Thus, the present paper describes the purification and physicochemical characterization of a novel trypsin inhibitor from J. Additionally, to gain better insights on the biological activity of this protein and to devise future use as a new therapeutic drugs, its inhibitory activity against the growth of the human pathogen bacteria Staphylococcus aureus and Salmonella enterica , as well as the ability of JcTI-I to inhibit the bacterial proteases were evaluated.
Swiss mice Mus musculus , 20—25 g, were from the animal house at UFC. Chromatographic matrixes were from GE Healthcare. All other chemicals and reagents used were of analytical grade. The method described by Bradford was used with BSA as standard.
Absorbance at nm was also used to detect the presence of protein in the chromatographic eluates. Trypsin inhibitory activity against bovine trypsin EC 3. The enzyme concentration used was established based on a dependent concentration assay and was that producing an absorbance between 0.
The enzymatic activities in the absence and presence of the inhibitor were evaluated by p -nitroanilide release from BAPNA measured at nm using a Biochrom Libra S spectrophotometer. One trypsin inhibitory activity unit TIU was defined as the decrease in 0.
Appropriate blanks for the enzyme, inhibitor and the substrate were also included in the assay along with the test samples. Defatted flour was extracted with 0. The protein content and trypsin inhibitory activity of this dialyzed extract were determined and it was further used for purification of the trypsin inhibitor as described below.
The clear supernatant obtained was dialyzed exhaustively against water Milli-Q grade , lyophilized, and assayed for antitrypsin activity. The TCA fraction 30 mg was dissolved in 0. After complete removal of the non-retained proteins with the equilibrating buffer, the proteins bound to the immobilized trypsin were eluted with 0. Fractions 1 mL were eluted at the flow rate of 0.
Samples were prepared in 0. Chromatography was carried out at a constant flow rate of 0. The column was previously calibrated with proteins of known molecular mass BSA, 66 kDa; egg albumin, 45 kDa; chymotrypsinogen, 25 kDa; ribonuclease, In addition, native mass was obtained by mass spectrometry analysis of JcTI-I 0. The data collection was performed using MassLynx 4.
To assess whether JcTI-I is composed of subunits covalently linked by disulphide bridges it was previously treated with 0. Mobile phases A and B consisted of 0. Readings were taken at nm. The carbohydrate content was estimated based on a calibration curve built with different concentrations of galactose. Briefly, after the electrophoresis run the gel was fixed in a solution of 7. The glycoprotein nature of the band was revealed by immersion of the gel in a solution of 0.
Phenylthiohydantoin PTH amino acids were detected at nm after separation on a reversed phase C 18 column 4. This was evaluated according to Klomklao et al. JcTI-I 0. The buffers used were: 0. After treatment the inhibitory activity against trypsin was determined as previously described and the residual inhibitory activity reported. The papain inhibitory assay was performed as described by Abe et al.
The chymotrypsin inhibitory activity was measured by the method of Erlanger et al. Twenty microliters of chymotrypsin 0. The velocity of enzyme reaction v was determined from progress curves using different BAPNA concentrations on the base of product concentrations measured at nm at a fixed time of a reaction.
To calculate K m Michaelis—Menten constant and V max maximal rate of the reaction and to study the pattern of inhibition, data were plotted as a function of BAPNA concentration using the usual non-linear curve fitting of Michaelis—Menten and linear Lineweaver—Burk, respectively. The antibacterial activity Hancock, of JcTI-I was tested in vitro against Salmonella enterica , Staphylococcus aureus , Bacillus subtilis , and Pseudomonas aeruginosa. Bacterial growth was monitored at nm, every hour within the incubation period, using an automated microplate reader Bio-TekElx The cell growth of both bacteria in the absence of JcTI-I was monitored as a blank control.
Experiments were run in triplicate. The capacity of JcTI-I to inhibit the secreted and endogenous bacterial proteases was done following the methodology described by Lima et al. The supernatants obtained, containing the protease from Salmonella enterica and Staphylococcus aureus , were pooled separately for every bacterium species and the serine protease activity as well as the inhibitory activity of JcTI-I assayed, according to the method previously described, using azocasein as substrate, bovine trypsin and PMSF as serine protease control inhibitor.
The hemolytic assay, performed in triplicate, was carried out as previously described Kim et al. Human red blood cells were collected in the presence of heparin, washed three times with phosphate-buffered saline PBS; 3. PBS 0. Toxic activity was defined as mortality observed in Swiss mice within 24 h after intraperitoneal injections of JcTI-I at varied concentrations.
