Quantitative investigation of the effect of surfactants and chaotropes on ascorbate peroxidase activity in strawberry and blackberry

Document Type : scientific research article

Authors

1 Associate Prof., Dept. of Biology, Payame Noor University, Tehran, Iran.

2 Corresponding Author, Associate Prof., Dept. of Agriculture, Payame Noor University, Tehran, Iran.

3 M.Sc. of Molecular and Cellular Biology, Kharazmi University, Tehran, Iran.

Abstract

Background and purpose: As one of the antioxidant enzymes, ascorbate peroxidase (APX) has an important role in the defense system of plants against environmental stresses and is effective in regulating the concentration of hydrogen peroxide (H2O2) in plant cells. APX decomposes hydrogen peroxide in order to prevent plant damage. During various environmental stresses, antioxidant enzymes such as APX increase in different plant parts such as fruit. Of course, the rate of biosynthesis of this antioxidant enzyme depends on the intensity and duration of stress in the plant. Considering the importance of the presence of this enzyme in plant structures, this study was conducted to investigate the maximum activity of ascorbate peroxidase enzyme at different levels of surfactants and chaotropes.
Materials and methods: This research was carried out in the research laboratory of Payam Noor University of Kurdistan. In order to prepare the extract from strawberries and blackberries, in the presence of 0.1 M citrate-phosphate buffer with pH 7 and 0.02 phenylmethanesulfonylfluoride solution as a protease inhibitor, the fruits were homogenized separately.

Results: Based on the results of this research, for the ascorbate substrate used in the APX measurement in the extracts prepared from strawberry (ST.APX) and blackberry (BM.APX), the optimal pH is 6.7 and the catalytic efficiency of BM.APX, is about 1.6 times higher than ST.APX was obtained. With the increase of ascorbate concentration in constant concentration of H2O2, the activity of BM.APX and ST.APX increases, so that the highest level of activity of both plant 0. 5 units per mg of protein. A further increase in substrate concentration was associated with substrate inhibition of BM.APX and ST.APX. The activity curve of APX in both plant species is hyperbolic, which follows the Michaelis-Menten kinetics, which indicates the high tendency of the enzyme to consume ascorbate. The activity of ST.APX and BM.APX is inhibited by kojic acid and it is non-competitive in both. Among the used chaotropes and surfactants, only SDS activated the activity of ST.APX and BM.APX, and the activities of ST.APX and BM.APX showed different sensitivities to the surfactants, chaotropes and kojic acid. Despite the fact that BM.APX showed higher activity than ST.APX in the presence of ascorbate and SDS, in the presence of other factors, the rate of inhibition of BM.APX was higher than ST.APX and showed higher percentages of inhibition. Urea and guanidine hydrochloride as reducing agents showed an inhibitory role on ST.APX and BM.APX. The activity of APX The results also showed that the activity of ST.APX and BM.APX was inhibited by kojic acid and it was non-competitive in both. Among the chaotropes and surfactants used, only SDS activated the activity of ST.APX and BM.APX and its maximum effect occurred at a concentration of 0.2 mM. On the other hand, in other factors, the rate of inhibition and reduction of BM.APX activity was higher than ST.APX, and with the exception of ascorbate peroxidase response to kojic acid and sodium calate in strawberry, the enzyme response followed a monophasic decreasing exponential








Conclusion: In this research, ascorbate peroxidase takes a more active form by following Michaelis Menten's kinetics and binding the substrate to the active site of the enzyme, and by performing enzyme catalysis, it causes ascorbate consumption in strawberries and blackberries. Ionic surfactants with an activation mechanism such as SDS and an inhibitory mechanism such as sarkosyl and sodium cholate have different effects on ascorbate peroxidase. Non-ionic surfactants such as kojic acid and chaotrope agents reduce the activity of ascorbate peroxidase in both plant species. The position of kojic acid is different from the position of ascorbate due to the non-competitive type of inhibition.

