Sephadex G-100, CM-Sephacel, LMW markers were procured from Pharmacia Fine Chemicals, Uppsala, Sweden and DEAE-cellulose, aprotinin, p-chloromercuribenzoate (PCMB), N-ethylmaleimide (NEM), phenylmethylsulfonylfluoride (PMSF), iodoacetamide, Soybean trypsin inhibitor, eserine, diisopropylflourophosphate (DIFP), bestatin and 1,10- phenanthroline, diethylpyrocarbonate (DEPC), CBZ-L-phenyl alanine and L-Leu-p-nitroanilide were purchased from Sigma Chemical company, USA. All other chemicals, unless otherwise specified, were of analytical grade and purchased from Specrochem (India) and Qualigens (India).
Horse gram (Macrotyloma uniflorum (Lam.) Verdc.) seeds were procured from Agricultural Farm of Andhra Pradesh Agricultural University, Rekulakunta, Anantapur, Andhra Pradesh, India.
Horse gram seeds were surface sterilized with 0.1% HgCl2 solution for 5 min, washed repeatedly with sterile water and soaked in 10 volumes of water for four hours. The imbibed seeds were set to germinate at room temperature (30 ± 2°C) at 12 h dark and 12 h light cycle for four days in sterile petri dishes lined with four layers of filter paper. Sterile conditions were maintained by including 20 ppm of streptomycin sulphate in the incubation medium (water). The cotyledons harvested for two days were used for the isolation of CPR HG.
Preparation of the enzyme extract
Cotyledons were ground thoroughly in a mortar adding four volumes of chilled 0.05 M Tris-HCl buffer, pH 7.2, containing 2 mM β-ME. The extract was filtered through four layers of cheesecloth and the filtrate was centrifuged at 10000 g for 15 min at 4°C. The supernatant was used for isolation of the enzyme. All separation procedures were carried out at 4°C, unless otherwise stated.
Purification of CPRHG from germinating horse gram cotyledons
The crude enzyme (endoproteinase) extract was prepared as described above from 100 g of cotyledons (day 2) was subjected to ammonium sulfate precipitation. The precipitate obtained between 30% - 60% ammonium sulfate saturation was collected and dissolved in minimal amount of extraction buffer and extensively dialyzed against 0.01 M Tris- HCl buffer, pH 7.2 containing 2 mM β-ME at 4°C (1:200 volume ratio with 2 h change upto 12 h). The dialysate was loaded on DEAE-cellulose column equilibrated with 0.01 M Tris- HCl buffer, pH 7.0 containing 2 mM β-ME. The bound proteins were eluted with a linear gradient of increasing ionic strength of potassium chloride (0.5 M) in equilibration buffer and 5 ml fractions were collected. The fractions (14-20) with higher enzymatic activity were pooled and brought to 60% saturation with pulverized ammonium sulfate at 4°C and centrifuged. The pellet was dissolved in minimal volume of 0.02 M sodium acetate buffer, pH 5.5 containing 2 mM β-ME and dialyzed against the same buffer at 4 °C for 12 hr. The dialysate was loaded on CM-Sephacel column equilibrated with 0.02 M sodium acetate buffer, pH 5.5 containing 2 mM β-ME. The unbound fractions were collected and concentrated by lyophilization and stored at -20°C. Criteria of purity at each stage was checked by 10% SDS-PAGE.
Assay of endoprotease
Endoprotease (EP-HG) activity was measured by using chromogenic substrate, azocasein, following the method described by Sarath et al  with slight modifications. 0.25 ml of 1% azocasein (prepared in 0.02 M sodium acetate buffer, pH 5.5 containing 2 mM β-ME) was mixed with 0.15 ml of enzyme extract or 150 μg of purified enzyme (1 mg/ml) and incubated at 40°C for 1 hr. The reaction was arrested by adding 1.2 ml of 10% TCA and mixed thoroughly. The contents were allowed to stand for 15 min and centrifuged for 5 min at 3000 rpm. 1.2 ml of the supernatant was transferred to a tube containing 1.4 ml of 1 M NaOH, mixed and the absorbance was read at 440 nm against the reagent blank. One unit of protease activity was defined as the amount of the enzyme required to produce an absorbance change of 1.0 in 1 cm cuvette under the conditions of the assay.