TCA, at 2. This TCA precipitated fraction was chromatographed on a trypsin-Sepharose 4B affinity column and the adsorbed proteins, eluted with 0. At this stage, the trypsin inhibitor was purified to The fraction obtained from the trypsin-Sepharose 4B affinity chromatography displaying trypsin inhibitory activity was composed of multiple protein bands as evaluated by SDS-PAGE Figure 1A ; insert: lane 3.
Therefore, this fraction was further purified on a Sephacryl S column Figure 1B. The eluted protein present in the major peak showed a specific activity of This purified protein is a trypsin inhibitor of J.
JcTI-I purification. A Affinity-chromatography Trypsin-Sepharose 4B column. A molecular mass of Moreover, JcTI-I has 6. In addition, other peptide sequences were obtained similar to 2S albumins, particularly from R. Mass spectrometry analysis of JcTI-I. TABLE 2. TABLE 3. However, at 2. Each point represents the mean of three estimates. JcTI-I was specific for inhibiting bovine trypsin, but did not inhibit significantly chymotrypsin and papain.
The data obtained for the studies conducted on protease—protease inhibitor interaction is depicted in Figure 4. Moreover, it is calculated that the stoichiometry of trypsin—protease inhibitor interaction is Kinetic studies of inhibition of trypsin by JcTI-I showed that plots of a fixed concentration of trypsin 8. Furthermore, calculated K i Figure 5 , insert was on average 0.
Stoichiometry of the JcTI-I-protease interaction. Bar indicates standard deviation from triplicate determinations. However, the protein inhibited the growth of Staphylococcus aureus and Salmonella enterica at all concentrations tested when compared to controls. Moreover, JcTI-I caused Control: 0. Assay was done in triplicate for every inhibitor concentration. JcTI-I did not promote hemolysis of human erythrocyte under the experimental conditions tested Figure 8. Assessment of the hemolytic effect of JcTI-I on human erythrocytes.
Control was the red cells incubated with 0. JcTI-I did not display toxic activity to mice, even at a concentration times greater than the MIC for Staphylococcus aureus and Salmonella enterica. JcTI-I is a Both the molecular mass and p I are similar to other seed trypsin inhibitors previously characterized as, for example, the It is plausible that under such mild conditions in which JcTI-I was dissolved in 0.
Alignments of JcTI-I sequence with other protein sequences showed that the highest similarity was verified with Jat c 1, a J. Several trypsin inhibitors show sequence similarities with the 2S albumin-like proteins, as two barley Bowman—Birk type trypsin inhibitor isoforms and the trypsin inhibitor of Brassica juncea seeds Terras et al. Protease inhibitors exhibit a considerable stability to high temperatures and to large pH variations Bhattacharyya et al.
Such stability was associated to disulphide bridges that are presumably responsible for the functional stability of Kunitz type protease inhibitors in the presence of physical and chemical denaturants such as temperature, pH, and reducing agents Kridric et al. JcTI-I interaction with trypsin occurred at the Similar result was found for a protease inhibitor from Moringa oleifera leaves Bijina et al. Resistance of some strains of Gram-positive and Gram-negative bacteria to conventional antibiotics has increased dramatically caused or induced by the widespread misuse and overuse of antibiotics and represents a serious threat to public health worldwide.
Indeed, extracts from leaves, root barks, latex, and various fruit parts of J. However, few reports are related to the antimicrobial activity of compounds present in the seed cake of J.
The seed cake of J. It is estimated that 1-tn per day of J. The use of protease inhibitors as novel therapeutics has previously been proposed Bhattacharjee et al. Therefore, take into consideration the huge amount of J. This was experimentally confirmed as JcTI-I behaved as a potent inhibitor of Staphylococcus aureus and Salmonella enterica growth.
Serine proteases have been implicated in the virulence of some bacterial strains Speranskaya et al. JcTI-I did have inhibitory activity against the serine proteases from the tested bacteria. These results are similar to that reported by Lima et al.
Even at such concentration, JcTI-I does not lysis the red cells. This result suggests that the mode of action of JcTI-I is not by disrupting cell membranes and it does not have toxic effects toward mammalian cells. In summary, we have isolated, purified, and characterized a novel trypsin inhibitor from J. The lack of hemolytic activity against human erythrocytes and toxic activity to mice together with resistance to heat treatment, pH, high salt concentrations, and putative resistance to proteases make JcTI-I a pharmacologically interesting and valuable drug for the design of a novel antibiotic medicament.
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