Protease Inhibitors from the seeds of Senna sophera: Isolation and Properties

 

K S Chandrashekharaiah^1*, K B Sangeetha¹, Rajiv Bharadwaj¹, N G Raju²

N Ramachandra Swamy³

¹Department of Biochemistry, Mangalore University, Chikka Aluvara, Kodagu-571232

²Department of Biotechnology, Karnataka State Open University, Mysore-570006

³Department of Biochemistry, Bangalore University, Bangalore-560001

 

ABSTRACT:

Need for protease inhibitors in medicinal and therapeutic applications have been well acknowledged by the research community. The natural flora has always been an extensive contributor for various compounds used in disorder treatment owing to its abundance and ease of isolation and purifications methodologies. Conventional protein purification methods such as salt precipitation, gel filtration chromatography on Sephadex G-10 and G-50 and RP-HPLC have been employed for the purification of protease inhibitors from the seeds of Senna sophera. Two protease inhibitors SSPI-1 and SSPI-2 were purified. The protease inhibitor SSPI-1 was purified to 16.11 fold with a recovery of 26.20% and showed a specific inhibitor activity of 31.12. SSPI-2 was purified to 61.08 fold with a recovery of 66.23 and showed a specific inhibitor activity of 24.51. Both the purified inhibitors were found to be stable in the pH range 2 – 10 and temperature between 4 – 65^oC.

 

 

 

INTRODUCTION:

Protease inhibitors are storage proteins of plants present in more amounts in the storage organs of plants and seeds. In plants, these proteinacious inhibitors involved in several functions such as regulation of proteases which hydrolyze proteins thereby preventing the unwanted proteolytic cleavage in the cell, act as defense proteins against insect pests and pathogens by binding to the active sites of proteases required for their growth and development thereby preventing crop loss or yield in agriculture¹.

 

Leguminous plants are the potential sources of these inhibitors and most of the inhibitors isolated and studied were serine protease inhibitors which play a significant role in many biological processes such as cell signaling, cell cycle progression, digestion, immune responses, blood coagulation, wound healing, cancer², apoptosis, hormone processing pathways, inflammation, hemorrhage, intracellular protein breakdown, transcription, cell invasion and also in the treatment of AIDS.^3-10 

 

The plant Senna sophera belong to leguminoseae is a shrub and popular due to its numerous medicinal values. All parts of the plant are used for various medicinal properties. The whole plant is purgative and febrifuge. It is used in homeopathic medicine. The roots are diuretic. It is also used in the treatment of osteoarthritis, asthma and allergic conditions. The pharmacological activities of the seeds of Senna sophera includes analgesic, anticonvulsant, antidiabetic, herbicidal and fungicidal effect. The seeds are used to reduce fevers. The boiled seeds are used for the treatment of Bright’s disease. An infusion of the bark is used in the treatment of diabetes. Extracts of all plant parts are used to treat epilepsy. The dried leaves have been shown to have insect repellent and insecticidal properties. Root bark and seeds are used for preparation of medicine. It has been used by ancient Indian physicians for its efficiency in respiratory disorder. In the present study, isolation and properties of trypsin inhibitory peptides were described.

 

MATERIAL AND METHODS:

Materials:

Senna alata seeds were collected from surroundings of Puthige panchayath of Kasaragod district of Kerala State. Bovine Serum Albumin, trypsin, BAPNA, acrylamide, bisacrylamide, Sephadex G-10 and Sephadex G-50 were purchased from Sigma chemical laboratory, USA. All the other chemicals and reagents used were of technical grade.

 

Methods:

Isolation and purification of protease inhibitors: 

Crude protein extract containing inhibitor proteins was prepared acetone powder prepared from soaked seeds of Senna sophera using 0.05M sodium phosphate buffer pH 7.0 by stirring over a magnetic stirrer for 1.5 hr at 4^o C as described by Chandrashekharaiah (2011)¹¹. It was subjected to centrifugation at 10000 rpm for 15 minutes. The supernatant obtained was collected and used as crude protein extract. The crude protein extract was subjected to 0 – 85% ammonium sulphate fractionation. The appropriate amounts of ammonium sulphate was weighed, powdered and added slowly to crude protein extract while stirring over magnetic stirrer at 4^oC until it dissolved completely. The solution was allowed to stand at 4^oC for 3 hours for precipitation followed by centrifugation at 10000 rpm for 30 minutes. The pelleted protein was dissolved in small volume of extraction buffer and subjected to separation on sephadex G-10 chromatography which was performed as described by Rajiv Bharadwaj and Chandrashekharaiah (2017)¹². The factions with trypsin inhibitor activity were pooled, concentrated and separated on Sephadex G-50 gel chromatography which was performed as described by Rajiv Bharadwaj and Chandrashekharaiah (2017)¹² using 0.05 M sodium phosphate buffer, pH. 7.0 as elution buffer. The flow rate was calibrated to 10 ml/hour and 2.0 ml fractions were collected. The trypsin inhibitor fractions were further fractionated on RP-HPLC in Reversed-phase octadecylsilica (C18) column in binary solvent system with binary gradient capability and a UV detector. Buffer A is 0.1% (v/v) TFA in water and Buffer B is 100% acetonitrile containing 0.1% (v/v) TFA.

