Circular Dichorism Research Articles

Fenugreek seed ethanolic extract inhibited formation of advanced glycation end products via scavenging reactive carbonyl intermediates

Senescence is a natural phenomenon of growing old. It accelerates under certain conditions like diabetes mellitus resulting in early decline of bodily functions, which can be avoided by many claimed functional foods. The present study aims to investigate the anti-aging ability of Fenugreek seeds (Trigonellafoenum-graecum); a common ingredient of Indo-Pak cuisines. Briefly, the Fenugreek seeds extract (FgSE) in concentrationsof0.1, 0.5 and 1 mg/ml inhibited the formation of Advanced Glycation End products (AGEs) and fructosamine adducts in Bovine serum albumin (BSA)/fructose model in vitro. The BSA conformational analysis via Circular Dichorism and Congo red assays showed that it preserves secondary structure of BSA in aforementioned model. Although mechanistic studies revealed insignificant lysine blocking ability of Fenugreek by OPA assay, however carbonyl entrapping was found to be 24%, 34% and 42% at 0.1, 0.5 and 1 mg/ml, respectively. In vivo model of High Fructose diet (HFD) induced glycation, FgSE treatment in doses of 10, 25 & 50 mg/kg markedly improved Escape latency (p < 0.01) and preserved cognition in Morris Water Maze. Our data further exhibits significant decrease of CML (Nε-carboxymethyl lysine) levels in serum and hippocampus byFgSE treatment in comparison with HFD group. Therefore, we deduced that FgSE prevents glycation-induced memory decline via entrapping the reactive carbonyl intermediates, formed during production of AGEs. Hence, as a promising functional food it slows down the harmful process of glycation and aging associated morbidities.

Pseudouridimycin-A Potent Nucleoside Inhibitor of the RNA Polymerase Beta Prime Subunit of Streptococcus pyogenes.

Streptococcus pyogenes (group A streptococcus, GAS), a Gram-positive bacterium, is a major cause of mild to severe life-threatening infections. Antibacterial resistance to penicillin and macrolides poses a major threat in the treatment of GAS and necessitates alternate drugs and newer antibiotics. In this direction, nucleotide-analog inhibitors (NIAs) have emerged as important antiviral, antibacterial, and antifungal agents. Pseudouridimycin (PUM), a nucleoside analogue inhibitor discovered from the soil bacterium Streptomyces sp., has proven to be effective against multidrug-resistant S. pyogenes. However, the mechanism of its activity remains elusive. In this study, subunits of the RNA polymerase of GAS have been identified as targets for PUM inhibition and the binding regions have been mapped to the N-terminal domain of the β' subunit, using computational methods. The antibacterial activity of PUM against macrolide-resistant GAS was evaluated. PUM showed effective inhibition at 0.1-1 μg/mL concentration, which was higher when compared to earlier reports. The molecular interaction between PUM and the RNA polymerase β'-N terminal subunit was investigated using isothermal titration calorimetry (ITC), circular dichorism (CD), and intrinsic fluorescence spectroscopy. The thermodynamic characterization by ITC showed an affinity constant of 6.175 × 105 M-1 denoting a moderate affinity. Fluorescence studies revealed that the interaction of protein-PUM was spontaneous in nature and follows a static quenching of tyrosine signals from the protein. The near- and far-UV CD spectral analysis concluded that PUM induced local tertiary structural changes in the protein, predominantly contributed by aromatic amino acids rather than notable changes in the secondary structure. Hence PUM could be a promising lead drug target for macrolide-resistant strains of S. pyogenes and enable eradication of pathogen in the host system.

Bidirectional Origami Inspiring Versatile 3D Metasurface (Adv. Mater. Technol. 8/2022)

Bidirectional Origami In article number 2200373, Changzhi Gu, Junjie Li, and co-workers demonstrate bidirectional origami based on focused ion beam (FIB)-induced stress for various of materials, and the Monte Carlo simulations show that the key factor of bidirectional origami relies on the sputtering ratio for materials. Furthermore, a 3D chiral metasurface composed of bidirectional folded split ring resonators is designed and fabricated to realize a giant circular dichorism of 0.78/0.85 (Experiment/Simulation).

