V.G.Meenu Krishnan,K.Murugan

Plant Molecular Biochemistry and Biology Lab,Department of Botany,University College,Thiruvananthapuram,695 034 Kerala,India

Abstract Double-headed protease inhibitor was isolated,purifie and characterized including its kinetics from the fruits of Solanum aculeatissimum Jacq.(SAPI).The inhibitor was purifie to homogeneity via four sequential step procedure,i.e.,salt precipitation to Sepharose affinit chromatography.The purity was confirme by reverse phase HPLC chromatography.The molecular mass was detected using size elution chromatography(22.2 kDa).SAPI inhibits trypsin and chymotrypsin simultaneously and the molar inhibitory ratio for trypsin and chymotrypsin was 1:1,i.e.,it functions as double headed PI of Bowman–Birk group.Purifie SAPI showed optimal specifi activities of 502 and 433.7 U/mg with trypsin and chymotrypsin,respectively.Overall, the fold of purificatio increased with remarkable yield.Native-PAGE showed four isoinhibitors (pI: 4.7, 5.2, 5.6 and 5.9).Dixon plots and Lineweaver–Burk double reciprocal plots revealed competitive mode of inhibition.High pH amplitude (2–12) and broad temperature range(10–80°C)were observed for SAPI.Circular dichroism spectrum of native SAPI displayed random structure with more β-sheets.Further,metal ions,detergents,oxidizing and reducing agents affected the inhibitory potential of SAPI in different levels.Chemical modificati n studies to analyze the key amino acid present in the reactive site of SAPI revealed the presence of lysine and tryptophan residue(s).

Keywords: Characterization;Chromatography;Solanum aculeatissimum;Inhibitors;Kinetic studies;Protease inhibitors

1.Introduction

Diverse types of protease inhibitors(PIs)have been isolated,characterized,and evaluated for their biological potentials.They are small molecular mass peptides capable of inhibiting endogenous proteases.Plant PIs fascinates the attention of researchers because of their increasing use in pharmaceutical and biotechnological industries.In the course of evolution,plants have evolved adaptive mechanisms that provide them significan resistance against diverse kinds of unfavorable conditions including insects and phytopathogens.The mode of action of PI is either via inactivating the hydrolase enzymes or depolarization of plasma membrane of the pathogens thereby inhibiting its growth and invasion.PIs are diverse viz., rice cysteine PIs, potato serine PIs, cowpea serine PIs, sweet potato PIs, corn cystatins, soybean kunitz PIs, tobacco PIs, bean α-amylase inhibitors and mustard trypsin inhibitors.Some are trailed for producing transgenic insect resistant plants.Ussuf et al.[1]produced transgenic tobacco plant with CPTI gene from cowpea.In addition, PIs have long been treated as antinutritional and therefore,reviewed their possible role as antimetastatic and antiinflammation Syed Rakashanda et al.[2]isolated and characterized serine protease inhibitor(LC-PI-I)fromLavatera cashmerianaseeds.It showed bactericidal potential against urinary tract infection,pneumonia and septicemia in humans.PIs were also employed as potential drugs in antiretroviral combination therapy which increases the expectancy in HIV patients[3].Satheesh and Murugan[4]also revealed the remarkable microbicidal potentiality of PIs from the leaves ofCoccinia grandisagainstKlebsiella pneumoniaeandAspergillus flavus.

Structurally, PIs belongs to diverse classes such as serine proteases, cysteine proteases, aspartate proteases and metalloproteases based on the active amino acid that was seated in the reaction centers.The most extensively analyzed protease inhibitors in plants were serine and cysteine protease inhibitors.Eight groups of plant serine protease inhibitors were reported based on their amino acid sequences [5].Soybean trypsin inhibitor (SKTI), protease inhibitor 1 (PIN1) and protease inhibitor 2 (PIN2) families were well studied among them.PIN2 family consists of four groups such as Kunitz, Bowman Birk, Potato I and Squash families.PIN2 enzymes show wound-inducible expression patterns in leaves and constitutive expression in fl wers [6].Meulenbroek et al.[7] purifie and characterized double-headed heterodimeric serine PI of Kunitztype fromSolanum tuberosum.

