PURIFICATION AND CHARACTERIZATION OF A TRYPSIN INHIBITOR FROM MURRAYA KOENIGII

Abstract

A protein with trypsin inhibitory activity was purified to homogeneity from the seeds of Murraya koenigii (Curry leaf tree) by ion exchange chromatography on DEAE sepharose column and gel filtration chromatography on HPLC. The molecular mass of the protein was determined to be 27 kDa by SDS-PAGE analysis under reducing conditions. However, MALDITOF analysis revealed the exact molecular mass of protein to be 21.4 kDa. This protein showed high affinity towards Cibacron blue 3GA and therefore, was purified in single step by affinity column using Cibacron blue 3GA with substantial increase in yield. The inhibitory activity of purified protein at different concentration against a fixed trypsin concentration was determined by measuring the hydrolytic activity towards synthetic substrate N-benzoyl-L-arginine ethyl ester (BAEE) and N-benzoyl-L-arginine p-nitronilide (BAPNA) and by natural substrate BSA. The purifiedprotein inhibitedbovine pancreatic trypsin completely at a molar ratio of 1:1.1. The trypsin inhibitory activity was also determined at different pH buffers by using BAEE as substrate and the maximum inhibition was observed at pH 8.0. The Ki value and mode of inhibition of the inhibitor was determined using BAPNA as a substrate. The Ki value obtained from a Dixon plot was found to be 7 x 10"9 Mandthe mode of inhibition was determined to be a competitive inhibitor. The inhibitory assay of purified protein against chymotrypsin was also performed by measuring the hydrolytic activity towards synthetic substrate N-benzoyl-Ltyrosine ethyl ester (BTEE) and natural substrate BSA. Although, the protein showed proteolytically resistant against chymotrypsin but it did not show any chymotrypsin inhibitory activity. Murraya koenigii trypsin inhibitor (MKTI) was found to be the most abundant protein in the mature seeds of Murraya koenigii plant. The quantity of protein was determined to be approximately 20o/o of total protein extracted by simple buffer extraction. The relative concentrations of the inhibitor, monitored during seed development and germination periods were obtained from variable band intensities on SDS-PAGE gels under reducing condition using Quantity One ID gel analysis software (version 4.5.2; Biorad). The electrophoretic patterns of different stages of seed development and germination showed most intense protein band at 27 kDa which expressed rapidly during mid maturation stage of seed development and was utilized during seed germination. Trypsin inhibitory activity, in soluble protein extract of seeds, was monitored during different stages of seed development and germination. During seed developmental stages, the residual trypsin activity decreased approximately from 94% to 6.8% against soluble extract of seeds collected at 7and 55 days respectively and during the different stages of germination process the residual trypsin activity increased approximately from 6.9 to 94.5% against the soluble extract of seeds obtained after seeding at 1and 22 days respectively. Considering the highest protein expression at mid-maturation stage and degradation during germination correlating with similar trypsin inhibitory activity patterns demonstrate the storage property ofMKTI. Amino acid sequence analysis was performed by Edman degradation and MALDI-TOFTOF studies. The N-terminal amino acid sequence analysis was performed by Edman degradation and the sequence of first 15 amino acids (He-Asp-Pro-Leu-Leu-Asp-Ile-Asn-Gly- Asn-Val-Val-Glu-Ala-Ala), in short sequence search, showed significant homology to aKunitztype chymotrypsin inhibitor from Erythrina variegata. The partial internal amino acid sequencing of MKTI was also performed by MALDI-TOF-TOF studies after tryptic digestion and alkyl reduction. In partial internal sequencing by MALDI-TOF-TOF, six peptides of varying length totaling 98 amino acid residues were obtained. These peptides exhibited li similarities to the sequences from proteinase inhibitors, storage proteins and homeodomain like proteins. The maximum sequence homology of MKTI was found to be with miraculin like proteinfrom Citrusjambhiri, a memberof Kunitz family. Solubility studies of Murraya koenigii trypsin inhibitor (MKTI) were carried out under different physicochemical conditions including temperature, pH, salts, detergents and organic solvents. For solubility studies, the protein (1 mg/ml) was incubated at different physicochemical conditions for different time periods and then centrifuged and supernatant and precipitate were collected separately for determination of protein concentration and inhibitory activity. The thermal stability studies showed that protein gradually precipitates irreversibly after heating above 50°C and around 50% of protein is precipitated on heating at 100°C for 30 minutes. Addition of salt further lowered the solubility of protein at higher temperatures. The