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Molecules (Basel, Switzerland) Sep 2018Plant peptidase inhibitors play essential roles in the defense systems of plants. A trypsin inhibitor (PHTI) with a molecular mass of 20.5 kDa was isolated from the...
Plant peptidase inhibitors play essential roles in the defense systems of plants. A trypsin inhibitor (PHTI) with a molecular mass of 20.5 kDa was isolated from the fresh roots of the medicinal herb, . The purification process involved ammonium sulfate precipitation, gel filtration chromatography on Sephadex G50, and ion-exchange chromatography on DEAE 650M. The PHTI contained 3.7% α-helix, 42.1% β-sheets, 21.2% β-turns, and 33% disordered structures, which showed similarity with several Kunitz-type trypsin inhibitors. Inhibition kinetic studies indicated that PHTI was a competitive inhibitor, with a K value of 3.01 × 10 M, indicating a high affinity to trypsin. The PHTI exhibited considerable stability over a broad range of pH (2⁻10) and temperatures (20⁻70 °C); however, metal ions, including Fe, Ba, Mn, and Al, could inactivate PHTI to different degrees. Results of fluorescence spectroscopy and circular dichroism showed that Fe could bind to TI with an association constant of 2.75 × 10⁵ M to form a 1:1 complex, inducing conformation changes and inactivation of PHTI. In addition, PHTI could inhibit the growth of the phytopathogens, and , through disruption of the cell membrane integrity. The present study extended research on proteins and makes PHTI an exploitable candidate as an antifungal protein for further investigation.
Topics: Amino Acid Sequence; Antifungal Agents; Caryophyllaceae; Chromatography, Gel; Circular Dichroism; Enzyme Stability; Fungi; Hydrogen-Ion Concentration; Kinetics; Microbial Sensitivity Tests; Molecular Weight; Phytochemicals; Plant Extracts; Plant Roots; Temperature; Trypsin Inhibitors
PubMed: 30231516
DOI: 10.3390/molecules23092388 -
Molecules (Basel, Switzerland) Jul 2022The utility of the marama bean (MB) as an alternative protein source to soybean (SB) can be limited by the high concentration of trypsin inhibitors (TI). The physical...
The utility of the marama bean (MB) as an alternative protein source to soybean (SB) can be limited by the high concentration of trypsin inhibitors (TI). The physical treatment of MB has the potential to ameliorate the antinutritional activities of TI and modify other chemical components. Thus, this study investigated the effects of physical treatments on the chemical components and trypsin inhibitor activity (TIA) of raw MB and SB. The bean substrates were subjected to each of the following treatment methods: (1) room temperature (20−22 °C) soaking for 24 h; (2) electric stove cooking at 100 °C for 10, 20, and 30 min; (3) steam autoclaving at a temperature of 110 °C and pressure of 7 pounds per square inch (psi), as well as a temperature of 121 °C and 7 psi for 5, 15, and 30 min; (4) pre-soaked autoclaving at 110 °C (7 psi) and 121 °C (17 psi) for 5, 15, and 30 min. Treated MB and SB had greater (p < 0.05) crude protein content than untreated samples. All the treatments (except 24 h soaking of MB) reduced (p < 0.05) the TIA and ash content. Marama and SB are similar in protein content, but their amino acids profile and TIA are quite different. Soaking for 24 h was less effective in reducing TIA in MB and SB, compared to the thermal methods, and it was detrimental to the ash and amino acids profile of the two beans. Soaking prior to autoclaving yielded beans with the lowest TI concentrations. In conclusion, thermal methods reduced the TI contents and modified the level of proximate components and amino acids profile of the beans.
Topics: Amino Acids; Cooking; Fabaceae; Hot Temperature; Glycine max; Trypsin Inhibitors
PubMed: 35889324
DOI: 10.3390/molecules27144451 -
Bioorganic & Medicinal Chemistry Aug 2020A range of guanidine-based pyridines, and related compounds, have been prepared (19 examples). These compounds were evaluated in relation to their competitive inhibition...