Bidirectional Origami Inspiring Versatile 3D Metasurface

AbstractMicro/nano origami is a fascinating 3D fabrication technology, showing a strong ability to control structural space degrees of freedom, but which are usually only able to achieve single‐direction origami and hence its controlling spatial orientation is still limited to a certain extent. Here, the bidirectional origami induced by focused ion beam irradiation is proposed to break through the freedom of structural space control and realize challenging 3D micro/nanofabrication. It is found that the FIB‐induced bidirectional deformation mainly relies on both materials and the ion doses, and the deformation degrees can be tuned by ion irradiated doses, which greatly contributes to construct large numbers of diverse 3D structures. Further, the underlying physics of FIB induced origami are discussed by Monte Carlo simulations along with experiments to reveal that the amounts of atoms sputtering determines the initial direction of deformation. This bidirectional origami exhibits unique capabilities in design and fabrication of versatile 3D metasurface devices. With this strategy, a 3D chiral metasurface composed of an array of bidirectional folded split ring resonators is achieved, showing a giant circular dichorism as high as 0.78/0.85 (Experiment/Simulation) in the mid‐infrared region. Such powerful bidirectional origami paves high efficiency approach to broaden 3D micro/nano photonics device.

Luminescence studies of binding affinity of vildagliptin with bovine serum albumin

Vildagliptin (VDG)is a frontier drug for diabetes mellitus. It is prescribed both in the monotherapy as well as in an amalgamation with other antidiabetic drugs. Drug-serum protein binding is an essential parameter which influences ADME properties of the drug. In current study, binding of VDG with serum protein (bovine serum albumin: BSA) was investigated using multi-spectroscopic techniques. A computational approach was also employed to identify the binding affinity of VDG with BSA at both Sudlow I and II sites. An enzyme activity assay specific for esterase was also investigated to know the post-binding consequences of VDG with BSA. Fluorescence spectra of BSA samples treated with VDG shows static quenching with binding parameters for VDG-BSA complex show single class of equivalent binding stoichiometry(n = 1.331) and binding constant 1.1 x 104M−1 at 298.15 K. The binding constant indicates important role of non-polar interactions in the binding process. Fluorescence resonance energy transfer (FRET) analysis of VDG absorption spectra and emission spectrum of BSA confirmed no significant resonance in energy transfer. Synchronous fluorescence of BSA after binding with VDG show maximum changes in emission intensity at tryptophan (Trp) residues. Post binding with VDG, BSA conformation changes as suggested by circular dichorism (CD) spectra of BSA and this lead to enhanced protein stability as indicated by a thermal melting curve of BSA. Communicated by Ramaswamy H. Sarma

Formulation and optimization of silymarin-encapsulated binary micelles for enhanced amyloid disaggregation activity

Silymarin (SLY) is a natural hydrophobic polyphenol that possesses antioxidant and amyloid fibril (Aβ1–42) inhibition activity, but its activity is hindered due to low aqueous solubility. In this study, SLY is encapsulated in binary micelle (SLY-BM) that has been utilized to enhance the Aβ1–42 fibril disaggregation. To enhance the aqueous solubility, SLY payload in micelles were optimized using Box–Behnken Design (BBD) to increase the efficiency of Aβ1–42 fibril disaggregation. BBD was used to investigate the effect of ratio of Solutol HS15:Poloxamer-188, amount of acetone and hydration volume on critical quality attributes, particle size, and entrapment efficiency for SLY-BM. Furthermore, SLY-BM was characterized for its physical and drug release properties. The Aβ1–42 fibril disaggregation and antioxidant studies were monitored using spectroscopic and microscopic techniques. BBD optimized the particle size <50 nm with %EE > 80%, and solubility factor of SLY-BM was enhanced to 460 folds than free SLY. Inhibitory concentration 50% (IC50) value of SLY-BM was 19.67 µg/mL compared to free SLY (30.06 µg/mL) in diphenylpicrahydrazyl assay. SLY-BM increased the Aβ1–42 disaggregation compared to free SLY observed via thioflavin-T assay, photon correlation spectroscopy, and circular dichorism. Further morphological evaluation of Aβ1–42 disaggregation was monitored by microscopy which showed that SLY-BM disaggregated the fibrils in 48 h. According to our findings, we concluded that SLY-BM micelles are potential candidates for delivery of neuroprotective agents.

Hydroxy-Porphyrin as an Effective, Endogenous Molecular Clamp during Early Stages of Amyloid Fibrillization.