solubility studies at different pH conditions showed that it is completely soluble at and above pH 7.5 and slowly precipitates below this pH at the protein concentration of 1 mg/ml. The solubility of protein decreases below pH 7.5 to around 15% at pH 4.0. The presence of increasing amounts of salts (both divalent and monovalent) helped in resolubilization of protein at lower pH. Only, (NH4)2S04 was showing the salting out effect above 2 M concentration in pH 8.0. The inhibitory activity of precipitated protein at lower pH and high molar (NH4)2S04 was completelyregained after resolubilization of protein in buffer of pH 8.0. Except ethylene glycol, all other organic solvents like ethanol, methanol, acetonitrile, dioxane and MPD precipitated MKTI at different percentages. Interestingly, the inhibitory activity of precipitated protein was almost completely regained after resolubilization in buffer ofpH 8.0. The thermal stability of this protein under in-vitro and in-vivo condition was investigated by incubating the purified protein and mature intact seeds respectively in different temperatures in ranging fi™ 30 to ,00°C. The inhibitory activity of purified MKTI decreased continuously with increasing temperature by 15% at 40T to 95% a, 90°C and sharp deciine in inhibitory activity was observed above 50°C .However, the inhibitory activity of MKTI was no, affected under in-vivo conditions at high temperature, even after heating to 100°C for 30 minutes. The structural stability of MKTI was examined by proteolysis studies with five proteases, trypsin, papain, proteinase K, chymotrypsin and pepsin. Native MKTI was found to be completely resist, ,o proteolysis for all times tested from 30 minutes to 24 hours a. protease/protein molar ratio of 1:50. Bovine Serum Albumin, used as positive control, was quite susceptible to proteolysis against al, five proteases a, 1:100 molar ratios. The thermal stability of MKTI towards proteolytic degradation was also explored by incubating the MKTI a, range of temperatures from 30 to 90°C for 30 minutes prior to digestion with proteases. The results demonstrated that the beat treated MKTI a, al, temperatures was comp.etely resistant to proteolytic degradation by trypsin, chymotrypsin, pepsin and papain but was found susceptible to proteolysis by proteinase Kwhen heat treated at 90°C. Circular dichroism studies, under native conditions, showed MKTI contains approximately 30.1% a-helix, 46% P-strand, 16.2% turn and 6.9% random-coil structure. There was remarkable change in the conformation of MKTI at higher temperatures. Agradual loss in a-helical content of MKTI was observed with increasing temperatures while P-strand structure almost remained unchanged. The loss of inhibitory activity and a-helical content ofthe inhibitor with rising temperatures may be correlated suggesting apossible role for a-helical structure in inhibitory function of the protein. These results suggest acompact structure for this inhibitor with stable 0-sheet structure forming the core while a-helical structure is present on surface. IV ANS fluorescence experiments were performed in different physicochemical conditions to monitor the extent of changes in native structure of MKTI and relate them to the inhibitory activity. The ANS fluorescence study showed a linear increase in fluorescence intensity with increase in temperature above 50°C was observed without showing any melting transition up to 90°C. In different pH conditions ranging from pH 2-12, the fluorescence intensity of ANS at 525 nm was found to be highest at pH 2.0 with sharp decrease from pH 3.0 - 5.0 and then remains almost constant till pH 12.0. In presence of different concentration ofNaCl at pH 2.0, the fluorescence intensity decreased till 1.5 Mand then remained stable up to 2.5 MNaCl concentration. However, further increase in salt concentration above 2.5 M resulted in an increase in fluorescence intensity. The addition of salts, therefore, solubilizes and stabilizes the MKTI till a particular concentration only. A minor increase in fluorescence intensity was observed with increasing concentration ofurea (0 to 9 M) and guanidine hydrochloride (0 to 8 M), suggesting that overall MKTI structure remains intact with only avery slight relaxation. Murraya koenigii has been crystallized by sitting-drop vapor diffusion method using PEG 8000 as precipitating agent. The crystals belong to the tetragonal space group P432i2, with unit-cell parameters a = b = 75.8, c = 150.9 A. The crystals contain two molecules in asymmetric unit with aVM value of 2.5 A3 Da'1. Diffraction was observed to 2.65 Aresolution and a complete data set was collected to 2.9 Aresolution. The structure was solved by molecular replacement method using the structure of Erythrina caffra Kunitz type trypsin inhibitor as a search model. The model fitted well in electron density and was refined to Rfactor of 41.7% with overall correlation coefficient of 49.2%.