A range of guanidine-based pyridines, and related compounds, have been prepared (19 examples). These compounds were evaluated in relation to their competitive inhibition of bovine pancreatic trypsin. Results demonstrate that compounds in which the guanidinyl substituent can form an intramolecular hydrogen bond (IMHB) with the pyridinyl nitrogen atom (6a-p) are better trypsin inhibitors than their counterparts (10-13) that are unable to form an IMHB. Among the compounds 6a-p, examples containing a 5-halo substituent were, generally, found to be better trypsin inhibitors. This trend was inversely related to electronegativity, thus, 1-(5-iodopyridin-2-yl)guanidinium ion 6e (K = 0.0151 mM) was the optimum inhibitor in the 5-halo series. Amongst the isomeric methyl substituted compounds, 1-(3-methylpyridin-2-yl)guanidinium ion 6h demonstrated optimum levels of trypsin inhibition (K = 0.0140 mM). In order to rationalise the measured enzyme inhibition, selected compounds were docked with bovine and human trypsin with a view to understanding active site occupancy and taken together with the K values the order of inhibitory ability suggests that the 5-halo 2-guanidinyl pyridine inhibitors form a halogen bond with the catalytically active serine hydroxy group.
Topics: Animals; Cattle; Guanidine; Hydrogen Bonding; Molecular Docking Simulation; Pyridines; Trypsin; Trypsin Inhibitors
PubMed: 32690267
DOI: 10.1016/j.bmc.2020.115612 -
Toxins Jul 2016Kazal-type serine proteinase inhibitors are found in a large number of living organisms and play crucial roles in various biological and physiological processes....
Kazal-type serine proteinase inhibitors are found in a large number of living organisms and play crucial roles in various biological and physiological processes. Although some Kazal-type serine protease inhibitors have been identified in leeches, none has been reported from Hirudinaria manillensis, which is a medically important leech. In this study, a novel Kazal-type trypsin inhibitor was isolated from leech H. manillensis, purified and named as bdellin-HM based on the sequence similarity with bdellin-KL and bdellin B-3. Structural analysis revealed that bdellin-HM was a 17,432.8 Da protein and comprised of 149 amino acid residues with six cysteines forming three intra-molecular disulfide bonds. Bdellin-HM showed similarity with the Kazal-type domain and may belong to the group of "non-classical" Kazal inhibitors according to its Cys(I)-Cys(II) disulfide bridge position. Bdellin-HM had no inhibitory effect on elastase, chymotrypsin, kallikrein, Factor (F) XIIa, FXIa, FXa, thrombin and plasmin, but it showed a potent ability to inhibit trypsin with an inhibition constant (Ki) of (8.12 ± 0.18) × 10(-9) M. These results suggest that bdellin-HM from the leech of H. manillensis plays a potent and specific inhibitory role towards trypsin.
Topics: Amino Acid Sequence; Animals; Leeches; Molecular Weight; Organic Chemicals; Protein Domains; Structure-Activity Relationship; Trypsin; Trypsin Inhibitors
PubMed: 27455325
DOI: 10.3390/toxins8080229 -
Biomedicine & Pharmacotherapy =... Dec 2021Erythrina poeppigiana belongs to Fabaceae family (subfamily Papillionoideae) and is commonly found in tropical and subtropical regions in Brazil. Herein, we described...
Erythrina poeppigiana belongs to Fabaceae family (subfamily Papillionoideae) and is commonly found in tropical and subtropical regions in Brazil. Herein, we described the purification and characterization of a new Kunitz-type inhibitor, obtained from E. poeppigiana seeds (EpTI). EpTI is composed by three isoforms of identical amino-terminal sequences with a molecular weight ranging from 17 to 20 kDa. The physicochemical features showed by EpTI are common to Kunitz inhibitors, including the dissociation constant (13.1 nM), stability against thermal (37-100 °C) and pH (2-10) ranging, and the presence of disulfide bonds stabilizing its reactive site. Furthermore, we investigated the antimicrobial, anti-adhesion, and anti-biofilm properties of EpTI against Gram-positive and negative bacteria. The inhibitor showed antimicrobial activity with a minimum inhibitory concentration (MIC, 5-10 µM) and minimum bactericidal concentration (MBC) of 10 µM for Enterobacter aerogenes, Enterobacter cloacae, Klebsiella pneumoniae, Staphylococcus aureus, and Staphylococcus haemolyticus. The combination of EpTI with ciprofloxacin showed a marked synergistic effect, reducing the antibiotic concentration by 150%. The increase in crystal violet uptake for S. aureus and K. pneumoniae strains was approximately 30% and 50%, respectively, suggesting that the bacteria plasma membrane is targeted by EpTI. Treatment with EpTI at 1x and 10 x MIC significantly reduced the biofilm formation and prompted the disruption of a mature biofilm. At MIC/2, EpTI decreased the bacterial adhesion to polystyrene surface within 2 h. Finally, EpTI showed low toxicity in animal model Galleria mellonella. Given its antimicrobial and anti-biofilm properties, the EpTI sequence might be used to design novel drug prototypes.
