Seed samples: Seeds of Phaseolus vulgaris have been collected from different geographical locations of Uttarakhand. Fifteen cultivars showing variation in seed coat colour, size and shape were selected. The seeds were authenticated and deposited in National Bureau of Plant and Genetic Research, New Delhi, India (Table 1).
Chemicals and Reagents: Sephadex G-50, PPA (Porcine pancreatic amylase) were purchased from Sigma (India). Protein markers were purchased from Genei (India) and others required chemicals from Himedia (India).
Crude extract Preparation: The extraction of seed proteins from seed flour was done according to the method described by literature3. 100 mg of finely grounded seed flour was taken, homogenised in extraction buffer and was incubated at 4ºC for 1hr. The homogenate was then centrifuged at 15000 rpm for 15 min at 4ºC. The supernatant was collected and stored in aliquots at -20°C for further analysis. The protein content was measured by method described by Bradford15
Amylase inhibitory activity: The amylase inhibitory activity was determined according to literature descriptions8 with some modifications. A soluble starch solution (0.4 ml,1 % w/v) was made in 80mM phosphate buffer (pH = 6.9) and a solution of PPA (0.2 ml, 0.001 % w/v) in 20mM acetate buffer (pH-4.5, containing 20mM CaCl2 and 10 mM NaCl) was added into it and then incubated for 15 min at 37ºC. The reaction was stopped by addition of 0.8ml of Dinitrosalicylic acid reagent (1gm DNS, 200 mg crystalline phenol and 50 mg of sodium sulphite dissolved in 1% NaOH). The contents were heated in a boiling water bath for 5 min, and after cooling it was diluted with 4ml of water. Absorbance of the mixture was read at 540 nm against blank prepared without using PPA. Amount of maltose produced was calculated from standard curve of maltose. The above method was also used to describe α-amylase inhibitor activity but PPA solution and purified inhibitor solutions (0.2ml) were pre-incubated for 15 min before addition of soluble starch solution. Alpha-amylase inhibitory activity was calculated according to equation shown below:
Inhibitory activity (%) = [Mo- Mi/ Mo] x 100
Where, Mo and Mi are amount of maltose (mg/ml) produced in absence and presence of inhibitor respectively, under the same conditions.
Heat stability: Heat stability was evaluated from literature descriptions13. Both the extracts (crude and purified) were incubated in a water bath at different temperatures ranges from 20ºC to 100ºC with the difference of 10ºC, after that amylase inhibitory activity was calculated
Purification of α-amylase inhibitor: Ammonium sulphate precipitation (80–100% saturation) of the crude protein extract was performed at 4°C. The precipitate was dissolved in 10mM Tris-HCl and was dialyzed against buffer in batches. The dialyzed material was stored at -20ºC till further analysis. The α-amylase inhibitor was fractionated by repeated size exclusion chromatography on a Sephadex G-50 column (26x1.2 cm).
Molecular identification: The polypeptides in the samples were fractionated using SDS-PAGE (15%) under reducing conditions and Native PAGE. The molecular weight of purified inhibitor was determined by using medium range protein marker (14.3–97.4 kd).
X-ray Crystallography: The purified protein sample was re-dissolved at a concentration of 10 mg/ml in double-deionised water. Crystallization was performed using VDX48 plates by the hanging-drop vapour-diffusion method.
Statistical analysis: Each sample was analysed in triplicates and the values were averaged. Data was assessed by analysis of variance (ANOVA), previous verification of normal distribution and variance homogeneity16 and mean comparison was done by using Duncan’s multiple range test using software R17