Amyloid fibril formation of proteins is of great concern in neurodegenerative disease and can be detrimental to the storage and stability of biologics. Recent evidence suggests that insulin fibril formation reduces the efficacy of type II diabetes management and may lead to several complications. To develop anti-amyloidogenic compounds of endogenous origin, we have utilized the hydrogen bond anchoring, π stacking ability of porphyrin, and investigated its role on the inhibition of insulin amyloid formation. We report that hydroxylation and metal removal from the heme moiety yields an excellent inhibitor of insulin fibril formation. Thioflavin T, tyrosine fluorescence, Circular Dichorism (CD) spectroscopy, Field emission scanning electron microscopy (FESEM) and molecular dynamics (MD) simulation studies suggest that hematoporphyrin (HP) having hydrogen bonding ability on both sides is a superior inhibitor compared to hemin and protoporphyrin (PP). Experiments with hen egg white lysozyme (HEWL) amyloid fibril formation also validated the efficacy of endogenous porphyrin based small molecules. Our results will help to decipher a general therapeutic strategy to counter amyloidogenesis.

OPTICAL NANOFIBER AS A SMART DETECTOR OF MYCOTOXINS IN BLOOD

The objective of this study is to use the optical fiber, an emerging device due to their biocompatibility, photo-sensitivity, as a biosensor which can detect the mycotoxin produced by the Aspergillus sp. found in food and feed product of animals. First, we have treated the Red Blood Corpuscles (RBCs) of human blood with different concentration of Aflatoxin B1 and Aflatoxin G1 (AflB1 and AflG1respectively) and after certain time slot, we have studied the changes of the RBC and its protein by Circular Dichorism spectroscopy (CD)and, Fluorescent Spectroscopy. From these Spectroscopic data, it has been found that there was a change in the structure of the Haemoglobin (Hb), supported by theReactive Oxygen Species (ROS), which gives the apoptotic dimension of the RBC. These experiments followed by the preparation of the nanoprobe by etching optical nanofibre with Hydrofluoric Acid (HF) upto certain extent. The prepared nanoprobe was then used to detect theAflB1and AflG1treated RBC.The outcome of the experiments it has been found that the optical nanofiber can detect a small variation of pH of the solution.

Biopolymer nanoparticle surface chemistry dictates the nature and extent of protein hard corona

Biopolymer nanoparticles functionalized with targeting ligands serve as carriers for a wide range of therapeutic payloads to desired sites of the body. Upon introduction into the blood stream, the nanoparticles tend to bind with the plasma proteins in its immediate vicinity, forming a protein corona. In this work, we have investigated the effects of differently functionalized PLGA nanoparticles on the formation of protein corona. It was observed that nanoparticles of uniform size range adsorbed protein in varying amounts and the final hard corona differed structurally with respect to nanoparticle surface functionalization. A combination of fluorescence spectroscopy and FTIR studies revealed that the association of plasma proteins with PLGA nanoparticles during corona formation was intricately guided by the nanoparticle surface chemistry. Densitometric analysis through one dimensional gel electrophoresis further showed that the plasma proteins present in the hard corona were different for each case of surface functionalization. The aggregation of plasma protein during corona formation as studied through Thioflavin T fluorescence and circular dichorism spectroscopy was also found to significantly differ with each nanoparticle type. It is evident that surface chemistry of biopolymeric nanoparticles defines the final corona form in the physiological environment, and this study on detailed understanding of nanoparticle-protein corona would help to develop nanomedicines for clinical applications.

Critical Assessment of the Interaction between DNA and Choline Amino Acid Ionic Liquids: Evidences of Multimodal Binding and Stability Enhancement.

Long-term storage and stability of DNA is of paramount importance in biomedical applications. Ever since the emergence of ionic liquids (ILs) as alternate green solvents to aqueous and organic solvents, their exploration for the extraction and application of DNA need conscientious understanding of the binding characteristics and molecular interactions between IL and DNA. Choline amino acid ILs (CAAILs) in this regard seem to be promising due to their non-cytotoxic, completely biobased and environment-friendly nature. To unravel the key factors for the strength and binding mechanism of CAAILs with DNA, various spectroscopic techniques, molecular docking, and molecular dynamics simulations were employed in this work. UV–Vis spectra indicate multimodal binding of CAAILs with DNA, whereas dye displacement studies through fluorescence emission confirm the intrusion of IL molecules into the minor groove of DNA. Circular dichorism spectra show that DNA retains its native B-conformation in CAAILs. Both isothermal titration calorimetry and molecular docking studies provide an estimate of the binding affinity of DNA with CAAILs ≈ 4 kcal/mol. The heterogeneity in binding modes of CAAIL-DNA system with evolution of time was established by molecular dynamics simulations. Choline cation while approaching DNA first binds at surface through electrostatic interactions, whereas a stronger binding at minor groove occurs via van der Waals and hydrophobic interactions irrespective of anions considered in this study. We hope this result can encourage and guide the researchers in designing new bio-ILs for biomolecular studies in future.

