C Partellus Gut Protease Lab Report

C. partellus gut protease and protease inhibitor activity assays. C. partellus gut protease activity was estimated using chromogenic substrate BApNA [Manasi A et al., 2005]. It was dissolved in the DMSO and a final concentration was made to 1mM in 1.0ml, Glycine-NaOH buffer (pH 9), assays were carried at 38°C for 30min [Manasi A et al., 2005]. The reaction was terminated by adding of acetic acid (30%) of 200µl [8, 9, Manasi A et al., 2005 ]. Supernatant (0.5ml) was added to 1M NaOH (0.5ml) and the absorbance of this solution was measured at 410nm [Manasi A et al., 2005]. One protease unit is defined as increases in 1 OD/min. An azocasein assay was carried out using 1% (w/v) solution of the substrate in buffer [Manasi A et al., 2005].
DEAE Sepharose fast flow anion exchange chromatography was used for partial purification of C. partellus gut proteases [Kumar, H.1997]. For the CPGPs, 50mM NaOH buffer, pH 9 was used to equilibrate the column. Gut homogenate equilibrated with the 50mM NaOH buffer, pH 9 buffer was loaded on the column and elution was carried out using a 0-0.6M NaCl gradient [Kumar, H.1997]. 2.0ml fractions were collected and analyzed for proteolytic activity using BApNA and SAAPFpNA. Fraction was dialyzed against distilled water and concentrated for further analysis. Protein content of gut and fraction was determined by Lowry’s method [7]. Ammonium sulphate precipitated (60-90% saturation) and I. batata extract (200 TI units, 40mg proteins) were loaded on 50ml capacity DEAE ion exchange column (1.6 × 12.4cm, bed volume 25ml) equilibrated with 50mM Tris-HCl (pH 8.0) buffer. The column was washed with 50mM Tris-HCl, pH 8.0 (200ml) and 2ml fractions (fraction no.1 to 30 was collected. Solution of 0.25M NaCl in 50mM Tris-HCl, pH 8 (30ml) (fraction no. 31 to 60) was then passed through the column followed by gradient of 0.25-0.4M in 50mM Tris-HCl, pH 8 (60ml) (fraction no. 61 to 91) and 25ml wash of 0.4M NaCl in 50mM Tris-HCl, pH 8.0 (fraction no.92 to 123) and 2ml fractions per tube were collected [Tamhane et al., 2005]. The fraction was analyzed at 280nm using PC-based spectrophotometer (JASCO 500 series) and plotted graph. Dot-blot assays of each fraction
Gel electrophoresis was carried out according to buffer system discussed by [11]. Complisation of electrophoresis gel was processed for activity of proteases detection by gel X-ray film contact print method (GXCP) [12]. After electrophoresis gel was incubated in 0.1M Glycine-NaOH buffer pH 9 for 7 to 8min then was placed on X-ray film [Padul, M.V. et al., 2012]. The gel was removed from X-ray film after 45min depending on the extent of hydrolysis of gelatin. The X-ray film was washed with warm water to observe CPGPs bands as hydrolyzed of gelatin against the background of gelatin [Padul, M.V. et al., 2012]. The X-ray film was then developed, after developing gut proteases bands against the dark opaque background [Padul, M.V. et al.,