ISOLATION AND PURIFICATION OF PEROXIDASE FROM SHOOTS OF TOMATO

Abstract

Utilizing starch-gel electrophoresis, peroxidases were identified in purified

extracts of the dwarf tomato shoot (Lycopersicon esculentum).A major peroxidase has been found in the tomato pericarp (Lycopersicon esculentum var. Tropic) of the ripe and green fruit. A purification scheme yielding this enzyme approximately 85% pure has been developed. The tomato enzyme resembles horseradish peroxidase (HRP) in a standard peroxidase assay and in its ability to be reduced to ferroperoxidase, to be converted to oxyferroperoxidase (compound III), and to form peroxidase complexes with hydrogen peroxide (compounds I and II). In contrast to the HRP, the tomato peroxidase fails to catalyze the aerobic oxidation of indole-3-acetic acid in the presence of 2,4-dichlorophenol and manganese. The tomato peroxidase can be resolved into two nonidentical subunits in the presence of dithiothreitol while HRP remains as a single polypeptide chain after such treatment. Dithiothreitol is oxidized in the presence of tomato or horseradish peroxidase with the enzymes accumulating in their oxyferroperoxidase forms during the oxidation reaction. Whereas HRP returns to its free ferric form at the end of the reaction, the tomato enzyme is converted into a form that absorbs at 470 nanometers. Molecular weight of peroxidase was measured 46kg/dalton and the protein was estimated 1.813 by the biuret test.

Introduction

A single peroxidase which has been shown to exist in tomato fruit extracts and

to exhibit some IAA oxidase activity (5, 8) has been implicated both in the production of ethylene (12, 17, 18) and in the destruction of the plant growth hormone IAA (8). Although it is not unusual to relate the action of the enzyme peroxidase with the control of the above hormones, in the case of tomato fruit it is premature to assume such a relationship because of insufficient information about the physical and catalytic properties of this peroxidase and the lack of a purification method which would allow a quantitative estimation and a complete isozyme composition of the noncovalently bound peroxidases of the fruit (22, 23). Spectral properties and pH optima of the tomato fruit peroxidase in catalyzing the oxidation of redogenic substrates in the presence of H202 have been previously reported by Evans (6). The present report deals with additional properties of the purified enzyme including its capability to be converted to complexes of higher oxidation states (compounds I, II, III) which are thought to be important for the IAA oxidase activity of peroxidases (13, 26). The enzyme HRP,3 a well studied peroxidase, was employed in order to compare some of its properties with similar propertiesof the tomato enzyme. This paper describes the isolation and partial purification of the major peroxidases from the extreme dwarf tomato shoot.

Materials and Methods

Plant Material: The extreme dwarf (dx/d1) tomato plant (Lycopersicon esculentum) was originally described by Rick and Butler ( 10) and descendents of Rick's stock were used in thes studies. There are no qualitative differences between dwarf and normal tomato peroxidases, but peroxidase activity is greater in the dwarf tomato plant (2). Isolation of Crude Peroxidases: Tomato shoot was homogenized in the presence of I liter of0.1 M phosphate buffer (pH 6.5) supplemented with 20 g of insoluble PVP from Calbiochem and 10 g of sodium ascorbate. The homogenate was filtered through a triple layer of cheesecloth, and the residue was washed twice with 100 ml of 0.1 M phosphate buffer (pH 6.5). The filtrates were combined and centrifuged at 2,000 g for 10 min. The supernatant, to be referred to as crude soluble peroxidase fraction, was stored at 0 C for further use. The pellet and the solid material on the cheesecloth were suspended in 200 ml of 0.2 M sodium maleate-0.2 M calcium chloride adjusted to pH 6.5. The suspension was then sonicated for 5 min and centrifuged at 2,000g for 10 min. Under this treatment peroxidases that were ionically bound to the tomato pulp were solubilized (17). These peroxidases remained in solution after,the supernatant was dialyzed twice against 4 liters of 5 mm phosphate buffer (pH 6.5) for 20 hr and will be referred to as crude ionically bound peroxidases. All steps of the above procedure took place at 4 C.