Euphosantianane A⁻D: Antiproliferative Premyrsinane Diterpenoids from the Endemic Egyptian Plant Euphorbia Sanctae-Catharinae.

Euphorbia species are rich in diterpenes. A solvent extraction of Euphorbia sanctae-catharinae, a species indigenous to the Southern Sinai of Egypt, afforded several premyrsinane diterpenoids (1–4) as well as previously reported metabolites (5–13) that included three flavonoids. Isolated compounds were chemically characterized by spectroscopic analysis. Identified compounds were bioassayed for anti-proliferative activity in vitro against colon (Caco-2) and lung (A549) tumor cell lines. Compound 9 exhibited robust anti-proliferative activity against A549 cells (IC50 = 3.3 µM). Absolute configurations for 8 versus 9 were determined by experimental and TDDFT-calculated electronic circular dichorism (ECD) spectra.

A new 4-arylflavan from the pericarps of Horsfieldia motleyi displaying dual inhibition against α-glucosidase and free radicals

In search for effective antidiabetic agents that simultaneously inhibit α-glucosidase and scavenge free radicals, Horsfieldia motleyi showed promising bioactivity according to the proposed criteria. Bioassay-guided isolation of pericarp extract yielded a new 4-arylflavan named myristinin G (6), whose gross structure and absolute configuration were verified by 2D NMR and electronic circular dichorism (ECD). Myristinin G (6) concomitantly inhibited α-glucosidases (IC50 107.0 and 126.9 μM) and free radicals (SC50 54.3 and 279.9 μM). Of interest, 6 inhibited sucrase through an uncompetitive manner, which is rare in nature.

Hypochlorous acid induced structural and conformational modifications in human DNA: A multi-spectroscopic study

Hypochlorous acid (HOCl) is generated by activated phagocytes at the site of inflammation. Exposure of DNA to HOCl results in base and nucleotide modifications causing DNA damage, which is one of the leading causes of various pathological conditions including carcinogenesis. In the present work, various biophysical techniques were used to study HOCl induced structural and conformational changes in human placental DNA. The HOCl modified DNA showed hyperchromicity, reduced fluorescence and decrease in melting temperature. Circular dichorism (CD) and Fourier transform infra-red (FT-IR) studies exhibited conformational changes and shift in band positions of DNA, respectively, suggesting structural changes. Agarose gel electrophoresis and scanning electron microscopy showed strand breakage and decreased aggregation. These results suggest that HOCl causes conformational and structural perturbations in mammalian DNA, which may consequentially lead to DNA mutations resulting in perturbation of epigenetic signals leading to cancer and autoimmune diseases.

Unraveling the thermodynamics, binding mechanism and conformational changes of HSA with chromolyn sodium: Multispecroscopy, isothermal titration calorimetry and molecular docking studies

Cromolyn sodium is an anti-allergic drug effective for treatment in asthma and allergic rhinitis. In this project, interaction of chromolyn sodium (CS) with human serum albumin (HSA) has been investigated by various techniques such as UV-vis, fluorescence, circular dichorism (CD), fourier transform infrared (FT-IR) spectroscopy, isothermal titration calorimetric (ITC) and molecular docking. The fluorescence quenching results revealed that there was static quenching mechanism in the interactions of CS with HSA. The binding constant (Kb), enthalpy change (ΔH°), entropy change (ΔS°) and Gibbs free energy change (ΔG°) were calculated. The negative values of TΔS° and ΔH° obtained from fluorescence spectroscopy and isothermal titration calorimetry, indicate that hydrogen bonding and van der Waal’s forces played major role in the binding process and the reaction is exothermic in nature. The binding constant (Kb) was found to be in the order of 104M−1 which depicts a good binding affinity of CS towards HSA. The conformational changes in the HSA due to interaction of CS were investigated from CD and FT-IR spectroscopy. The binding site of CS in HSA was sub-domain IIA as evident from site probing experiment and molecular docking studies.