Determination of total protein, Trypsin and trypsin inhibitor activity:

Total Protein was determined from the crude protein extract, fraction of sephadex G-10 and G-50 chromatography according to the method of Lowry et al., (1951)¹³. Trypsin activity was measured with substrate Nα-Benzoyl-D,L-arginine 4-nitroanilide hydrochloride (BAPNA) according to the modified photometric method of Kakade et al., (1969)¹⁴.  The substrate concentration of 0.919 mM BAPNA was prepared in dimethylsulfoxide (DMSO) followed by 1:100 dilution in 50 mM Tris-HCl buffer, pH 8.2 containing 20 mM CaCl₂ (40mg BAPNA in  2 mL of  dimethylsulfoxide (DMSO) and diluted to 100 ml with 50 mM Tris-HCl buffer, pH 8.2 containing 20 mM CaCl₂) prior to enzyme assay.  The trypsin assay was carried out by incubating enzyme with BAPNA for 10 min at 37 °C followed by the addition of 30 % acetic acid to arrest the reaction. The absorbance of p-nitroanilide liberated was measured at 410 nm against an appropriate blank.  One trypsin (TU) unit is arbitrarily defined as an increase in absorbance of 0.01 at 410 nm under conditions of assay.

 

The trypsin inhibitor assay was performed by incubating trypsin with an aliquot of inhibitor for 10 min at 37 °C. The reaction was started by the addition of BAPNA followed by incubation for 10 min and the reaction was arrested by the addition of 30 % acetic acid.  The residual trypsin activity was measured at 410 nm against an appropriate blank.  The trypsin inhibitory unit (TIU) is defined as the number of trypsin units inhibited under the assay conditions.

 

Polyacrylamide gel electrophoresis:

An anionic disc gel electrophoresis was carried out essentially according to the method of Davis and Ornstein (1964)¹⁵. A discontinuous gel system consisting of 8% separating gel and4% spacer gel was used. The electrophoresis was carried out in cold applying a current of 20 – 25 mA for 4 hours using tris – glycine (pH 8.3) as electrode buffer and bromophenol blue as marker dye. After the electrophoresis, the proteins were stained with CBB R – 250 for 1 hour and distained using 7 % acetic acid.

 

Effect of pH and temperature:

The effect of pH on the activity of the partially purified Senna sophera trypsin inhibitor was studied as described by Chandrashekharaiah (2013)³ using different buffers such as Glycine – HCl buffer (0.2M, pH 2), Sodium acetate buffer (0.2 M, pH 4), Sodium citrate buffer (0.2 M, pH 5.5), Sodium phosphate buffer (0.2 M, pH 6.5), Tris – hydrochloride buffer (0.2 M, pH 8.0), Sodium borate   buffer (0.2 M, pH 10). Similarly, the pH stability was determined by preincubating the partially purified Senna sophera trypsin inhibitor with above buffers for 30 min. Trypsin inhibitor assay was performed as described earlier. The effect of temperature on the activity partially purified Senna sophera trypsin inhibitor was studied at different temperatures ranging between 0 - 90 ºC as described by Chandrashekharaiah (2013)³. The temperature stability of partially purified Senna sophera trypsin inhibitor was studied by pre-incubating, the trypsin inhibitor at different temperatures (0 - 90 ºC) for 30 min. The incubated samples were rapidly cooled and assayed at room temperature. Trypsin inhibitor assay was performed as described earlier.

 

RESULTS AND DISCUSSION:

Purification and properties of protease inhibitors:

The purification of protease inhibitors from the seeds of Senna sophera was performed at 4⁰C unless otherwise stated. the crude protein extract of Senna sophera was subjected to 0 – 85% ammonium sulphate fractionation. The precipitated protein was dissolved in small volume of elution buffer (0.025M sodium phosphate buffer, pH 7.0) and fractionated on sephadex G-10 chromatography. Two protein peak fractions were obtained, fraction-I, II and III (Fig.1). Among three protein peak fractions, fraction-I containing protease inhibitor activity were pooled, concentrated and separated on sephadex G-50 chromatography. Two protein peak fractions were obtained on sephadex G-50 chromatography,fraction –I and II (Fig.2). Fraction –I containing protease inhibitor activity were further purified by RP-HPLC (Fig.3). Two protease inhibitors SSPI-1 and SSPI-2 (Fig.4). The protease inhibitor SSPI-1 was purified to 16.11 fold with a recovery of 26.20% and showed a specific inhibitor activity of 31.12. SSPI-2 was purified to 61.08 fold with a recovery of 66.23 and showed a specific inhibitor activity of 24.51.

 

Several protease inhibitors have been isolated and purified from various sources  including wheat, soybean, maize, chick pea, potato,  tomato,  Moringa oleifera,  Dimorphandra mollis etc., employing conventional protein purification techniques.^16-19  Konala Geetha et al (2013)²⁰ purified protease inhibitor from the seeds of prickly chaff flower (Achyranthus aspera) to a near homogeneity with 15% recovery using ammonium sulphate fractionation, chromatography on DEAE-Cellulose and Sephadex G-100. Effect of pH on both the purified SSPI-1 and SSPI-2 showed that both stable between pH 2 – 10. Similarly, both the protease inhibitors were retained more than 50% inhibitory activity in the temperature range 4 – 65^oC indicated that both are temperature stable.

 

CONCLUSION:

SSPI-1 and SSPI-2 protease inhibitors were isolated and purified from the seeds of Senna sophera employing conventional protein purification techniques. Both the inhibitors were found to be stable between pH range of 5 - 8 and temperature stable between 4 – 65^oC.

 

ACKNOWLEDGEMENT: 

The authors are grateful to Mangalore University for providing research facilities.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 24.09.2017          Modified on 19.11.2017

Accepted on 20.02.2018       ©AandV Publications All right reserved