Isolation and characterization of peroxidase from the leaves of Ricinus communis.
Multiple forms of peroxidase (E.C. 22.214.171.124) are widely distributed in plants, microbes, and animal tissues representing a huge family of heme containing enzymes [1,2] and have been used in a great number of analytical applications  such as clinical diagnosis (blood sugar and cholesterol), immunoassays (ELISA kit), biosensor construction , food processing and food storage, treatment of waste water containing phenols and aromatic amines , bio-bleaching processes, lignin degradation in fuel, production of dimeric alkaloids, oxidations, and biotransformation of organic compounds .
Plant peroxidases are found in tonoplast and plasmalemma, inside and outside the cell wall  and it occur in the soluble, as well as, ionically bound forms on the cellular walls. They oxidize several substrates in the presence of hydrogen peroxide and usually contain a protoporphyrin IX prosthetic group  and several authors have reviewed its properties and physiological roles in fruits and vegetables. Many studies have been done on amino-acid sequencing and heme structure of peroxidases [9,10], Peroxidase has been implicated in metabolic processes such as ethylene biogenesis, cell development and membrane integrity , defense mechanism toward pathogens [12,13] and various abiotic stresses, including metal ions and UV stress , salt , air pollution damage , cold tolerance , control of cell elongation, polymerization of extension , generation of reactive oxygen species , hydrogen peroxide scavenging .
These enzymes can participate in a number of oxidation and biodegradation reactions associated with changes of flavour, color, texture, and the nutritional quality of food [21,22]. The control of the activity of Peroxidase (POD) and polyphenoloxidase (POP) is of great importance in the processing of fruits, vegetables, and its products [22,23,24].
The present study was undertaken to characterize the peroxidases from the leaves of Ricinus communis and their kinetic studies.
Material and Methods
Experimental Plant Materials
The plant material used in this investigation was Ricinus communis (castor). The fresh, healthy and young leaves were collected from the botanical garden of D. D. U. University, Gorakhpur, U. P., India, washed, cut into small pieces and used for extraction of peroxidases.
Preparation of crude enzyme and solvent precipitation
To asses the total peroxidases activity from wild Ricinus plant was extracted by homogenizing 25 grams leaves with mortar and pestle added with 100mM sodium acetate buffer, pH 6.0 with addition of Polyclar aT (1.0g/10 g of tissue) as phenolic scavenger . The homogenate was filtered through four layers of cheesecloth were centrifuge for 20 min at 16,000g at 4 [degrees]C. The clear supernatant was use for peroxidase activity assay according to the method of Neves and Lourenco . All procedures were carried out at 4 [degrees]C.
The crude enzyme was precipitated with a double volume of chilled absolute ethanol and centrifuged at 15,000 rpm for fifteen minutes. Pellets were collected, dissolved in 10 mL of deionised water, and used as a source of ionically bound peroxidases.
Peroxidase activity was determined by a change in absorbance at 470 nm, due to oxidation of o-dianisidine (250 mM) in the presence of hydrogen peroxide (500 nm) in 1.0 mL of reaction mixture .
One unit of enzyme activity is defined as the amount of enzyme producing a 0.001-absorbance change per minute under the standard assay conditions.
Study of pH optima
Enzyme activity as a function of pH was determined using sodium acetate buffer (pH 4.0-5.0), Sodium phosphate buffer (pH 6.0-7.0) and Tris-HCl buffer (pH 8.0-9.0). POD activity was assayed under standard conditions and relative activity was studied.
Effect of temperature and thermal stability
The effect of temperature on peroxidases was measured in the range of 30-80 [degrees]C. The enzyme was incubated for 10 min at different temperatures and aliquots were withdrawn at regular time intervals and assayed for the per cent age relative activity.
Determination of the effect of phenolic compounds, metal ions and amino acids
The enzyme was incubated with derivatives of hydroxycinemic acid such as ferulic acid (0.02-0.08 [micro]M), caffeic acid (1.0-4.5 [micro]M), a hydroxybenzoic acid derivative like protocatechuic acid (1.0-4.5 [micro]M) and growth hormones like indol 3-acetic acid (1.04.5 [micro]M) with fixed enzyme concentrations.
The effects of metal cations [Fe.sup.2+], [Cu.sup.2+], [Mn.sup.2+], [Mg.sup.2+] and [Zn.sup.2+] (1.0-2.0 [micro]M), derivatives of hydroxycinemic acid such as ferulic acid (0.02-0.08 [micro]M), caffeic acid (1.0-4.5 [micro]M) and a hydroxybenzoic acid derivative like protocatechuic acid (1.0-4.5 [micro]M). Growth hormones like indol 3-acetic acid (1.0-4.5 [micro]M) and different amino acids (D-Alanine, DL-valine, DL-methionine, L-proline and L-cysteine) were also checked for their effect on the activity of peroxidases (added at different concentration in the reaction mixture). The relative activity was measure at standard assay conditions with fixed enzyme concentrations and the per cent relative activity was calculated.