Exploring thermodynamic parameters and the binding energetic of berberine chloride to bovine serum albumin (BSA): Spectroscopy, isothermal titration calorimetry and molecular docking techniques

The present study reveals a mechanistic approach of binding mode of berberine chloride with bovine serum albumin (BSA) using isothermal titration calorimetry (ITC), fluorescence quenching, circular dichorism (CD), UV–vis absorption spectroscopy and molecular docking techniqes. Thermodynamic profile clearly demonstrated that the binding was endothermic and spontaneous process. The major binding force involved in complexation of berberine chloride to BSA was hydrophobic interactions. Static quenching is the foremost fluorescence quenching mechanism between BSA and berberine chloride and there is one binding site in BSA for berberine chloride. Molecular docking and competitive binding probes denoted that the binding of berberine chloride to BSA primarily took place in site I i.e. sub-domain IIA. Additionally, the alteration of the BSA secondary structure in the presence of berberine chloride were confirmed by UV–vis, circular dichroism spectral analysis. All these results demonstrated that berberine chloride can bind to BSA.

Characterization of the Wild-Type and Truncated Forms of a Neutral GH10 Xylanase from Coprinus cinereus: Roles of C-Terminal Basic Amino Acid-Rich Extension in Its SDS Resistance, Thermostability, and Activity.

A neutral xylanase (CcXyn) was identified from Coprinus cinereus. It has a single GH10 catalytic domain with a basic amino acid-rich extension (PVRRK) at the C-terminus. In this study, the wild-type (CcXyn) and C-terminus-truncated xylanase (CcXyn-Δ5C) were heterologously expressed in Pichia pastoris and their characteristics were comparatively analyzed with aims to examine the effect of this extension on the enzyme function. The circular dichorism analysis indicated that both enzymes in general had a similar structure, but CcXyn-Δ5C contained less α-helices (42.9%) and more random coil contents (35.5%) than CcXyn (47.0% and 32.8%, respectively). Both enzymes had the same pH (7.0) and temperature (45°C) optima, and similar substrate specificity on different xylans. They all hydrolyzed beechwood xylan primarily to xylobiose and xylotriose. The amounts of xylobiose and xylotriose accounted for 91.5% and 92.2% (w/w) of total xylooligosaccharides (XOS) generated from beechwood by CcXyn and CcXyn-Δ5C, respectively. However, truncation of the C-terminal 5-amino-acids extension significantly improved the thermostability, SDS resistance, and pH stability at pH 6.0-9.0. Furthermore, CcXyn-Δ5C exhibited a much lower Km value than CcXyn (0.27 mg/ml vs 0.83 mg/ml), and therefore, the catalytic efficiency of CcXyn-Δ5C was 2.4-times higher than that of CcXyn. These properties make CcXyn-Δ5C a good model for the structure-function study of (α/β)8-barrel-folded enzymes and a promising candidate for various applications, especially in the detergent industry and XOS production.

Iridoid and phenylpropanoid glycosides from the roots of Lantana montevidensis

A new iridoid glycoside; 6-O-β-D-xylopyranoside-shanzhiside methyl ester (1) along with six known compounds; shanzhiside methyl ester (2), lamalbid (3), geniposidic acid (4), theveside (5), verbascoside (6) and arenarioside (7) were isolated from the roots of Lantana montevidensis. The structures of the compounds were determined through 1D and 2D NMR spectroscopic data analysis, HRESIMS, electronic circular dichorism and UPLC-UV/MS method. The total extract, chloroformic (F1) and aqueous (F2) fractions together with the isolated compounds were tested for their antimicrobial, antiprotozoal, antiplasmodial, anti-inflammatory, monoamine oxidase inhibition and cell viability activities in addition to free radical scavenging activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The phenylpropanoid compounds (6 and 7) resulted in a potent antioxidant activity. Total methanolic extract together with the aqueous fraction (F2) showed decrease in reactive oxidative stress with 57 and 66%, respectively, while the chloroformic fraction (F1), together with the total methanolic extract, showed a decrease in iNOS with IC50 values 5 and 30 μg/mL, respectively. Compounds 1, 2, 3, 6, and 7 showed inhibition in the reactive oxidative stress with values 50, 60, 57, 63, and 52%, respectively. Both F1 and F2 fractions demonstrated measurable inhibition of MCF-7 breast cancer cell growth, with IC50 value 0.3 mg/mL. Compounds 2 and 7 showed mild monoamine oxidase inhibition. None of the tested compounds showed antimicrobial, antiplasmodial or antiprotozoal activity.