Keywords

Main Subjects


1.Dixon, D. P., Cummins, I., Cole, D. J., & Edwards, R. (1998). Glutathione-mediated detoxification systems in plants. Current Opinion in Plant Biology, 1, 258-266.
2.Hosseini, Z., & Pourakbar, L. (2013). Investigation of interaction between zinc and organic acid (malic acid, citric acid) on antioxidant responses in Zea mays L. Iranian Journal of Plant Biology, 5 (16), 1-12. [In Persian]
3.Tavakoli Zanyani, F., Shabani, L., & Razavizadeh, R. (2013). Activation of defense responses under isothiocyanate stress in oilseed rape plantlets. Iranian Journal of Plant Biology, 5 (16), 81-92 [In Persian]
4.Hasanuzzaman, M., Anwar, M., Teixeira da Silva, J., & Fujita, M. (2012). Plant response and tolerance to abiotic oxidative stress: Antioxidant defense is a key factor.Springer, New York, USA, pp. 261-351.
5.Johnson, S. M., Doherty, S. J., & Croy, R. R. D. (2003). Biphasic superoxide generation in potato tubers: a self-amplifying response to stress. Plant Physiology, 13, 1440-1449.
6.Kawano, T. (2003). Roles of the reactive oxygen species-generating peroxidase reactions in plant defense and growth induction. Plant Cell Reports, 829-837.
7.Peltzer, D., Dreyer, E., & Polle, A. (2002). Differential temperature dependencies of antioxidative enzymes in two contrasting species. Plant Physiology and biochemistry, 40, 141-150.
8.Chaves, M. M., Maroco, J. P., & Pereira, J. S. (2003). Understanding plant responses to drought –from genes to the whole plant. Function of Plant Biology, 30, 239-264.
9.Asada, K. (2006). Production and scavenging of reactive oxygen species in chloroplasts and their functions, Plant Physiology, 141, 391-396.
10.Bajpai, D. (2007). Laundry detergents: an overview. Journal of Oleo Science, 56 (7), 327-340.
11.Lomax, E. G. (1996). Amphoteric surfactants. CRC Press.
12.Schick, M. J. (1987). Nonionic surfactants. Physical Chemistry. CRC Press.
13.Hosseini, Z., & Pourakbar, L. (2013). Investigation of interaction between zinc and organic acid (malic acid, citric acid) on antioxidant responses in Zea mays L. Iranian Journal of  Plant Biology, 5 (16), 1-12. [In Persian]
14.Kielkopf, C. L., Bauer, W., & Urbatsch, I. L. (2020). Bradford Assay for Determining Protein Concentration. Cold Spring Harbal Protocol, 1 (4), 102269.
15.Nakano, Y., & Asada, K. (1987). Purification of ascorbat peroxidase in spinach choloroplasts, its inactivation in Ascorbate-Depleted medium and reactivation by monodehydroascorbate radica, Plant Cell Physiology, 28 )1), 131-140.
16.Kavousi, H. R., & Barandeh, F. (2017). Effect of Cadmium on changes of some enzymatic and none enzymatic antioxidant defense systems in lentil seedlings (Lens culinaris Medik.), Iranian Journal of Pulses Research, 7 (2), 125-137.
17.SAS. (2009). Statistical analysis system, Version: 9.2. Carry NC.
18.Motulsky, H. (1999). Analyzing data with GrapPad Prism, A companion to GraphPad Prosm version 3, GraphPad software, Inc.
19.Khaliliaqdam, N., & Talebzade, S. J. (2022). Prediction of Rate of Leaf Appearance, Leaf Area Index and Growth Stages in Corn and Sunflower plants. Journal of Crop Production, 15 (1), 205-228.