Most of the serine PIs have been isolated and characterized from Fabaceae,Cucurbitaceae,Solanaceae,Euphorbiaceae and Poaceae families.There are only limited works of purificatio and characterization of these inhibitors from Solanaceae.WildSolanumspecies represents repository of protease inhibitor(PIs)diversity and their co-evolutionary trends with the insect proteases ensure its dynamic role.Solanum aculeatissimumJacq.is commonly known as the African nightshade or Dutch egg plant and is used by local vendors for curing many diseases including viral.In this juncture, the present work targets PIs fromS.aculeatissimumin terms of isolation, purificatio and characterization including kinetics.

2.Material and methods

2.1.Purification of SAPI

S.aculeatissimumJacq.fruits were obtained from Munnar hills of Western Ghats,Kerala.100 g fresh fruits were homogenized with 250 mL of saline Tris buffer (20 mmol/L Tris, pH 8.0;0.15 mol/L NaCl)containing 1%polyvinylpyrrolidone(1:6,w/v) and filtere through chilled 4-fold muslin cloth and further, centrifuged for 15 min at 10,000×g.The entire protocol was carried at 4°C[8].The crude PI extract was fractionated by 20–90%(NH4)2SO4precipitation.

Low ammonium sulfate concentration precipitate proteins with less hydrophilic regions.Meanwhile proteins with more hydrophilic regions are concentrated and precipitated with higher (NH4)2SO4saturation.The beaker containing 100 mL of protein solution was placed in a cooling water bath on top of a magnetic stir plate.This was accomplished by placing the beaker within another beaker containing water–ice slurry.(NH4)2SO4precipitation was carried out at different saturation as 0–20, 20–40, 40–60, 60–80 and 80–90% by adding 5.35,11.45, 36.6, 52.3 and 61.7 g salt slowly and gentle agitation on magnetic stirrer, respectively.This step was completed in 5–10 min.The stirring was continued for 10–30 min after all salt has been added.Resultant solution was centrifuged at10,000×gfor 10 min at 4°C.The supernatant was decanted and the precipitate was resuspended in 1–2 pellet volumes of cold extraction buffer.The(NH4)2SO4was removed by the process of dialysis using the extraction buffer stirred gently with magnetic stirrer to improve solute exchange and the dialysis buffer was changed once in 3 h for 4–5 times.

The dialyzed protein showing high protease inhibition activity was subjected to DEAE cellulose exchanger column,pre-equilibrated with 20 mmol/L Tris buffer with pH 8.0.3 mL protein fractions were eluted using linear gradient of NaCl(0.02–0.50 mol/L)at a f ow rate of 0.5 mL/min.Fractions eluted with 0.18–0.24 mol/L NaCl were pooled,dialyzed,lyophilized and loaded(1.0 mg/mL)to Sephadex G-50 superfin from Pharmacia column.

Suspend the gel (Sephadex G-50) in large volume of water until it is fully swollen (heating in water bath for 2–4 h).Plug the bottom of column tube with glass wool and keep the column upright.Pour small volume of buffer into the column to avoid air bubbles in the plug immediately followed by the slurry in to the column.Keep until the gel settles down to the desired height by gravitational force.Place a suitable filte circle on top of the gel bed.Equilibrate the column thoroughly by passing through the column buffer(25 mmol/L Tris–HCl,pH 8.0)and adjust the fl w rate.Apply the ammonium sulfate dialysate in column buffer onto the top of the bed (the sample volume should preferably be limited to 1–3%of the total bed volume).Now connect the buffer line to the elution buffer(25 mmol/L Tris–HCl,pH 8.0)to develop the chromatogram.Open the column outlet tubing and allow the sample to penetrate into bed.Protein molecules pass through the gel space while small molecules distribute between the solvent inside and outside the gel and then pass through the column at a slower rate.Wash the remaining sample from the column wall by applying small amounts of buffer from a Pasteur pipette.Collect 5 mL fractions by continuously adding buffer.The effluen emerging out of the column can be routed through a suitable spectrophotometer to monitor the absorbance and the data recorded.The amount of protein is expressed as mg/mL of protein.The volume of mobile phase required to elute a particular solute is known as the elution volume while the corresponding time for elution of the solute at a given fl w rate is known as the retention time.The elution is continued(usually 2–3 times bed volumes of buffer)until the absorbance monitor reaches the baseline value.

