloride concentration, the structure undergoes peroxidation. Structural changes have been associated with a decrease in enzyme activity to nearly zero in all three isozymes. By binding to the active site of the enzyme, zinc ion plays the role of peroxidase inhibitor and negative structural changes in the direction of non-acceptance and peroxidase substrate catalysis. The non-linear regression analysis of the activity of all three enzymes APX-LI, APX-LII and APX-F at different temperature concentration levels following a two-piece function showed that the slope of the increasing of enzyme activity in the first part of the APX-LI model is higher than APX- LII and APX-F and this indicates that APX-LI is more sensitive to ambient temperature in blackberry.
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