20.Ishikawa, T., Yoshimura, K., Tamoi, M., Takeda, T., & Shigeoka, S. (1997). Alternative mRNA splicing of 3'-terminal exons generates ascorbate peroxidase isoenzymes in spinach (Spinacia oleracea) chloroplasts. Biochemical Journal, 328, 795-800.
21.Lu, Z., Takano, T., & Liu, S. (2005). Purification and characterization of two ascorbate peroxidases of rice (Oryza sativa L.) expressed in Escherichia Coli. Biotechnology Letter, 27, 63-67.
22.Li, C., Huang, W. Y., Wang, X. N., & Liu, W. X. (2013). Oxygen Radical Absorbance Capacity of Different Varieties of Strawberry and the Antioxidant Stability in Storage. Molecules, 18, 1528-1539.
23.Rabia, Sh., Arifa, Sh., & Rakesh, K. M. (2011). Cyclic voltammetry and viscosity measurements of aggregated assemblies of anionic surfactants with nonionic surfactants and triblock copolymers. Colloid and Polymer Science, 289, 43-51.
24.Wang, S. Y., & Lin, H. S. (2000). Antioxidant activity in fruits and leaves of blackberry, raspberry, and strawberry varies with cultivar and developmental stage. Journal of Agriculture and Food Chemistry, 48 (2), 140-146.
25.Dąbrowska, G., Kata, A., Goc, A., Szechyńska-Hebda, M., & Skrzypek, E. (2007). Characteristics of the plant ascorbate peroxidase family. Acta Biologica Cracoviensia Series Botany, 49 (1), 7-17.
26.Espin, J. C., & Wichers, H. J. (1999). Activation of a latent mushroom (Agaricus bisporus) tyrosinase isoform by sodium dodecyl sulfate (SDS). Kinetic properties of the SDS‐activated isoform. Journal of Agriculture and Food Chemistry, 47 (9), 3518‐25.
27.Mohammad, A. (2017). Alsenaidy. Biophysical evaluation of amyloid fibril formation in bovine cytochrome c by sodium lauroyl sarcosinate (sarkosyl) in acidic conditions. Journal of Molecular Liquids, 241, 722-729.
28.Saeidian, Sh., Keyhani E., & Keyhani, J. (2007). Effect of Ionic Detergents, Nonionic Detergents, and Chaotropic Agents on Polyphenol Oxidase Activity from Dormant Saffron (Crocus sativus L.) Corms. Journal of Agriculture and Food Chemistry, 55 (9), 3713-3719.
29.Alexander, N. P., Hiner, J. N., Rodriguez-Lopez, M. B., Arnao Emma, L. R., Francisco, G. C., & Manuel, A. (2000). Kinetic study of the inactivation of ascorbate peroxidase by hydrogen peroxide. Biochemistry Journal,348, 321-328.
30.Gasowska, B., Kafarski, P., & Wojtasek, H. (2004). Interaction of mushroom tyrosinase with aromatic amines,
o‐diamines and oaminophenols. Biochimica Biophysica Acta, 4 (3), 170‐177.
31.Shweta, S., Santosh, K., & Tonmoy, R. (2016). Aggregation Behaviour of Sodium Cholate and Sodium Deoxycholate under the Influence of Drug (Disprine) in Aqueous Solution at Various Temperatures. Science Research, 4 (1), 112-119.

32.Yadav, P., Yadav, T., Kumar, S., Rani, B., Kumar, S., & Jain, M. (2014). Partial purification and characterization of ascorbate peroxidase from ripening ber (Ziziphus mauritiana L) fruits. African Journal of Biotechnology,
13 (31), 3323-3331.

33.Sanchez Ferrer, A., Rodriguez‐Lopez, J.N., Garcia‐Canovas, F., & Garcia‐Carmona, F. (1995). Tyrosinase: a comprehensive review of its mechanism. Biochimica Biophysica Acta, 22 (1), 1-11.
34.Saeidian, S. (2016). Kinetic investigations of peroxidase in roots of Gundelia Tournefortii. Experimental Animal Biology, 5 (2), 1-11.