Active fractions of 0.5 mL with fl w rate of 1 mL/3 min were collected.The column fractions with SAPI activity were dialyzed,concentrated and loaded onto sepharose affinit column equilibrated with 100 mmol/L phosphate buffer (pH 7.6) containing 100 mmol/L NaCl.The adsorbed SAPI was eluted with 100 mmol/L HCl.The purity was checked by reverse phase HPLC(C18 column)at a f ow rate of 1.0 mL/min with 100%solvent A(0.1%trifluoroaceti acid(TFA)in water)for 10 min and a linear gradient(0–100%)of solvent B(0.08%TFA in 80%acetonitrile)over 45 min.Apparent molecular weight was obtained by Sephadex G-50 gel filtratio column (0.1 mol/L phosphate buffer,pH 7.6)calibrated with known molecular weight proteins(14.3–43 kDa).

2.2.Protease inhibitor activity assay

SAPI activity was determined by estimating the residual hydrolytic activity of trypsin and chymotrypsin toward the substrates BAPNA (N-benzoyl-L-arginine-p-nitroanilide) and BTPNA (N-benzoyl-L-tyrosyl-p-nitroanilide), respectively, at pH 8.0 after pre-incubation with inhibitor [9].One trypsin or chymotrypsin unit is referred as 1 μmol of substrate hydrolyzed per min of reaction.One inhibitor unit was recorded as the quantity of inhibitor needed to inhibit 50% of the corresponding enzyme activity.Protein content was measured as per the method of Bradford[10]using BSA by Coomassie blue staining.

2.3.SDS,Native-PAGE and isoelectric focusing

Molecular mass and purity of PI was evaluated by SDS-PAGE[11]and Native-PAGE as per the protocol of Felicioli et al.[12].The molecular mass was further confirme by size elution chromatography.In 2-D electrophoresis, IEF was performed with Ampholine polyacrylamide gel plates from Pharmacia(pHrange 3–11) together with Pharmacia broad-range pIcalibration kit containing proteins with various isoelectric points ranging from 3 to 10.Proteins were stained with either CBB R250(0.1%)or silver nitrate method.

2.4.Effect of temperature and pH

The effect of temperature on trypsin or chymotrypsin inhibitory activity was evaluated by incubating SAPI for 30 min in water bath at temperature range of 10–100°C,and then cooled before testing for residual inhibitory activity.

To measure the pH stability, SAPI was mixed with buffers such as 100 mmol/L sodium citrate (pH 2–3), 100 mmol/L sodium acetate (pH 4–5), 100 mmol/L sodium phosphate (pH 6–7),100 mmol/L Tris–HCl(pH 7–8)and 100 mmol/L sodium bicarbonate(pH 9–12).After incubation in the specifi buffer for 60 min at 37°C,the trypsin and chymotrypsin inhibitory activities were assayed as described above.Further,extreme alkaline and acidic effects on the inhibitory activity of SAPI were also analyzed by incubating SAPI with 6 N HCl and 1 N NaOH.

2.5.Kinetic analysis

Kinetic analysis of SAPI activity was carried out following the protocol of Dixon plot analysis [13].Concentrations such as 4, 5, 6, 8, 10 and 12 nmol/L were used to determine the inhibition constant (Ki).The PI inhibitory potential was analyzed for trypsin and chymotrypsin using BAPNA (0.005 and 0.01 mmol/L)and BTPNA(0.5 and 1 mmol/L).The initial slope‘v’was determined for each inhibitory concentration.The reciprocal velocity (1/v) versus PI for each substrate concentration,(S1)and(S2),was also plotted.Single regression line for each(S)was obtained and theKiwas calculated from the intersection of the two lines.The velocity of the reaction was expressed as 1/v(OD247mM/min/mL)?1.The mode of inhibition was determined using Lineweaver–Burk plots, in which the inverse of the initial rate was plotted against the inverse of the substrate concentration in the presence or absence of PI.

2.6.Effect of metal ions,detergents,oxidizing and reducing agents in PI activity

Role of metal ions such as Na+, Ca2+, Mg2+, Zn2+, Cu2+,Fe3+, Mn2+, NI2+, Hg2+, Ba2+, Cd2+, M06+and Al3+on the activities of PI was evaluated by incubating the PI along with 1,5, 10 and 20 mmol/L concentrations of the respective cations in the SAPI solution for 30 min followed by measuring the trypsin/chymotrypsin inhibitory activities as described earlier.

Further, the effect of non-ionic and ionic detergents such as Triton X 100, SDS and Tween-80 (0.5 and l% each w/v)on PI activity was determined by incubating PI in each of the detergents for 30 min and dialyzed against 0.01 mol/L phosphate buffer pH 7.0.The residual inhibitory activities were calculated.

