Molecular weight of the enzyme inhi

Molecular weight of the enzyme inhibitor: SDS Polyacrylamide gel electrophoresis was carried out using Phosphorylase-b (97.4 kDa), bovine serum albumin (66 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), trypsin inhibitor (22.1 kDa) and lysozyme (14.3 kDa) as standard proteins for calibration. The molecular weight of the inhibitor was determined using the calibration curve.

Effect of pH on the stability of the inhibitor: Solutions of the inhibitor (1mg ml-1) in 10 mM buffers of five pH values (pH 3.0, glycine-HCl; 5.0, sodium citrate; 7.0, sodium phosphate; 9.0, Tris-HCl; 12.0, glycine-NaOH) were kept at 50C for 24h. Aliquots of the inhibitor were diluted with 0.1M phosphate buffer, pH 7.6 and assayed for trypsin inhibitory activity (TIA).

Effect of temperature on the stability of the inhibitor: The inhibitor solutions (100µg ml-1) were separately incubated in a water bath at various temperatures for 10 min and then quickly cooled in ice and appropriate aliquots were assayed for trypsin inhibitory activity (TIA).

Kinetic measurements: The amidolytic activity of trypsin (50 µg) was determined with various concentrations of BAPNA (1.2to5.0µmol) in the absence of the inhibitor. The assays were then repeated in the presence of 5 and 15 µg of the inhibitor. The Ki values were calculated from Lineweaver-Burk plots.

Competition experiments: 50 µg trypsin was separately incubated with 5, 10, 15 and 20 µg of SNTI for 10 minutes at 37oC in 1ml phosphate buffer, pH7.6. Suitable aliquots of all the samples were taken and assayed for residual trypsin activity using BAPNA as a substrate. Studies on complex formation: The trypsin-SNTI complex was isolated by gel filtration on Sephadex G-100. To form the complex, 2 mg of SNTI was incubated with 5 mg of trypsin at 37oC for 15 minutes”. Excess trypsin was used to make sure that all the inhibitor is complexed, such a mixture was applied onto a column of Sephadex G-100 at 5oC previously calibrated with SNTI and trypsin run separately and the absorbance was monitored at 280 nm. Trypsin and Trypsin inhibitory activities are monitored in the fractions collected.

Fourier Transform Infra-Red Spectroscopy (FTIR): The solid state FTIR spectra are recorded in the middle infrared (4000 cm-1to400 cm-1) on Perkin Elmer. The sample for FTIR analysis are prepared by grinding the dry blended powders of trypsin inhibitor with powdered KBr and then compressed to form discs.

Database and Sequence information: To demonstrate the inhibitory activity of SNTI on mid gut proteases, computational approach has been applied where in the proteases from larval guts of two insects viz., H. armigera and S. frugiperda were considered as these organisms commonly cause damage to agricultural fields. The sequence information for SNTI was taken from MALDI-TOF analysis done by Rachel et al., (2012) [35] and the gut proteases of H. armigera and S. frugiperda were retrieved from NCBI protein data base [36].

Homology modeling of SNTI and Threading based modeling of insect gut proteases: Primarily all the three sequences are subjected to PDB BLAST for template identification. The templates are identified based on homology and the homologous template was used for homology modeling of SNTI sequence. For the insect gut proteases no homologous templates were available, hence they are subjected for modeling using threading approach. In threading approach folds (secondary structure) of the protein are considered and templates are identified from fold library. Based on the folds of template identified the target protein is modeled. Prime module from Schrodinger suite (Schrodinger 2011) was used for modeling proteins by Homology and Threading approaches.

Structure Validation: The predicted structures are subjected for validation to ERRAT and PROCHECK servers. The validations by PROCHECK were done based on the stereo chemical quality, hydrogen bonding energy and torsion angles [37]. Based on the interaction of atoms with respect to amino acid residues ERRAT validates the predicted protein structure by separating correct and incorrect determined structures [38].

Binding Site prediction: The active site of all the three modeled proteins were predicted using SiteMap [39]. SiteMap determines primary binding site on a receptor by calculating the sites on protein surface by searching the grid points called site points. Then the contour site maps are generated, producing hydrophobic and hydrophilic maps.

Protein-Protein Docking: To understand role of SNTI in inhibiting proteases, modeled SNTI and gut proteases were subjected for protein-protein docking using Piper [40]. Prior to docking the proteins are subjected for protein preparation to optimize the molecule using PrepWizard. For protein-protein docking gut proteases were set as ligands and docked separately with receptor SNTI. Number of ligand rotation to probe were set for 10000 rotation and for each dock ten poses were retrieved. After docking, the best pose was selected and then these complex structures were again set for optimization by PrepWizard. These prepared complexes are set for analysis of protein-protein interaction using protein interaction option in Bioluminate [41].

