The cotyledons of day 2 germinating horse gram seeds were used for isolation of endoprotease, since the activity was maximal at this point during the four-day period of germination 7. Maximum yield of protease activity observed with 0.05 M Tris-HCl buffer pH 7.2, containing 2 mM of β-ME among other buffers (acetate, borate, phosphate buffers) used. Change in pH, alterations in molarity or omission of β-ME in the extraction buffer substantially lowered the yield of the activity (data not shown). The protease activity was measured by using the chromogenic substrate, azocasein. The results of the four step purification of CPRHG were summarized in Table 1. The CPRHG was initially precipitated with (30% - 60%) ammonium sulphate and dialyzed and subjected to DEAE cellulose column. The bound enzyme was eluted with a linear gradient of KCl (0 - 0.5 M). The fractions 14151617181920 contained 40% of enzyme activity with specific activity of 4.80 were pooled and concentrated by ammonium sulphate (Fig. 1). The final efficient step of purification is the fractionation on CM-Sephacel. Under provided conditions, 12% of the loaded enzyme was not adsorbed to the column and eluted, that was found to be homogeneous on 10% SDS-PAGE (Fig. 2).

The purified CPRHG was found to be homogeneous by the detection of a single polypeptide by SDS-PAGE which is further supported by single precipitin band on immunodiffusion of the crude extract (result not shown). Immunoblot analysis of the purified CPRHG also conform the monoreactivity of the antiserum (Fig 3 lane 5). The size of the purified CPRHG was estimated to be 30.2 K Da on Sephadex G-100 column and the same was also supported by SDS-PAGE (Fig. 2). However, on isoelectrofocussing (IEF) two isoforms were observed with different pI values of 5.85 and 6.1 (Fig 4).

The amino acid analysis of CPRHG was shown relatively fewer numbers of Histidines, sulfur containing amino acids (Cys and Met) and higher number of aspartic acid (Asp), glutamic acid (Glu) and serine (Table 2).

Effect of pH on CPRHG was carried out using azocasein as substrate and found the enzyme was active at acidic pH optimum, 5.5 with azocasein (Fig 5), 4.0 with hemoglobin and 5.2 with gelatin and BSA (data not shown). The activity was stable in the pH range 5.0- 6.0 (Fig 6) and also observed gradual loss of activity outside this pH range. However, the temperature vs activity profile showed an increased activity with temperature and exhibited maximal activity at 40°C (Fig 7). The enzyme was fairly stable at 40°C (Fig 8) and also exhibits linear proteolytic activity up to 4 h at this temperature (Fig 9).

Table 3 showed the comparative hydrolytic activity of the purified CPRHG with endogenous (seed proteins) and exogenous proteins (BSA, casein, gelatin and hemoglobin). Seed protein degradation was remarkably higher than the gelatin, casein, BSA and hemoglobin. The enzyme was unable to cleave Leu-p-nitroanilide and CBZ-L-phenylalanine. Digestion of endogenous proteins by CPR HG on time course study through SDS-PAGE shows no appreciable degradation within first 90 min. However, the disappearance of polypeptides of 96, 81, 66, 60, 58 and 21 k Da and appearance of new polypeptides of 73 and 59 k Da and intensification of 56 and 29 kDa bands were observed on SDS-PAGE with increasing digestion time (Fig 10).

The protease activity was clearly inhibited at lower concentrations of Zn²⁺, Hg²⁺ and Cu²⁺ (0.5 mM). However, metal ions like Ni²⁺, Co²⁺and Pb²⁺ were shown an inhibition on above 1 mM. Mn²⁺ at higher concentrations (above 1 mM) stimulated the activity. Metal chelating agents such as 1, 10 phenanthroline and EDTA had no effect (Table 4 &5). The CPRHG activity was neither inhibited by aspartyl modifying reagent (pepstatin) nor by serine modifying reagents (DIPF, soybean trypsin inhibitor, eserine, PMSF and aprotinin). However, the activity was completely inhibited by sulfhydryl modifying reagents (pCMB and iodoacetamide). We also observed the complete inhibition by histidyl modifying reagent (DEPC). These results suggested that the CPRHG is a cysteine protease, with cysteine and histidine residues at the active site (Table 5). Addition of sulfhydryl reagents (cysteine, dithiothreitol, glutathione and β-ME) reverses the strong inhibition by pCMB (Table 6 &7).

The polyclonal anti-CPRHG could recognize 81, 41, 30 and 27 k Da polypeptides from crude extract of germinating cotyledons. Presence of HMW 81 k Da protein in the first 2 days of germination and disappeared by day 3. The 41 k Da polypeptide was noticed in the extracts from days 0-3. The intensity of 30 k Da endoprotease (CPR HG) in the cotyledons of germinating seeds increased up to day 2 and declined later. Similar profile was also noticed for 27 k Da polypeptides (Fig 3). These results revealed that the enzyme CPRHG synthesized as zymogen (preproenzyme with 81 K Da) and processed to a 40 K Da proenzyme and then to the 30 K Da active enzyme which is further degraded to a 27 K Da product.

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.

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.

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.

Endoprotease (EP-HG) activity was measured by using chromogenic substrate, azocasein, following the method described by Sarath et al 41 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.

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 42.

IEF and 2D electrophoresis of purified enzyme was carried out according to the method of O' Farrel 43.

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 44.

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.

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.

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).

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 44.

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.

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.

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 45. Presence of exopeptidase activity was checked by using L-Leu-p-nitroanilide and CBZ-L-phenylalanine as described by Chrispeels and Boulter 46 and Sarath et al. 41, respectively.

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.