Effect of oxidizing agents such as hydrogen peroxide and dimethyl sulfoxide on the activity of PI were studied by incubating them with 0.5, 1, 2, 3 and 4% (v/v) for 30 min and subsequently,the residual inhibitory activities were estimated.

Similarly, the effect of reducing agents like dithiothretol(DTT) andβ-mercaptoethanol on the activities of PI was analyzed by incubating SAPI with 1, 2 and 3 mmol/L for 3 h and their residual inhibitory activities were estimated.

2.7.Circular Dichroism(CD)

Jasco spectropolarimeter equipped with stopped fl w chamber and thermostated cell holder with 1 nm bandwidth,scanning speed of 50 nm/min at cell length of 0.2 cm and at 25°C temperature (over three accumulations) in 10 mmol/L Tris–HCl, pH 8 are the parameters employed for analyzing the native structural features [14].Different CD spectral measurements were recorded for native SAPI and also,SAPI exposed to(i)thermopH extremities and (ii) treated with 6 mol/L guanidine (Gdn)HCl and DTT over the range of 190–250 nm(protein solutions of 0.5 mg/mL) following the methodology of Leach and Fish[15].The molar ellipticity was noted and subsequently,the percent of secondary structure was predicted using the program k2d[16,17].

2.8.Chemical modification of amino acid residues of SAPI

To analyze the amino acids at the reactive site of inhibitor molecule related with its PI activity was measured using specifi chemical modifier and the effect of modifier on the anti proteolytic activity of the inhibitor molecule was determined.Patthy and Smith [18] protocol was used to analyze arginine residues modificatio using1,2-cyclohexanedione(CHD).SAPI in 50 mmol/L borate buffer(pH 9.0)was incubated with 15-fold molar excess of CHD and the reaction vial was flushe with nitrogen and kept at 37°C for 2 h.The reaction was terminated by the addition of 5% acetic acid.Lysine residues were modifie using succinic anhydride following the protocol of Haynes et al.[19].PI in 0.1 mol/L sodium carbonate buffer(pH 8.0)was incubated with 10-fold molar excess of succinic anhydride at 30°C for 120 min.The reaction was terminated by the addition of 10%SDS followed by 0.2 mL of 1 N HCl.Tyrosine residues were modifie using N-acetylimidazole (NAI) following the method of Yu et al.[20].SAPI in 50 mmol/L Tris–HCl(pH 7.5)was incubated with 60-fold molar excess of NAI at 37°C for 120 min.The reaction was arrested by adding excess NAI and dialyzed for 300 min at 4°C against 50 mmol/L Tris–HCl (pH 7.5).Tryptophan residues modificatio was performed using N-bromosuccinimide (NBS) as per the method of Spande andWitkop [21].PI in 50 mmol/L sodium acetate buffer (pH 4.0)was incubated with 60-fold molar excess of NBS at 37°C for 120 min.In all these cases residual inhibitory activities against trypsin and chymotrypsin were measured.

Table 1 Different concentrations of ammonium sulfate precipitation of PI from S.aculeatissimum.

3.Results and discussion

3.1.Purification of the S.aculeatissimum protease inhibitor(SAPI)

Protease inhibitor activities from different plant parts ofS.aculeatissimumwere evaluated.Fruits displayed the maximum PI activity compared to leaves, stem and seeds, i.e., 54% for trypsin and 48%chymotrypsin inhibitory activity.Subsequently,crude protease inhibitor(Pl)obtained from fruits was purifie to homogeneity through ammonium sulfate precipitation followed by chromatographies such as DEAE cellulose ion exchange,Sephadex G-50 and sepharose affinit chromatography.

SAPI was concentrated by (NH4)2SO4precipitation with varying concentrations ranging from 0–20,20–40,40–60,60–80 and 80–90%saturation.The concentrated proteins were desalted by dialysis against the buffer 20 mmol/L Tris–HCl,pH 8.2 and the protein concentrations were determined by Lowry’s method[22].The concentrated proteins had the protein contents as given in Table 1.80–90%saturated(NH4)2SO4fraction yielded 367 mg/g protein compared to 0–20% saturation (874 mg/g).Similarly, the concentrated and desalted (NH4)2SO4fractions were assayed for SAPI activity and the amount of SAPI in the 0–20% fraction was negligible compared to fractions of 80–90%.The (NH4)2SO4precipitation resulted 1.49 and 1.51 fold of purificatio compared to the crude extract(Table 1).

