Circular Dichroism Studies Research Articles

Naphthodithiophene Diimide Based Chiral π‐Conjugated Nanopillar Molecules

AbstractThe synthesis, structures, and properties of [4]cyclonaphthodithiophene diimides ([4]C‐NDTIs) are described. NDTIs as important n‐type building blocks were catenated in the α‐positions of thiophene rings via an unusual electrochemical‐oxidation‐promoted macrocyclization route. The thiophene–thiophene junction in [4]C‐NDTIs results in an ideal pillar shape. This interesting topology, along with appealing electronic and optical properties inherited from the NDTI units, endows the [4]C‐NDTIs with both near‐infrared (NIR) light absorptions, strong excitonic coupling, and tight encapsulation of C60. Stable orientations of the NDTI units in the nanopillars lead to stable inherent chirality, which enables detailed circular dichroism studies on the impact of isomeric structures on π‐conjugation. Remarkably, the [4]C‐NDTIs maintain the strong π–π stacking abilities of NDTI units and thus adopt two‐dimensional (2D) lattice arrays at the molecular level. These nanopillar molecules have great potential to mimic natural photosynthetic systems for the development of multifunctional organic materials.

Naphthodithiophene Diimide Based Chiral π-Conjugated Nanopillar Molecules.

The synthesis, structures, and properties of [4]cyclonaphthodithiophene diimides ([4]C-NDTIs) are described. NDTIs as important n-type building blocks were catenated in the α-positions of thiophene rings via an unusual electrochemical-oxidation-promoted macrocyclization route. The thiophene-thiophene junction in [4]C-NDTIs results in an ideal pillar shape. This interesting topology, along with appealing electronic and optical properties inherited from the NDTI units, endows the [4]C-NDTIs with both near-infrared (NIR) light absorptions, strong excitonic coupling, and tight encapsulation of C60 . Stable orientations of the NDTI units in the nanopillars lead to stable inherent chirality, which enables detailed circular dichroism studies on the impact of isomeric structures on π-conjugation. Remarkably, the [4]C-NDTIs maintain the strong π-π stacking abilities of NDTI units and thus adopt two-dimensional (2D) lattice arrays at the molecular level. These nanopillar molecules have great potential to mimic natural photosynthetic systems for the development of multifunctional organic materials.

Protein binding and cytotoxic activities of monomeric and dimeric oxido-vanadium(V) salan complexes: Exploring the solution behavior of monoalkoxido-bound oxido-vanadium(V) complex

Three ONNO donor tetradentate diamino bis(phenolato) “salan” ligands, N, N′-dimethyl-N, N′-bis-(5-chloro-2-hydroxy-3-methyl-benzyl)-1,2-diaminoethane (H2L1), N, N′-dimethyl-N, N′-bis-(5-chloro-2-hydroxy-3-isopropyl-6-methyl-benzyl)-1,2-diamino-ethane (H2L2) and N, N′-bis-(5-chloro-2-hydroxy-3-isopropyl-6-methyl-benzyl)-1,2-diaminocyclohexane (H2L3) have been synthesized by following Mannich condensation reaction. Reaction of these ligands with their corresponding vanadium metal precursors gave one oxidomethoxidovanadium(V) [VVOL1(OCH3)] (1) and two monooxido-bridged divanadium (V, V) complexes [VVOL2–3]2(μ-O) (2–3). The complexes were characterized by IR, UV–vis, NMR and ESI mass spectrometry. Also, the structure of all the complexes (1–3) was confirmed by the Single-Crystal X-ray diffraction analysis, which revealed a distorted octahedral geometry around the metal centres. The solution behavior of the [VVOL1(OCH3)] (1) reveals the formation of two different types of V(V) species in solution, the structurally characterized compound 1 and its corresponding monooxido-bridged divanadium (V, V) complex [VVOL1]2(μ-O), which was further studied by IR, and NMR spectroscopy. The electrochemical behavior of all the complexes was evaluated through cyclic voltammetry. Interaction of the salan-V(V) complexes with human serum albumin (HSA) and bovine serum albumin (BSA) were analysed through fluorescence quenching, UV–vis absorption titration, synchronous fluorescence, circular dichroism studies, and förster resonance energy transfer (FRET). Finally, the in vitro cytotoxicity of the complexes was investigated against MCF-7 and HT-29 and NIH-3T3 cell lines. Cytotoxicity value of complexes in both MCF-7 and HT-29 follows the same trend that is 3 > 1 > 2 which is in line with protein binding affinity of the complexes.