Determination of Kinetic Constants
The apparent Km and Vmax were determined from the Lineweaver-Burk plot by following the optimum pH and temperature conditions.
Result and Discussion
Effect of pH
Ricinus Peroxidases showed the maximum percent relative activity at pH 5.0 and it get decreased when pH increases Dubey et al.,[ 27] has also showed the similar result i.e. acidic pH ranged 5-7 in four different variety of apple peroxidases (Fig.1). Optimum level of peroxidases was also reported from various vegetable sources , apoplastic peroxidases from several plant species [28,29] at acidic pH and Cassia didymobotrya peroxidase at pH 5.5 . Estimation of secondary structural elements at various pH values indicated that there is a maximal reduction of beta-strands and beta-turns at pH 5.5 causing the heme to be further exposed to the solvent and increasing the overall conformational flexibility of the protein .
Effect of Temperature
Ricinus peroxidases showed maximum activity at 60 [degrees]C, it started decreasing while moving towards high temperature and a sharp decline in relative activity was also observed at 80 [degrees]C (Fig. 2). The influence of temperature at 60 [degrees]C and above induces the protein globule unfolding and loosing of peroxidase activity [31,32]. Effect of temperature on structural and functional properties of horseradish peroxidases was established that temperatures ranged 20-55oC causes reversible conformation and occurrence of changes of the hemoprotein molecule, which is related to consequent unfolding and folding of the protein globule.
Effect of effectors (Metal ions, phenolic compounds, growth hormone and amino acids)
A wide variety of proteins and enzymes incorporate metal ions or metal complex into their overall structure and trigger to enhance their activity. [Fe.sup.2+], [Cu.sup.2+], [Mn.sup.2+], [Zn.sup.2+] and [Mg.sup.2+] were reported to be the potent activators 586, 193, 145,103 and 145 per cent relative activity at 4.0 [micro]M concentration (Fig.3).
The derivatives of hydroxybenzoic acid (protoctatechuic acid) act as potent inhibitor for Ricinus peroxidases and show the following 43, 20, 8.0, 5.0 and 0.0 per cent relative activity at different concentration 1.0, 2.0, 3.0, 4.0 and 4.5 [micro]M. Ferulic acid (derivative of hydroxycinnamic acid) stimulated the enzyme activity at very low concentration i.e. 0.04-0.08 [micro]M, however the per cent relative activity of other derivative of hydroxycinnamic acid (caffeic acid) was recorded to be 164, 179,174, 164 and 152 and this value was somewhat similar in case of a growth hormone (Indol 3-acetic acid) with the value of 128, 116, 118, 141 and 125 activates of the Ricinus peroxidase at given concentration 1.0, 2.0, 3.0, 4.0 and 4.5 [micro]M respectively (Fig. 4).
DL-methionine and DL-valine worked as inhibitors for Ricinus peroxidase at 4.0 mM concentration and showed the following per cent relative activity 63 and 80 while L-cyteine worked as a potent inhibitor at a very low concentration 0.4 mM. However D-alanine and L-proline are activate the of Ricinus peroxidase activity at same concentration 115 and 128 (Fig. 5).
The Vmax/Km value for o-dianisidine was 680 units/min/mL and for [H.sub.2][O.sub.2] 6.5 x [10.sup.5] units/min/mL for Ricinus, respectively. The ratios Vmax/Km indicated a preferential action of the enzyme for hydrogen peroxide, compared to o-dianisidine, which was in contrast to peroxidases from apple , papaya , and kiwifruit .
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The Ricinus peroxidases were studied on native-PAGE (6.0%) and were observed that the acetone precipitated peroxidases showed seven distinct bands on the gel.
This research is the part of M. Sc. thesis work and authors are highly thankful to D. D. U. University, Gorakhpur, U. P. India for providing research grant.
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P. Kumar (1), M. Kamle (1), J. Singh (2) and D. P. Rao (3)*
(1) Central Institute for Subtropical Horticulture, P.O.-Kokori, Rahmankhaera, Lucknow- 227 107, U.P., India
E-mail: firstname.lastname@example.org, E-mail: email@example.com
(2) Department of Biotechnology, Punjab University, Chandigarh- 600 014, India
(3)* Department of chemistry, DAV college, kanpur, U.P., India