Subsequent to 90% (NH4)2SO4precipitation, the elution fractions of DEAE ion exchange chromatography revealed one major and a minor protein peaks.Pooled active fractions from 0.18 to 0.24 mmol/L NaCl (fractions: 9–12) were dialyzed and showed 93.2 TlU and 90.2 ClU for trypsin chymotrypsin inhibitory activities respectively.The fold of purificatio for Sephadex G-50 column (52.7 and 51.8), followed by affin ity column chromatography were significantl correlated with single prominent elution peaks.The purity of PI was further checked by RP-HPLC with retention time of 10 min in 50 mmol/L Tris–HCl buffer, pH 8.0, coinciding with the protein peak(Fig.1).Thus,purifie SAPI yielded specifi activity of 502 TlU and 433.7 CIU U/mg, with low protein content of 0.95 mg.Overall,the specifi activity increased about 92.6 and 82.9 folds with 9.8 and 8.77%yield with respect to trypsin and chymotrypsin respectively(Table 2).

Fig.1.RP-HPLC chromatogram of purifie SAPI using C-18 column.

The present purificatio profil of SAPI was comparable with PI isolated from seeds ofDerris trifoliatayielding only 57-fold of purificatio with Q sepharose[13].Chaudhary et al.[23]also purifie trypsin inhibitor from seeds ofPutranjiva roxburghiiby acid precipitation, cation-exchange and anion-exchange chromatography with low yield.Meanwhile,Prasad et al.[9]purifie Bowman–Birk proteinase inhibitor from seeds ofVigna mungo.The fold of purificatio was 55.61 following DEAE cellulose,trypsin-Sepharose 4B column and sephadex G-50 chromatography.The above purificatio profil results suggest that the fold and recovery of protein can be increased through various chromatographies.The observation related with SAPI was commendable when compared with PIs fromD.trifoliata, P.roxburghiiandV.mungo.

3.2.Molecular mass and isoinhibitor evaluation

SDS-PAGE electrophoretic separation of SAPI showed a single prominent band of 22.2 kDa mass (Fig.2a).In agreement,size elution chromatography also revealed the same mass.The molecular mass of purifie PI from the seeds ofAdenantherapavoniawas 20 kDa [24].Luo et al.[25] expressed an 18 kDa recombinant PIN2b PI fromS.americanuminEscherichia coliand was a potent inhibitor against serine proteinases.Kansal et al.[26] purifie PI fromCicer arietinumthat showed single band in SDS-PAGE corresponding to the molecular mass of 30 kDa.The mass of PI isolated fromS.aculeatissimumwas higher thanA.pavoniaandS.americanumbut lower thanC.arietinum.

Table 2 Purificatio profil of S.aculeatissimum PI.

Isoelectric focusing of SAPI revealed the presence of four isoinhibitors (Fig.2b) The isoinhibitors had pIof 4.7, 5.2, 5.6 and 5.9 respectively (Fig.2c).Isoinhibitors with strong acidic traits are physiologically important as part of the evolutionary survival strategies evolved by the host plant against pests and pathogens [27].PIs generally belong to multiple gene family and the possibility of hydrolysis results in the formation of large number of isoinhibitors.Domoney et al.[28] suggested that post-translational modificatio of the gene product during the ontogeny of seed leads to isoinhibitor formation.Lens culinaris[29]andVigna unguiculata[30]also revealed the existence of isoinhibitors.Campos et al.[31] also reported unusual diverse isoinhibitors with complete amino acid sequence fromPhaseolus acutifolius.Similarly, BBI class PI from black gram seeds revealed fie different isoinhibitors in 2-D electrophoresis[9].

3.3.Inhibitor kinetics and constant

Fig.2.(a)SDS-PAGE of purifie SAPI.M:Marker;1:crude;2:salt precipitated;3:ion exchange;4:gel filtration 5:affinit chromatography.(b)Native-PAGE of SAPI showing isoinhibitors.(c)2-D gel showing isoinhibitors of SAPI.The protein were separated in IEF using 3–11 pH non linear strips.

Fig.3.Protease inhibitory activity of S.aculeatissimum showing residual inhibition activity in percent as function of the inhibitor dose at a f xed concentration using BAPNA and BTPNA as substrate.