Kết quả (Việt) 1:

Trọng lượng phân tử của chất ức chế enzyme: SDS Polyacrylamide gel điện được tiến hành bằng cách sử dụng Phosphorylase-b (97.4 kDa), bò huyết thanh albumin (66 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), chất ức chế trypsin (22,1 kDa) và lysozyme (14.3 kDa) như là tiêu chuẩn protein cho hiệu chuẩn. Trọng lượng phân tử của chất ức chế được xác định bằng cách sử dụng đường cong hiệu chuẩn.Ảnh hưởng của pH sự ổn định của chất ức chế: các giải pháp của chất ức chế (1mg ml-1) trong 10 mM bộ đệm của năm pH giá trị (pH 3.0, glycine-HCl 5.0, natri citrat 7.0, Dinatriyfosfat; 9.0, Tris-HCl; 12.0, glycine-NaOH) được lưu giữ tại 50C cho 24h. Aliquots của chất ức chế được pha loãng với 0.1M phosphat đệm, pH 7.6 và assayed cho trypsin ức chế hoạt động (TIA).Ảnh hưởng của nhiệt độ ổn định của chất ức chế: giải pháp chất ức chế (100µg ml-1) đã được một cách riêng biệt ủ trong một bồn tắm nước ở các nhiệt độ khác nhau cho 10 phút và sau đó một cách nhanh chóng làm lạnh trong nước đá và aliquots thích hợp đã được assayed cho trypsin ức chế hoạt động (TIA).Phép đo động: hoạt động amidolytic của trypsin (50 μg) đã được xác định với các nồng độ khác nhau của BAPNA (1.2to5.0µmol) trong sự vắng mặt của chất ức chế. Các thử nghiệm đã được lặp đi lặp lại sau đó sự hiện diện của 5 và 15 µg của chất ức chế. Ki giá trị đã được tính toán từ Lineweaver-Burk lô.Competition experiments: 50 µg trypsin was separately incubated with 5, 10, 15 and 20 µg of SNTI for 10 minutes at 37oC in 1ml phosphate buffer, pH7.6. Suitable aliquots of all the samples were taken and assayed for residual trypsin activity using BAPNA as a substrate. Studies on complex formation: The trypsin-SNTI complex was isolated by gel filtration on Sephadex G-100. To form the complex, 2 mg of SNTI was incubated with 5 mg of trypsin at 37oC for 15 minutes”. Excess trypsin was used to make sure that all the inhibitor is complexed, such a mixture was applied onto a column of Sephadex G-100 at 5oC previously calibrated with SNTI and trypsin run separately and the absorbance was monitored at 280 nm. Trypsin and Trypsin inhibitory activities are monitored in the fractions collected.Fourier Transform Infra-Red Spectroscopy (FTIR): The solid state FTIR spectra are recorded in the middle infrared (4000 cm-1to400 cm-1) on Perkin Elmer. The sample for FTIR analysis are prepared by grinding the dry blended powders of trypsin inhibitor with powdered KBr and then compressed to form discs.Database and Sequence information: To demonstrate the inhibitory activity of SNTI on mid gut proteases, computational approach has been applied where in the proteases from larval guts of two insects viz., H. armigera and S. frugiperda were considered as these organisms commonly cause damage to agricultural fields. The sequence information for SNTI was taken from MALDI-TOF analysis done by Rachel et al., (2012) [35] and the gut proteases of H. armigera and S. frugiperda were retrieved from NCBI protein data base [36].
Homology modeling of SNTI and Threading based modeling of insect gut proteases: Primarily all the three sequences are subjected to PDB BLAST for template identification. The templates are identified based on homology and the homologous template was used for homology modeling of SNTI sequence. For the insect gut proteases no homologous templates were available, hence they are subjected for modeling using threading approach. In threading approach folds (secondary structure) of the protein are considered and templates are identified from fold library. Based on the folds of template identified the target protein is modeled. Prime module from Schrodinger suite (Schrodinger 2011) was used for modeling proteins by Homology and Threading approaches.

Structure Validation: The predicted structures are subjected for validation to ERRAT and PROCHECK servers. The validations by PROCHECK were done based on the stereo chemical quality, hydrogen bonding energy and torsion angles [37]. Based on the interaction of atoms with respect to amino acid residues ERRAT validates the predicted protein structure by separating correct and incorrect determined structures [38].

Binding Site prediction: The active site of all the three modeled proteins were predicted using SiteMap [39]. SiteMap determines primary binding site on a receptor by calculating the sites on protein surface by searching the grid points called site points. Then the contour site maps are generated, producing hydrophobic and hydrophilic maps.

Protein-Protein Docking: To understand role of SNTI in inhibiting proteases, modeled SNTI and gut proteases were subjected for protein-protein docking using Piper [40]. Prior to docking the proteins are subjected for protein preparation to optimize the molecule using PrepWizard. For protein-protein docking gut proteases were set as ligands and docked separately with receptor SNTI. Number of ligand rotation to probe were set for 10000 rotation and for each dock ten poses were retrieved. After docking, the best pose was selected and then these complex structures were again set for optimization by PrepWizard. These prepared complexes are set for analysis of protein-protein interaction using protein interaction option in Bioluminate [41].