Direct evidence to control the magnetization in Fe3O4 thin films by N2 ion implantation: a soft X-ray magnetic circular dichroism study

Direct evidence to control the magnetization in Fe3O4 thin films by N2 ion implantation: a soft X-ray magnetic circular dichroism study

Manipulating hydrophobicity of polyester nanofiber mats with egg albumin to enhance cell interactions

AbstractA hybrid of poly‐l‐lactic acid (PLA) and poly‐ε‐caprolactone (PCL) system was designed using hydrophilic generally regarded as safe (GRAS) protein, egg albumin (EA), and fabricated as nanofiber mats (NM) to facilitate improved cell interactions and functionality. Our studies include, preparation and analysis of physicochemical properties of NM. Surface morphology of NM was smooth with the diameter ranging from 250 to 400 nm. The contact angle of NM decreased from 80 to 45° with the increase in EA concentration. The rate and extent of swelling was increased 3‐folds with the addition of EA. Release studies of NM showed maximum amount of MTz was released with the increase in MTz concentration (>85%). The MTz interaction with EA and structure stability of EA was confirmed from fluorescence and circular dichroism studies. NM showed increase in inhibition of bacterial growth of Staphylococcus aureus and Escherichia coli with the increase in MTz concentration. Cell viability of the NM was >80% and also, the cell proliferation increased as EA content increased. NM hemolytic activity was less than 5% suggesting compatibility. Hence, results concluded that EA had regulated hydrophobicity, promoted cell interactions, and proliferation and therefore, NM is considered safe for tissue regeneration.

Role of Conformational Change and Glucose Binding Sites in the Enhanced Glucose Tolerance of Agrobacterium tumefaciens 5A GH1 β-Glucosidase Mutants.

β-Glucosidases are often inhibited by their reaction product glucose and a barrier to the efficient lignocellulosic biomass hydrolysis to glucose. We had previously reported the mutants, C174V, and H229S, with a nearly 2-fold increased glucose tolerance over the wild type (WT), H0HC94, encoded in Agrobacterium tumefaciens 5A (apparent Ki,Glc = 686 mM). We report our steady-state and time-resolved intrinsic fluorescence spectroscopy, circular dichroism, and isothermal titration calorimetry (ITC) studies to further understand increased glucose tolerance. Changes in the mutants' emission intensity and the differential change in quenching rate in the absence and presence of glucose reflect changes in protein conformation by glucose. Time-resolved lifetime and anisotropy measurements further indicated the microenvironment differences across solvent-exposed tryptophan residues and a higher hydrodynamic radius due to glucose binding, respectively. ITC measurements confirmed the increase of glucose binding sites in the mutants. The experiment results were supported by molecular dynamics simulations, which revealed significant variations in the glucose-protein hydrogen-bonding profiles. Protein structure network analysis of the simulated structures further indicates the mutants' conformation change than the WT. Computational studies also indicated additional glucose binding sites in mutants. Our results indicate the role of glucose binding in modulating the enzyme response to glucose.

Molecular aspects of the interaction of acid phosphatase with TiO2 nanoparticles: Kinetic and multispectroscopic studies

In this investigation, the structure and stability of acid phosphatase against TiO2 nanoparticles were investigated via enzyme activity assay, fluorescence spectroscopy, thermal stability, and circular dichroism spectroscopy techniques. The acid phosphatase activity showed that the TiO2 nanoparticles combination inhibited the enzyme activity in the form of a non-competitive inhibition. The value of dissociation constant (Ki) was computed to be 0.258 at 310K. By raising the nanoparticles concentration, the thermal stability was reduced from 342 to 338 K. Far-UV circular dichroism studies also illustrated that TiO2 nanoparticles could alter the secondary structure of acid phosphatase through an increase in the content of the α-helix structure (from 10.8% to 14.9%) and a decrease in the β-sheet (from 30.2% to 27.4%). The Stern-Volmer constant was declined from 6.4 ×104 to 5.5 ×104 M−1 with raising the temperature. It was also revealed that TiO2 nanoparticles decreased the intrinsic fluorescence of acid phosphatase via the static quenching mechanism. The binding process was spontaneous, and the thermodynamic parameters also demonstrated the van der Waals and hydrogen bond interactions between TiO2 nanoparticles and acid phosphatase. Therefore, TiO2 nanoparticles could influence acid phosphatase stability and activity.