Molar inhibitory ratio of SAPI against trypsin and chymotrypsin was found to be similar.The inhibitory graphs increased steadily up to 85% and subsequent linear extrapolation revealed 1:1 trypsin/chymotrypsin-SAPI complex(Fig.3).Most of the BBIs were known to inhibit both trypsin and chymotrypsin due to the presence of two different reactive sites[32].However, PIs show higher affinit toward trypsin compared with chymotrypsin as reported fromApios americanatubers andLupinus albusseeds [33,34], i.e., 1:2 stoichiometry.The dissociation constant(Ki)value and mode of inhibition of SAPI were determined from Dixon plot.The results showed that the SAPI shows competitive inhibition where two lines corresponding to each substrate intersect above thex-axis,a feature of competitive inhibition.The inhibition constants(Ki)against both trypsin (BAPNA hydrolyzing units) and chymotrypsin(BTPNA hydrolyzing units) activity were 1.6×10?10and 1.45×10?10mol/L respectively(Figs.4 and 5).The data supports the double headed nature of SAPI,i.e.,it belongs to BBI group of PIs.Bhattacharyya and Babu [35] obtained more or less similar inhibition constants (Ki) forDerris trifoliataPI against both trypsin(tosyl–arginyl–methyl ester hydrochloride hydrolyzing units)and chymotrypsin(n-benzoyl tyrosine ethyl ester hydrolyzing units).The activities were 1.7×10?10and 1.25×10?10mol/L for trypsin and chymotrypsin respectively.In addition,Kivalues recorded for PIs isolated from other legumes were also at par with SAPI [22,35].In spite of thesimilarities, theKivalue forPutranjiva roxburghiiwas found to be 1.4×10?11mol/L which clearly suggests that PI was highly potent against trypsin only[23].Earlier,highKivalues of 5.3×10?10,4.0×10?10,2.5×10?10,1.7×10?10mol/L have been reported for plant trypsin inhibitors fromPeltophorum dubium[36]andArchidendron ellipticum[37],respectively.

Table 3 Residual inhibitory activities of SAPI against trypsin (TI) and chymotrypsin(CI)at different temperatures and pH.

3.4.Stability studies

In temperature sensitivity studies,trypsin and chymotrypsin inhibitory activities related with SAPI was assayed at temperature ranging from 10 to 100°C.Residual inhibitory activities were retained remarkably up to 70°C suggesting its thermal stability nature.Above 70°C temperature there was a decrease in the inhibitory activities.Further, at 80°C it retained 74%and 69%inhibitory activities toward trypsin and chymotrypsin.However, the activities decreased drastically above 80°C, i.e.,60%–75%loss of inhibitory activities at 90°C.Thus,the transition midpoint for SAPI lies close to 85 and 80°C for trypsin and chymotrypsin respectively (Table 3).Moreover, SAPI showed higher trypsin/chymotrypsin inhibition following exposure to 1 N NaOH(93%and 89%)compared to 6 N HCl,i.e.,63%and 59%.

Figs.4 and 5.Dixon plot for the determination of the dissociation constant(Ki)value of SAPI at two different doses of BAPNA and BTPNA.The reciprocal of the velocity were plotted against different doses of SAPI.

Table 4 Effect of various metal ions on SAPI residual inhibitor activity.

Subsequently, the inhibitory activity of SAPI against both trypsin and chymotrypsin was tested at different pH between 2.0 and 12.0.However, only a marginal loss in inhibitory activity alongthepHgradient4–10wasnoticed.Thepossibleoccurrence of cysteine fractions forming disulfid bonds may lead for this remarkable stability in structural conformation and inhibitory properties of SAPI.However, it showed maximum residual inhibitory activities at pH 8.0.Further, it maintained over 55%–88%and 50%–76%of its trypsin/chymotrypsin inhibitory activitiesforacidic(pH3–6)whereas100%–56%and44%–45%for trypsin and chymotrypsin, respectively with alkaline (pH 8–11) range.The stability toward temperature and pH of the inhibitor may be due to its structural peculiarities including reversible denaturation via transient intermediate and also by the presence of aromatic amino acid molecules generally implicated in energy transfer and disulfid bonds.