Ammonium Sulfate Precipitation: Ammonium sulfate precipitation was used as a purification step for the crude soluble peroxidase fraction but not for the crude ionically bound peroxidases. In the former case, solid ammonium sulfate was added slowly with stirring to the crude soluble peroxidase fraction to 40%o saturation, and the resulting precipitate was removed by centrifugation. The supernatant carrying most of the peroxidase activity was then brought to 85% saturation. The precipitate collected by centrifugation, was dissolved in 50 ml of distilled H20 and dialyzed twice against 4 liters of 5 mm phosphate buffer (pH 6.5) for 20 hr. Inactive precipitate was removed by centrifugation. All steps of the above procedure were carried out at 4 C. The presence of inhibitors in the crude extract had been removed by the precipitation followed by dialysis. Dialysis Dialysis is a very small technique used extensively to separate macromolecules from smaller molecules. Here the solution containing sample and phosphate buffer was taken in a dialysis bag which allows only small molecules and ions to pass through but larger molecules like proteins are held back. The method was commonly used for removing salts from proteins. The dialysis bag was boiled for 10 minutes in a beaker of water containing sodium sulphate (2%) and EDTA (1M). Then the bag was taken out and rinsed in distilled water. The bag was boiled in a beaker of water containing 1mM EDTA. The bag was cooled. One end of the bag was tied and checked for leakage. The dialysis bag was diluted with sample and then tied at other end. The bag was then suspended in a beaker with 500 ml distilled water and kept overnight at 4C. The water was changed the next day and bag was suspended for 3 more hours later the bag was removed and the sample was transferred to lyophilization flask.

 SDS Page: Standard proteins and purified enzymes were incubated in 1 % SDS and 01 % -

mercaptoethanol in sample buffer for 2 h at 37C. Staining was performed with 1 % amido black in 7% acetic acid. Excess stain was removed and replaced by by 7% acetic acid with frequent changes. After destaining, gels were stored in 2 % acetic acid. 1] 1x SDS gel loading buffer:
2] 1x Triss glycine electrophoresis buffer: Stacking gel: (5%) Resolving Gel: (10%) Staining solution: Destaining solution:

 Peroxidase Assay:

Peroxidase assay was carried out in a reaction

mixturecontaining 46 mL phosphate buffer of pH 6, 0.320 mL of H202 (Substrate) and 0.850 mL guaiacol (chromogen). The absorbance of the coloured complex was read on spectrophotometer at 470 nm wavelength after 3 min. of reaction interval (Ambreen et al., 2000). Protein contents of the enzyme extract at all steps were measured by biuret method (Gornall et aI., 1949).

1. Molecular weight of peroxidase enzyme: 46 kg/Dalton

References

CHANCE, B. AND A. C. MAEHLY. 1955. Assay of catalases and peroxidases. 11. In: Methods in Enzymology. Vol. II. S. Colowick and N. Kaplan, eds. Academic Press, New York. p 764-75. EVANS, J. J. AND N. A. ALLDRIDGE. 1965. The distribution of peroxidases in extreme dwarf and normnal tomato. Phytochemistry 4: 449-503. HOSOYA, T. 1960. Turnip peroxidase. I. Purification and physicochemical properties of multiple components in turnip peroxidase. J. Biochem. 47: 369-81. KAWASHIMA, N. AND I. URITANI. 1965. Some nproperties of peroxidase produced in sweet potato infected by the black rot fungus. Plant Cell Physiol. 6: 247-65. KON, S. AND J. R. WHITAKER. 1965. Separation and partial characterization of the peroxidases of nFicus glabrata latex. J. Food Sci. 30: 977-85. KONDO, K. AND Y. MORITA. 1952. Studies on phytoperoxidase (2). Isolation and purification of nsweet potato peroxidases and their n absorption nspectra. Bull. Res. Inst. Food Sci., Kyoto Univ. n10: 33-45. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, AND R. J. RANDALL. 1951. Protein measurement with the Folin phenol reagent.n J. Biol. Chem. 193:n 265-75. MACNICOL, P. K. 1966. Peroxidases of the Alaska pea (Pisum sativum L.). Arch. Biochem. Biophys. n117: 347-56. RACUSEN, D. AND M. FOOTE. 1966. Peroxidase isozymes in bean leaves by preparative disc electroplhoresis. Can. J. Botany 44: 1633-38. RICK, C. M. AND L. BUTLER. 1956. Cytogenetics nof the tomato. Advan. Genet. 8: 267-382.