isolation and purification of peroxidase from shoots of of tomato

7/29/2019 Isolation and Purification of Peroxidase From Shoots of OF TOMATO

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ISOLATION AND PURIFICATIONOF PEROXIDASE FROM SHOOTS

OF TOMATO


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Abstract

Utilizing starch-gel electrophoresis, peroxidases were identified in purified

extracts of the dwarf tomato shoot (Lycopersicon esculentum).A majorperoxidase has been found in the tomato pericarp (Lycopersicon esculentumvar. Tropic) of the ripe and green fruit. A purification scheme yielding thisenzyme approximately 85% pure has been developed. The tomato enzymeresembles horseradish peroxidase (HRP) in a standard peroxidase assay and in

its ability to be reduced to ferroperoxidase, to be converted tooxyferroperoxidase (compound III), and to form peroxidase complexes withhydrogen peroxide (compounds I and II). In contrast to the HRP, the tomatoperoxidase fails to catalyze the aerobic oxidation of indole-3-acetic acid in thepresence of 2,4-dichlorophenol and manganese. The tomato peroxidase can beresolved into two nonidentical subunits in the presence of dithiothreitol whileHRP remains as a single polypeptide chain after such treatment. Dithiothreitol

is oxidized in the presence of tomato or horseradish peroxidase with theenzymes accumulating in their oxyferroperoxidase forms during the oxidationreaction. 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 wasestimated 1.813 by the biuret test.


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Introduction

A single peroxidase which has been shown to exist in tomato fruit extracts andto exhibit some IAA oxidase activity (5, 8) has been implicated both in theproduction of ethylene (12, 17, 18) and in the destruction of the plant growthhormone IAA (8). Although it is not unusual to relate the action of the enzymeperoxidase with the control of the above hormones, in the case of tomato fruit itis premature to assume such a relationship because of insufficient information

about the physical and catalytic properties of this peroxidase and the lack of apurification method which would allow a quantitative estimation and acomplete isozyme composition of the noncovalently bound peroxidases of thefruit (22, 23). Spectral properties and pH optima of the tomato fruit peroxidasein catalyzing the oxidation of redogenic substrates in the presence of H202have been previously reported by Evans (6). The present report deals withadditional properties of the purified enzyme including its capability to be

converted to complexes of higher oxidation states (compounds I, II, III) whichare 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 tocompare some of its properties with similar propertiesof the tomato enzyme.This paper describes the isolation and partial purification of the majorperoxidases from the extreme dwarf tomato shoot.


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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 noqualitative differences between dwarf and normal tomato peroxidases, but peroxidase activity isgreater 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. Thehomogenate 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 at2,000 g for 10 min. The supernatant, to be referred to as crude soluble peroxidase fraction, wasstored at 0 C for further use. The pellet and the solid material on the cheesecloth weresuspended in 200 ml of 0.2 M sodium maleate-0.2 M calcium chloride adjusted to pH 6.5. Thesuspension was then sonicated for 5 min and centrifuged at 2,000g for 10 min. Under thistreatment 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 5mm phosphate buffer (pH 6.5) for 20 hr and will be referred to as crude ionically boundperoxidases. All steps of the above procedure took place at 4 C.


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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 peroxidasefraction 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 distilledH20 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 theprecipitation 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 adialysis bag which allows only small molecules and ions to pass through but larger moleculeslike 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 sodiumsulphate (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 ofthe bag was tied and checked for leakage. The dialysis bag was diluted with sample and thentied at other end. The bag was then suspended in a beaker with 500 ml distilled water andkept overnight at 4C. The water was changed the next day and bag was suspended for 3more hours later the bag was removed and the sample was transferred to lyophilizationflask.


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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 % aceticacid.

1] 1x SDS gel loading buffer:

2] 1x Triss glycine electrophoresis buffer: Stacking gel: (5%)

Resolving Gel: (10%)

Staining solution:

Destaining solution:


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Peroxidase Assay:

Peroxidase assay was carried out in a reactionmixturecontaining 46 mL phosphate buffer of pH 6,

0.320 mL of H202 (Substrate) and 0.850 mLguaiacol (chromogen). The absorbance of thecoloured complex was read on spectrophotometer at470 nm wavelength after 3 min. of reaction interval

(Ambreen et al., 2000). Protein contents of theenzyme extract at all steps were measured by biuretmethod (Gornall et aI., 1949).


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


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References

CHANCE, B. AND A. C. MAEHLY. 1955. Assay of catalases and peroxidases. 11. In: Methods inEnzymology. 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 dwarfand normnal tomato. Phytochemistry 4: 449-503.

HOSOYA, T. 1960. Turnip peroxidase. I. Purification and physicochemical properties ofmultiple components in turnip peroxidase. J. Biochem. 47: 369-81.

KAWASHIMA, N. AND I. URITANI. 1965. Some nproperties of peroxidase produced in sweetpotato infected by the black rot fungus. Plant Cell Physiol. 6: 247-65.

KON, S. AND J. R. WHITAKER. 1965. Separation and partial characterization of theperoxidases of nFicus glabrata latex. J. Food Sci. 30: 977-85.

KONDO, K. AND Y. MORITA. 1952. Studies on phytoperoxidase (2). Isolation and purificationof nsweet potato peroxidases and their n absorption nspectra. Bull. Res. Inst. Food Sci., KyotoUniv. n10: 33-45.

LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, AND R. J. RANDALL. 1951. Proteinmeasurement 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 discelectroplhoresis. Can. J. Botany 44: 1633-38.

RICK, C. M. AND L. BUTLER. 1956. Cytogenetics nof the tomato. Advan. Genet. 8: 267-382.

isolation and purification of peroxidase from shoots of of tomato

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