3.5.Effect of metal ions,detergents,oxidizing and reducing agents on protease inhibitor activity

The role of monovalent and divalent cations was evaluated by incubating SAPI with 1,5,10 and 20%metal ions.Zn2+,Hg2+,Mn2+,iron and copper enhanced the residual inhibitory activity(%)of trypsin and chymotrypsin when compared to the control.Meanwhile, Pb2+and Cd2+declined the activities (Table 4).Metal ions play key role in maintaining the structural integrity of PIs.Side chain carboxylates of glutamic and aspartic fractions may involve in binding of cations to metalloproteins.Further,they also act as chelating agents.

Similarly, ionic and nonionic detergents except SDS have negative impact in PI activities,i.e.,1%SDS increased the residual activities to 145%and 140%for trypsin and chymotrypsin respectively when compared to the control.Triton X 100 and Tween 80 decreased the inhibitory activities remarkably (i.e.,55%and 60%;70–76%trypsin and chymotrypsin respectively)(Table 5).

Detergents are commonly employed for solubilizing protein from lipid membranes/other biological molecules and also for maintaining the solubility of proteins in the solution.The enhanced inhibitory activity by SDS compared to control suggests its role as stabilizer for PI.The hydrophobic nature of SDS apparently caused rearrangement of peptide backbone conformation leading to helix-formation with more hydrophobic residues exposed and consequently available to associate with the detergent[38].

On the other hand,reduction in SAPI activity with Triton X 100 and Tween 80 may be due to multiple factors such as (a)linking with specifi binding points of native proteins(b)cooperative association between protein and many detergent molecules without major conformational change (c) cooperative association with conformational changes in the protein such that the native structure is destroyed and replaced by an extended rodlike conformation with a moderately high content ofα-helix,in which most of the hydrophobic residues were presumably exposed for association with the detergent.Decline of function without a concomitant loss or alter in structure suggests that certain pivotal amino acids needed for inhibiting enzyme activity and the reactive site residues are affected[39].

Oxidizing agents reduced trypsin and chymotrypsin protease inhibitor activities gradually with concentration, i.e., 0.5% of DMSO,decreased the activities to 33%and 37%while,at 4%it was only 7%and 10%with trypsin and chymotrypsin,respectively.Similarly,69%and 58.6%with 0.5%H2O2(Table 6).It was proposed that methionine oxidation is the common means for regulating the activity of proteins.Thus,oxidation could be the key factor for the regulation of PI activity[40].

Further,the residual inhibitory activities of PI increased withβ-mercaptoethanol (1.0–2.0 mmol/L).For example, PI activity with 1 mmol/L was 110 and 100%; at 2 mmol/L 119 and 112% for trypsin and chymotrypsin, respectively.Meanwhile,DTT(1.0 mmol/L)for 3 h on the residual inhibitory activities of SAPI was 77%and 68%for trypsin and chymotrypsin,respectively,while with 2.0 mmol/L DTT it retained the activities 49%and 38%for trypsin and chymotrypsin,respectively.Further,the activities declined drastically(Table 7).

Disulfid bonds are significan in stabilizing the native protein structural conformation and inhibitory properties,which in turn maintain/resist change in pH and temperature.This sort of stability was more prominent in Bowman–Birk inhibitors(contain higher number of cysteine residues) compared withKunitz PIs.Therefore,the effect of DTT reduction on the trypsin and chymotrypsin inhibitory activity of SAPI was evaluated.There was a reduction in the inhibitory activity against trypsin(77%)and chymotrypsin(68%)at 1 mmol/L DTT(Table 7).Further,as the concentration of DTT increased from 3 mmol/L,the inhibitory activity of SAPI was remarkably declined (trypsin– 20%; chymotrypsin – 11%).The probable reason may be due to the combined effect of factors such as reduction in the hydrophobic interactions that play a crucial role in holding together the protein tertiary structure and the direct interaction with the protein molecule.However,β-mercaptoethanol induced the inhibitory activity up to 2 mmol/L and subsequently reduced.The decline in SAPI activity may be due to intra molecular disulfid bridges in the reactive site loops of inhibitor which are presumably responsible for the functional stability[41].

Table 5 Effect of detergents on inhibitor activity of SAPI.

Table 6 Effect of oxidizing agents on protease inhibitor activity.

Table 7 Effect of reducing agents on protease inhibitor activity.

