Studies on the Effect of Organophosphate, Dichlorvos on the Esterases of seeds of an Underutilized Legume, Mucuna pruriens

 

Kempohalli S. Chandrashekharaiah

Department of Biotechnology, PESIT, BSK III Stage, Bangalore-560085, Karnataka, India

*Corresponding Author E-mail: chandrasks@pes.edu

 

The esterases were isolated from the seeds of eight varieties/accessions of an underutilized legume, Mucuna and studied. The seeds of all the varieties of Mucuna exhibited esterase.  Among the different varieties analyzed for esterase activity, Mucuna pruriens exhibited highest ester hydrolyzing activity (4.7µmoles/min) and specific activity (0.018) while Mucuna cochinensis showed the lowest esterase activity (0.309µmoles/min) and specific activity (0.0017). Germination of Mucuna pruriens has been carried out and the change in esterases was monitored. Effect of organophosphate such as dichlorvos on the activities of esterases was analyzed. The esterases are completely inhibited by dichlorvos. The Mucuna seed esterases are found to be carboxylesterases.

 

 

INTRODUCTION:

Organophosphorus pesticides (OPs) are one of the most common classes of chemicals used for the control of insects on plants because of their high efficacy and broad spectrum of activity. As a result, OP residues are likely to occur in plants. The inappropriate and illegal usage of OPs further increases the risk of both human and plant exposure. Metabolic detoxification of insecticides is an important toxicokinetic mechanism for any organism to reduce the toxic effects of insecticides. Three enzyme systems are generally recognised as the major detoxification systems involved in insecticide resistance in insects. These are esterases, glutathione S-transferases, and cytochrome P450-dependent monooxygenases¹.

 

Esterases are widely distributed hydrolytic enzymes catalyze the hydrolysis of various types of ester bonds. These esterases include phosphoric monoester hydrolases, phosphoric diester hydrolases, thioester hydrolases, sulphuric ester hydrolases and carboxyl ester hydrolases. These occur in multiple molecular forms and exhibit broad substrate specificity. The physiological roles of these esterases are multifaceted. They are involved in fruit ripening, abscission, cell expansion, reproduction as well as hydrolysis of ester containing xenobiotic molecules. Esterases play an important role in agrochemical efficacy and detoxification^{2, 3} by interacting with agrochemicals,such as  Ops⁴.

 

Mucuna is one of the lesser known underutilized legume, grown as a minor food crop by tribal and ethnic groups of Asia and Africa. Seeds of Mucuna constitute source of food for tribals and some ethnic groups of Asia and Africa. The immature pods and leaves serve as vegetables, while seeds as condiment and main dish by ethnic groups in Nigeria⁵. The genus Mucuna belongs to the family fabaceae (leguminosae), include up to 150 species. They are annual or perennial legumes of pan tropical distribution. More than 15 varieties/accessions are available in southern India. Mucuna seeds are relatively good source of protein and have a relatively favorable amino acid composition. They contain high amounts of certain minerals, including Ca (calcium), Mg (magnesium) and Fe (iron) ^{6, 7}. Mucuna seeds are a promising source of protein and to meet the protein demands in developing countries like India, there is a need to source proteins from underutilized legume, Mucuna^8.

 

In the present study, the esterases and the effect of dichlorvos on the activity of esterases of Mucuna have been discussed.

 

MATERIALS AND METHODS:

Materials:

The seeds of Mucuna pruriens were collected from Siddara Betta hills, Koratagere, Tumkur District, Karnataka, India.

 

Chemicals:

Acrylamide, N, N, Methylene bisacrylamide, 1-naphthyl acetate, 1-naphthyl propionate, bovine serum albumin, fast blue RR salt, fast blue B salt (diazo blue B) were obtained from Sigma Chemical Company, St. Louis, MO, USA. All other chemicals were analytical grade.

 

Preparation of acetone powder and enzyme extract:

Acetone powder (10%) was prepared according to the method of Wetter (1957)⁹ by blending soaked and dehulled seeds of Mucuna in chilled acetone for 1 -3 minutes, followed by  filteration using suction pump, dried at 37^oC and stored at 4⁰C until further use.  A 10% extracts of the acetone powder was prepared using 50 mM sodium phosphate buffer pH 7.0 by stirring over a magnetic stirrer for 2 hr at 4⁰C, followed by centrifugation at 10,000 rpm for 15 min at 4⁰C. The crude enzyme extract was subjected to ammonium sulphate precipitation (0 – 80%). Solid ammonium sulphate was added slowly with constant stirring over magnetic stirrer at 4⁰C to obtain 80% saturation. The solution was allowed to stand for 1 hr at 4⁰C, centrifuged at 10,000 rpm for 30 minutes. The protein pellet obtained was dissolved in small volume of buffer and dialyzed against sodium phosphate buffer pH 7.0. After dialysis, the precipitated proteins were removed by centrifugation at 10,000 rpm for 30 min. The dialyzed fraction was assayed for esterase activity.

 

Esterase assay:

Esterase activity was determined by the method of Gomori (1953)¹⁰ as modified by Van Asperen (1962)¹¹. A typical assay mixture contained 5 ml of 0.3 mM substrate solution (a stock solution of 30 mM 1-naphthyl acetate was prepared in acetone and diluted to 100 fold with 0.05 M sodium phosphate buffer, pH 7.0) and 1 ml of enzyme extract. The reaction mixture was incubated for 15 minutes at 27⁰C and the reaction was stopped by the addition of 1 ml DBLS reagent (2 parts of 1% diazo blue B and 5 parts of 5% sodium lauryl sulphate). The reaction mixture was allowed to stand for 30 minutes and the absorbance was read at 600 nm. A calibration curve was prepared using 1-naphthol.