Topics: Animals; Anti-Bacterial Agents; Bacteria; Biofilms; Ciprofloxacin; Drug Synergism; Erythrina; Microbial Sensitivity Tests; Moths; Plant Extracts; Seeds; Trypsin Inhibitors
PubMed: 34656058
DOI: 10.1016/j.biopha.2021.112198 -
Protein Science : a Publication of the... Sep 2015The development of effective protease therapeutics requires that the proteases be more resistant to naturally occurring inhibitors while maintaining catalytic activity....
The development of effective protease therapeutics requires that the proteases be more resistant to naturally occurring inhibitors while maintaining catalytic activity. A key step in developing inhibitor resistance is the identification of key residues in protease-inhibitor interaction. Given that majority of the protease therapeutics currently in use are trypsin-fold, trypsin itself serves as an ideal model for studying protease-inhibitor interaction. To test the importance of several trypsin-inhibitor interactions on the prime-side binding interface, we created four trypsin single variants Y39A, Y39F, K60A, and K60V and report biochemical sensitivity against bovine pancreatic trypsin inhibitor (BPTI) and M84R ecotin. All variants retained catalytic activity against small, commercially available peptide substrates [kcat /KM = (1.2 ± 0.3) × 10(7) M(-1 ) s(-1) . Compared with wild-type, the K60A and K60V variants showed increased sensitivity to BPTI but less sensitivity to ecotin. The Y39A variant was less sensitive to BPTI and ecotin while the Y39F variant was more sensitive to both. The relative binding free energies between BPTI complexes with WT, Y39F, and Y39A were calculated based on 3.5 µs combined explicit solvent molecular dynamics simulations. The BPTI:Y39F complex resulted in the lowest binding energy, while BPTI:Y39A resulted in the highest. Simulations of Y39F revealed increased conformational rearrangement of F39, which allowed formation of a new hydrogen bond between BPTI R17 and H40 of the variant. All together, these data suggest that positions 39 and 60 are key for inhibitor binding to trypsin, and likely more trypsin-fold proteases.
Topics: Amino Acid Sequence; Animals; Cattle; Drug Resistance; Kinetics; Models, Molecular; Molecular Dynamics Simulation; Point Mutation; Protein Binding; Protein Conformation; Protein Engineering; Structure-Activity Relationship; Trypsin; Trypsin Inhibitor, Kazal Pancreatic; Trypsin Inhibitors
PubMed: 26106067
DOI: 10.1002/pro.2732 -
Journal of Enzyme Inhibition and... Dec 2019The increase in non-communicable chronic diseases has aroused interest in the research of adjuvants to the classic forms of treatments. Obesity and metabolic syndrome... (Review)
Review
The increase in non-communicable chronic diseases has aroused interest in the research of adjuvants to the classic forms of treatments. Obesity and metabolic syndrome are the main targets of confrontation because they relate directly to other chronic diseases. In this context, trypsin inhibitors, molecules with wide heterologous application, appear as possibilities in the treatment of overweight and obesity due to the action on satiety related mechanisms, mainly in the modulation of satiety hormones, such as cholecystokinin. In addition, trypsin inhibitors have the ability to also act on some biochemical parameters related to these diseases, thus, emerging as potential candidates and promising molecules in the treatment of the obesity and metabolic syndrome. Thus, the present article proposes to approach, through a systematic literature review, the advantages, disadvantages and viabilities for the use of trypsin inhibitors directed to the treatment of overweight and obesity.
Topics: Dose-Response Relationship, Drug; Humans; Metabolic Diseases; Molecular Structure; Obesity; Structure-Activity Relationship; Trypsin; Trypsin Inhibitors
PubMed: 30734596
DOI: 10.1080/14756366.2018.1542387 -
Journal of Agricultural and Food... Jun 2023The greater awareness of consumers regarding the sustainability of food chains has shifted part of the consumption from animal protein sources to vegetable sources....