3.6.Structural analysis of SAPI by circular dichroism(CD)

Secondary and tertiary structure of SAPI revealed by CD Spectra at 190–250 and 250–300 nm were analyzed at pH 7.5.Interestingly,65%β-pleated,14%spiralαhelix and 20%random coil are noticed.Prominent CD spectra at 260 followed by a minor shoulder at 285 nm further supports the disulfid bridges and aromatic residues in the protein[42](Fig.6a and b).

Temperature above 80°C resulted in marginal deformation in the natural structure of SAPI.Structural changes including reversibility revealed the fl xibility of the PI against temperature induced changes(Fig.6c).Similarly,thespectraatlow(2–4)and high pH(10 and 12)corroborates with activity assays,i.e.,reduction in the residual activities in the extreme acidic pH(possibly due to the electrostatic repulsion leads to loss ofβ-sheet structure).In addition,at pH 8 the activity increased suggesting its molding back to its native conformation(Fig.6d).The presence of sulfur containing amino acids may be the reason for this stability displayed by the SAPI.

The structural modificatio with DTT incubation(2 mmol/L)at far-UV CD spectra was also analyzed.Significan variations were seen in far-UV spectrum of native and reduced SAPI,i.e.,loss of the bandwidth between 205 and 215 nm and minor reduction in ellipticity between 235 and 245 nm band.These may be due to the chirality occurred in the cysteine bonds.The losses in theβ-sheets with concomitant modificatio in the helical content of the inhibitors were the changes noticed.Conformational modification due to loss of disulfid bonds altered the protein stability and also marginally the reactive sites as revealed by the retention of 49%trypsin and 38%chymotrypsin inhibitory activities, respectively after 3 h incubation with the reductant DTT(2 mmol/L)(Fig.6e).Alkylation using iodoacetamide further altered the secondary structure which in turn confirm the break occurred in the disulfid bonds.

Gross structural conformational changes were noticed when SAPI was incubated with 6 N GHC for 120 min at 80°C.Significant reduction was noticed in the amplitude of broad negative ellipticity band between 280 and 240 nm in comparison to the native SAPI in the near UV spectral zone.Detailed analysis suggests that the complete loss of secondary structure was basically due to the complete conversion of theβ-sheets to helical and random structural components.Many studies on other PIs strongly support the effect of GHC on PI.

Fig.6.(a and b)CD spectral analysis of SAPI revealing secondary and tertiary model.(c)Effect of different temperatures on far-UV CD spectra of SAPI.(d)Effect of pH(2–12)on far-UV CD spectra of SAPI.(e)Far-UV CD spectra of native SAPI reduced with 2 mmol/L DTT and reduced SAPI is alkylated with iodoacetamide.

3.7.Amino acid modification in SAPI

Different amino acids were individually modifie using specifi chemical modifier and the effect of amino acid modification on PI activity was determined.Succinic anhydride(lysine)or NBS(tryptophan)modifying agents positively affected trypsin PI, i.e., 68% and 100% loss.In spite,modificatio of arginine or tyrosine residues leads only marginal reduction in its inhibitory activity.

Meanwhile, tryptophan residue modification also resulted in the reduction of chymotrypsin inhibitory activity.However,no remarkable changes of chymotrypsin inhibitory activities were noticed with modification of lysine,arginine or tyrosine residues.The presence of amino acid residues lysine/tryptophan at N-terminal and aspartic/glutamic acid in the carboxy terminus are suggested to be responsible for self-association of monomers to form stable dimers and any deviation in these molecules results in to monomer formation [9].Replacement of lysine/tryptophan from the reactive site of PI may restrict the formation of oligomers and it was retained as monomers.Tsybina et al.[43] reported that change in lysine and arginine residue present at the reactive site of PI from buckwheat seeds reduces the inhibitory activity.Similarly,tryptophan of PI plays key role in maintaining the hydrophobicity and efficien y of the binding region with its target compound[44].

4.Conclusion

Thus, the present work unveils the PI from the fruits ofS.aculeatissimumwhich inhibits trypsin and chymotrypsin in a molar ratio 1:1.SAPI exhibit remarkable stability at temperature and wide range of pH.CD spectral analysis revealed the presence of secondary structure and random coils.Chemical modificatio studies indicated that lysine and tryptophan in the reactive site of PI which play key role in the protease inhibition mechanism.Future studies are warranted in this direction to completely elucidate the molecular and structural characteristic features of PI fromS.aculeatissimum.

Acknowledgement

The authors acknowledge the Department of Science and Technology,Govt.of India for providing the INSPIRE fellowship related with this work.