 

Protein assay:

The protein content in the extract and fraction was determined by the method of Lowry et al.(1951)¹² using bovine serum albumin as standard.

 

Electrophoresis:

An anodic disc gel electrophoresis was carried out according to the method of Ornstein and Davis (1964)¹³ using 7.5% separating gel and 4% spacer gel. The electrode chambers were filled with electrode buffer (tris-glycine buffer, pH 8.3). The samples suitably diluted with (100 µg protein) with 20% glycerol containing bromophenol blue, were loaded on to each sample well and subjected to electrophoresis at 4⁰ C applying a current of 2 mA /sample well for 4 hours.

 

Staining for esterase activity:

Esterase activity on polyacrylamide gels was detected according to the method of Hunter and Markert (1957)¹⁴. Fast-blue RR was used for simultaneous coupling with 1-naphthol moiety released upon hydrolysis of 1-naphthyl acetate. The gels were removed from plates and stained for esterase activity using 100 ml of 0.05 M phosphate buffer pH 7.0 containing 40 mg of fast-blue RR and 20 mg of 1-naphthyl acetate (dissolved in 2 ml of acetone) for 20 minutes at room temperature.

 

Staining for proteins:

Proteins were stained on polyacrylamide gels using 0.5% solution of coomassie brilliant blue R-250 in 25% methanol and 7.5% acetic acid in water for 1 hour and was destained in 25% methanol and 7.5% acetic acid in water for overnight.

 

Inhibition studies:

Inhibition studies were carried out colorimetrically by pre-incubating the enzymes with different concentrations (1 x 10^-3 to 1 x 10 ^-10 M) of dichlorvos for 30 minutes at 27⁰C prior to the addition of substrate. The control tubes contain 1% (w/v) Triton X-100 instead of inhibitor. Stock solutions of different inhibitors were prepared in 1% Triton X-100 and these were serially diluted to get required the concentrations of the inhibitors.

 

Inhibition studies on gels:

To study the effect of inhibitor, the gels was pre-incubated in inhibitor solution (dichlrvos, stock solution was prepared in acetone diluted to required concentrations using 0.05 M sodium phosphate buffer, pH 7.0) for 30 minutes at 37⁰C (1 x 10^-3M). After the incubation, the gel was rinsed with distilled water and substrate-dye solution.

 

Results and Discussion

Analysis of esterases from the seeds of Mucuna showed that all the varieties of Mucuna exhibited the esterase activity. However, among the different varieties analyzed, Mucuna pruriens exhibited highest ester hydrolyzing activity and specific activity while Mucuna cochinensis MP9 showed the lowest esterase activity and specific activity (Table 1).

 

The esterase activity and protein band pattern obtained for different varieties of Mucuna are shown in Figs 1 (a) and 1 (b) respectively. The esterase band pattern was similar in all the varieties tested, but showed marked changes in the number and intensity of esterolytic bands. The enzyme was numbered from the anodic end. A total of four esterolytic bands were observed in the varieties tested. The protein banding pattern revealed the presence of total 11 bands on anionic polyacrylamide gel electrophoresis.

 

In the present investigation, electrophoretic protein profiles of different accessions of the same subspecies showed identical or similar patterns, confirming the stability of seed storage proteins within these subspecies. However, considerable variation of protein patterns was observed among the seeds of different varieties of Mucuna pruriens, Mucuna cochinensis and Mucuna hirsute. This could be correlated to different geographical origins. Esterase isozyme pattern revealed a sharp distinction among, Mucuna pruriens, Mucuna cochinensis and Mucuna hirsute according to the number and loci of allelic bands.

Table 1: Protein and esterase profile of seeds of different varieties of Mucuna

 

 

 

The esterases are inhibited by dichlorvos (Fig.2). Many studies have revealed that esterases are a heterogeneous group of enzyme with overlapping substrate specificities, as well as inhibitor sensitivities and molecular properties¹⁵. Generally, the carboxylesterases which exhibit complete inhibition with organophosphates and active towards short chain carboxylic esters are classified as carboxylesterases¹⁶. Based on the above criteria, in our studies, Mucuna seed esterases were found to be carboxylesterases.

 

Fig. 1a: Electrophoretic pattern of esterases isolated from 1) Mucuna hirsute 2) Mucuna cochinensis 3) Mucuna utilis IC25333 4) Mucuna sps. IIHR MP5 5) Mucuna pruriens MP7 6) Mucuna cochinensis MP9 7) Mucuna sps. NRC 8) Mucuna pruriens

 

Fig. 1b: Electrophoretic pattern of total proteins isolated from 1) Mucuna hirsute 2) Mucuna cochinensis 3) Mucuna utilis IC25333 4) Mucuna sps. IIHR MP5 5) Mucuna pruriens MP7 6) Mucuna cochinensis MP9 7) Mucuna sps. NRC 8) Mucuna pruriens

 

Fig.2: Inhibition of esterases of Mucuna by organophosphate, dichlorvos

 

CONCLUSION:

The esterases from the seeds of different varieties of Mucuna are isolated. The seeds of all the varieties of Mucuna exhibited esterase activity.  Effect of organophosphate such as dichlorvos on the activities of esterases was analyzed. The esterases are completely inhibited by dichlorvos and  the Mucuna seed esterases are found to be carboxylesterases.

 

ACKNOWLEDGEMENT:

The author is thankful to Principal and Management of PESIT for research support and IIHR, Bangalore for providing seeds of different varieties of Mucuna.

 

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Received on 19.01.2013         Modified on 05.02.2013

Accepted on 08.02.2013         © AJRC All right reserved