Copper(II), cobalt(II) and nickel(II) complexes of lapachol: synthesis, DNA interaction, and cytotoxicity

Three novel copper(II), cobalt(II), and nickel(II) complexes of lapachol (Lap) containing 110-phenanthroline (phen) ligand, [M(Lap)2(phen)] (M=Cu(II), 1, Co(II), 2, and Ni(II), 3), have been synthesized and characterized using, elemental analysis and spectroscopic studies. Their interactions with calf thymus DNA (CT DNA) were investigated using viscosity, thermal denaturation, circular dichorism, fluorescence quenching, and electronic absorption spectroscopy. The DNA cleavage abilities of 1–3 have been studied, where cleavage activity of copper complex 1 is more than the complexes 2 and 3. The in vitro cytotoxic potential of the complexes 1–3 against human cervical carcinoma (HeLa), human liver hepatocellular carcinoma (HepG-2), and human colorectal adenocarcinoma (HT-29) cells indicated their promising antitumor activity with quite low IC50 values in the range of .15–2.41 μM, which are lower than those of cisplatin.

Insights into amyloid-like aggregation of H2 region of the C-terminal domain of nucleophosmin

Nucleophosmin (NPM1) is a multifunctional protein involved in a variety of biological processes including the pathogenesis of several human malignancies and is the most frequently mutated gene in Acute Myeloid Leukemia (AML). To deepen the role of protein regions in its biological activities, lately we reported on the structural behavior of dissected C-terminal domain (CTD) helical fragments. Unexpectedly the H2 (residues 264–277) and H3 AML-mutated regions showed a remarkable tendency to form amyloid-like assemblies with fibrillar morphology and β-sheet structure that resulted as toxic when exposed to human neuroblastoma cells. More recently NPM1 was found to be highly expressed and toxic in neurons of mouse models of Huntington's disease (HD). Here we investigate the role of each residue in the β-strand aggregation process of H2 region of NPM1 by performing a systematic alanine scan of its sequence and structural and kinetic analyses of aggregation of derived peptides by means of Circular Dichorism (CD) and Thioflavin T (Th-T) assay. These solution state investigations pointed out the crucial role exerted by the basic amyloidogenic stretch of H2 (264–271) and to shed light on the initial and main interactions involved in fibril formation we performed studies on fibrils deriving from the related Ala peptides through the analysis of fibrils with birefringence of polarized optical microscopy and wide-angle X-ray scattering (WAXS). This analysis suggested that the presence of branched Ile269 conferred preferential packing patterns that, instead, appeared geometrically hampered by the aromatic side-chain of Phe268. Present investigations could be useful to deepen the knowledge of AML molecular mechanisms and the role of cytoplasmatic aggregates of NPM1c+.

Allium sativum Protease Inhibitor: A Novel Kunitz Trypsin Inhibitor from Garlic Is a New Comrade of the Serpin Family.

PurposeThis study was aimed to purify and characterize the Protease inhibitor (PI) from a plant Allium sativum (garlic) with strong medicinal properties and to explore its phytodrug potentials.MethodsAllium sativum Protease Inhibitor (ASPI) was purified using ammonium sulphate fractionation and Fast Protein Liquid Chromatography on anion exchanger Hi-Trap DEAE column. The purified protein was analyzed for its purity and molecular weight by SDS PAGE. The confirmation of presence of trypsin inhibiting PI was performed by MALDI TOF-TOF and analyzed by MASCOT database. The ASPI was further investigated for its kinetic properties and stability under extreme conditions of pH, temperature and chemical denaturants. Secondary structure was determined by Circular Dichorism (CD) spectroscopy.ResultsASPI of ~15 kDa inhibited trypsin and matched "truncated kunitz Trypsin Inhibitor (Glycine max)" in MASCOT database. The purified ASPI showed 30376.1371 U/mg specific activity with a fold purity of 159.92 and yield ~93%. ASPI was quite stable in the range of pH 2–12 showing a decline in the activity around pH 4–5 suggesting that the pI value of the protein as ASPI aggregates in this range. ASPI showed stability to a broad range of temperature (10–80°C) but declined beyond 80°C. Further, detergents, oxidizing agents and reducing agents demonstrated change in ASPI activity under varying concentrations. The kinetic analysis revealed sigmoidal relationship of velocity with substrate concentration with Vmax 240.8 (μM/min) and Km value of 0.12 μM. ASPI showed uncompetitive inhibition with a Ki of 0.08±0.01 nM). The Far UV CD depicted 2.0% α -helices and 51% β -sheets at native pH.ConclusionsTo conclude, purified ~15 kDa ASPI exhibited fair stability in wide range of pH and temperature Overall, there was an increase in purification fold with remarkable yield. Chemical modification studies suggested the presence of lysine and tryptophan residues as lead amino acids present in the reactive sites. Therefore, ASPI with trypsin inhibitory property has the potential to be used as a non-cytotoxic clinical agents.