Determination of amino acid composition
The protein was hydrolyzed with 6N HCl for 24 h at 110°C in an evacuated sealed tube. The hydrolysate was filtered through Whatman No.1 filter paper and the filtrate was evaporated to dryness in a flash evaporator and the amino acid composition was analyzed on model 119 CL Beckman amino acid analyzer. Cysteine residues were measured by performic acid method .
IEF and 2D electrophoresis of purified enzyme was carried out according to the method of O' Farrel .
Determination of MW of CPRHG
The molecular weight of the purified protease was determined by Sephadex G-100 gel filtration (2.4 × 132 cm column, flow rate 2 ml/min) and also through SDS-PAGE .
Effect of pH and temperature on enzyme
The effect of pH on enzyme activity was determined by carrying experiment at different pH using sodium acetate buffer (pH 3.5 - 5.5), sodium phosphate buffer (pH 5.6- 7.0) and Tris-HCl buffer (pH 7.2 - 9.2). The enzyme activity was assayed as described. The effect of temperature on the enzyme activity was also determined at different temperatures ranging from 10-80°C for 1 hr in 0.02 M sodium acetate buffer pH 5.5, containing 2 mM β-ME using the same assay as described.
Storage stability of enzyme
The storage stability of the of the purified enzyme (CPRHG) was investigated by storing the enzyme at different temperatures, -10°C, 4°C and 30°C and its stability for a week was tested by withdrawing an aliquots of the enzyme at different intervals of time and was assayed for the enzyme activity.
Time course study
The purified CPRHG and azocasein were taken as described earlier and incubated at 40°C for 4 hr. Aliquots were withdrawn from incubation mixture at different intervals of time from 0 - 4 hrs and the azo compound liberated was measured as described.
Polyclonal antibodies against the CPRHG were raised in rabbits by injecting 100 μg of the purified protein. Antibodies were used for immunoinhibition, immunodiffusion and immunoblot to check their specificity (44).
Western blot analysis for developmental pattern of CPRHG in the cotyledons of germinating horse gram seedlings
Extracts of the cotyledons from germinating seeds harvested at daily intervals for 4 days were subjected to SDS-PAGE and the proteins were transferred on to a nitrocellulose paper. The proteins on nitrocellulose paper were treated with primary antibody (anti-CPRHG) and successively with secondary antibody (Ig-alkaline phosphotase). The bands were visualized by using NBT and BCIP in Tris HCl buffer, pH 9.0, containing 10 mM MgCl2 and 100 mM NaCl .
Effect of metal ions, selected inhibitors and sulfhydryl reagents
150 μg of CPRHG in 0.02 M sodium acetate buffer pH 5.5 was preincubated with 10 μl of different metal ions, selected inhibitors and sulfhydryl reagents at varied concentrations for 1 hr at room temperature and the assay was initiated by the addition of azocasein and the activity assayed as described.
Effect of -SH reagents on pCMB treated EP- HG
pCMB (10 mM) treated protease was reincubated prior to the assay with sulfhydryl reagent (5 mM and 10 mM) for 1 hr at room temperature and the activity was assayed as described. The proteolytic activity was compared with the control experiment containing all the components except the SH-reagent.
Hydrolysis of endogenous and exogenous substrates
The relative digestibility of endogenous and exogenous proteins by the purified CPRHG was measured by incubating 1 ml of 1% protein (dry seed protein, BSA, casein, hemoglobin, gelatin) prepared in an appropriate buffer mixed with 0.15 ml of the purified CPRHG (1 mg/ml) and incubated at 40°C for 1 hr and the amino acids released were estimated by ninhydrin method . Presence of exopeptidase activity was checked by using L-Leu-p-nitroanilide and CBZ-L-phenylalanine as described by Chrispeels and Boulter  and Sarath et al. , respectively.
In vitro digestion of seed proteins (SDS-PAGE analysis)
Suitable volumes of dry seed protein extract (1%) and CPRHG (1 mg/ml) were incubated as described, at various intervals aliquots were withdrawn from the digestion mixture, mixed with 2× sample buffer, boiled for 3 min and subjected to SDS-PAGE.