The greater awareness of consumers regarding the sustainability of food chains has shifted part of the consumption from animal protein sources to vegetable sources. Among these, of relevance both for human food use and for animal feed, is soy. However, its high protein content is unfortunately accompanied by the presence of antinutritional factors, including Kunitz's trypsin inhibitor (KTI). Now there are few analytical methods available for its direct quantification, as the inhibitory activity against trypsin is generically measured, which however can be given by many other molecules and undergo numerous interferences. Therefore, in this work, a direct label-free liquid chromatography-mass spectrometry (LC-MS) method for the identification and quantification of trypsin Kunitz inhibitor KTI3 in soybean and derivative products has been developed. The method is based on the identification and quantification of a marker peptide, specific for the protein of interest. Quantification is achieved with an external calibration curve in the matrix, and the limit of detection and the limit of quantification of the method are 0.75 and 2.51 μg/g, respectively. The results of the LC-MS method were also compared with trypsin inhibition measured spectrophotometrically, highlighting the complementarity of these two different pieces of information.
Topics: Animals; Humans; Trypsin Inhibitor, Kunitz Soybean; Trypsin; Tandem Mass Spectrometry; Trypsin Inhibitors; Chromatography, Liquid
PubMed: 37220219
DOI: 10.1021/acs.jafc.3c01173 -
Acta Crystallographica. Section F,... Jun 2011A Kunitz-type trypsin inhibitor protein (CPTI) purified from chickpea seeds was estimated to have a molecular mass of 18 kDa on SDS-PAGE. The IC(50) value of CPTI was...
A Kunitz-type trypsin inhibitor protein (CPTI) purified from chickpea seeds was estimated to have a molecular mass of 18 kDa on SDS-PAGE. The IC(50) value of CPTI was determined to be 2.5 µg against trypsin. The inhibitory activity of CPTI is 114 TIU (trypsin inhibitory units) per milligram of protein, which is high compared with those of other known Kunitz-type trypsin inhibitors from legumes. CPTI crystallized in three different orthorhombic crystal forms: P2(1)2(1)2 form A, P2(1)2(1)2 form B and P2(1)2(1)2(1). The crystals of P2(1)2(1)2 form A, with unit-cell parameters a = 37.2, b = 41.2, c = 104.6 Å, diffracted to 2.0 Å resolution at the home source and to 1.4 Å on beamline BM14 at the ESRF. Data were also collected from crystals grown in the presence of iodine. The Matthews coefficient for these crystals was calculated to be 2.37 Å(3) Da(-1), corresponding to a solvent content of 42%. The other two crystal forms (P2(1)2(1)2 form B and P2(1)2(1)2(1)) diffracted comparatively poorly.
Topics: Cicer; Crystallization; Crystallography, X-Ray; Plant Proteins; Seeds; Trypsin Inhibitors
PubMed: 21636920
DOI: 10.1107/S1744309111015338 -
Acta Biochimica Et Biophysica Sinica May 2015A proteinaceous inhibitor against trypsin was isolated from the seeds of Artocarpus heterophyllus Lam. by successive ammonium sulfate precipitation, ion-exchange, and...
A proteinaceous inhibitor against trypsin was isolated from the seeds of Artocarpus heterophyllus Lam. by successive ammonium sulfate precipitation, ion-exchange, and gel-filtration chromatography. The trypsin inhibitor, named as AHLTI (A. heterophyllus Lam. trypsin inhibitor), consisted of a single polypeptide chain with a molecular weight of 28.5 kDa, which was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel-filtration chromatography. The N-terminal sequence of AHLTI was DEPPSELDAS, which showed no similarity to other known trypsin inhibitor sequence. AHLTI completely inhibited bovine trypsin at a molar ratio of 1:2 (AHLTI:trypsin) analyzed by native polyacrylamide gel electrophoresis, inhibition activity assay, and gel-filtration chromatography. Moreover, kinetic enzymatic studies were carried out to understand the inhibition mechanism of AHLTI against trypsin. Results showed that AHLTI was a competitive inhibitor with an equilibrium dissociation constant (Ki) of 3.7 × 10(-8) M. However, AHLTI showed weak inhibitory activity toward chymotrypsin and elastase. AHLTI was stable over a broad range of pH 4-8 and temperature 20-80°C. The reduction agent, dithiothreitol, had no obvious effect on AHLTI. The trypsin inhibition assays of AHLTI toward digestive enzymes from insect pest guts in vitro demonstrated that AHLTI was effective against enzymes from Locusta migratoria manilensis (Meyen). These results suggested that AHLTI might be a novel trypsin inhibitor from A. heterophyllus Lam. belonging to Kunitz family, and play an important role in protecting from insect pest.
Topics: Artocarpus; Electrophoresis, Polyacrylamide Gel; Seeds; Trypsin Inhibitors
PubMed: 25851516
DOI: 10.1093/abbs/gmv022