Structural exploration of Y-domain reveals its essentiality in HEV pathogenesis

Hepatitis E virus (HEV) is a major causative agent of hepatitis E infections across the globe. Although the essentiality of HEV nonstructural polyprotein (pORF1) putative Y-domain (Yd) has been established in viral pathogenesis, its structural-functional role remains elusive. The current research discusses the novel exploration on Yd protein expression, purification, biophysical characterization and structure-based docking analysis. The codon optimized synthetic gene and optimized expression parameters i.e., 5 h induction with 0.25 mM IPTG at 37 °C, resulted in efficient production of Yd protein (~40 kDa) in E. coli BL21(DE3) cells. Majority of the recombinant Yd (rYd) protein expressed as inclusion bodies was solubilized in 0.5% N-lauroylsarcosine and purified using Ni-NTA chromatography. Circular dichroism (CD) and UV visible absorption spectroscopic studies on Yd revealed both secondary and tertiary structure stability in alkaline range (pH 8.0–10.0), suggesting correlation with its physiological activity. Thus, loss in structure at low pH perhaps play crucial role in cytoplasmic-membrane interaction. The biophysical data were in good agreement with insilico structural analyses, which suggested mixed α/β fold, non-random and basic nature of Yd protein. Furthermore, due to Yd protein essentiality in HEV replication and pathogenesis, it was considered as a template for docking and drug-likeness analyses. The 3D modeling of Yd protein and structure-based screening and drug-likeness of inhibitory compounds, including established antiviral drugs led to the identification of top nine promising candidates. Nonetheless, in vitro studies on the predicted interaction of Yd with intracellular-membrane towards establishing replication-complexes as well as validations of the proposed therapeutic agents are warranted.

Control of Peptide Amphiphile Supramolecular Nanostructures by Isosteric Replacements.

Supramolecular nanostructures with tunable properties can have applications in medicine, pharmacy, and biotechnology. In this work, we show that the self-assembly behavior of peptide amphiphiles (PAs) can be effectively tuned by replacing the carboxylic acids exposed to the aqueous media with isosteres, functionalities that share key physical or chemical properties with another chemical group. Transmission electron microscopy, atomic force microscopy, and small-angle X-ray scattering studies indicated that the nanostructure's morphologies are responsive to the ionization states of the side chains, which are related to their pKa values. Circular dichroism studies revealed the effect of the isosteres on the internal arrangement of the nanostructures. The interactions between diverse surfaces and the nanostructures and the effect of salt concentration and temperature were assessed to further understand the properties of these self-assembled systems. These results indicate that isosteric replacements allow the pH control of supramolecular morphology by manipulating the pKa of the charged groups located on the nanostructure's surface. Theoretical studies were performed to understand the morphological transitions that the nanostructures underwent in response to pH changes, suggesting that the transitions result from alterations in the Coulomb forces between PA molecules. This work provides a strategy for designing biomaterials that can maintain or change behaviors based on the pH differences found within cells and tissues.

Interaction between DNA, Albumin and Apo-Transferrin and Iridium(III) Complexes with Phosphines Derived from Fluoroquinolones as a Potent Anticancer Drug

A group of cytotoxic half-sandwich iridium(III) complexes with aminomethyl(diphenyl)phosphine derived from fluoroquinolone antibiotics exhibit the ability to (i) accumulate in the nucleus, (ii) induce apoptosis, (iii) activate caspase-3/7 activity, (iv) induce the changes in cell cycle leading to G2/M phase arrest, and (v) radicals generation. Herein, to elucidate the cytotoxic effects, we investigated the interaction of these complexes with DNA and serum proteins by gel electrophoresis, fluorescence spectroscopy, circular dichroism, and molecular docking studies. DNA binding experiments established that the complexes interact with DNA by moderate intercalation and predominance of minor groove binding without the capability to cause a double-strand cleavage. The molecular docking study confirmed two binding modes: minor groove binding and threading intercalation with the fluoroquinolone part of the molecule involved in pi stacking interactions and the Ir(III)-containing region positioned within the major or minor groove. Fluorescence spectroscopic data (HSA and apo-Tf titration), together with molecular docking, provided evidence that Ir(III) complexes can bind to the proteins in order to be transferred. All the compounds considered herein were found to bind to the tryptophan residues of HSA within site I (subdomain II A). Furthermore, Ir(III) complexes were found to dock within the apo-Tf binding site, including nearby tyrosine residues.

Magnetic anisotropic of thermally evaporated FeNi thin film: A soft X‐ray magnetic circular dichroism study

FeNi film was fabricated on Cu/MgO (001) substrates using thermal evaporation technique of a FeNi target to realize the formation of an L10‐FeNi phase. The magnetic anisotropy of FeNi film was systematically investigated by means of X‐ray absorption spectroscopy (XAS) and X‐ray magnetic circular dichroism (XMCD) techniques. Our L‐edge XAS and XMCD analysis is performed for 30‐nm FeNi film grown on Cu/MgO (001) substrate. The present experimental results of XAS and XMCD indicate that ~5% Fe atoms are oxidized. Our findings also showed that the orbital magnetic moment of Fe has a significant incident X‐ray angle dependence that exhibits a maximum value at the out‐of‐plane direction corresponding perpendicular to the plane. The origin of anisotropy is successfully explained by element‐specific XMCD technique. The enhancement in magnetic anisotropy energy (MAE) is due to orbital magnetic moments of Fe through spin–orbit interaction. The uniaxial anisotropy energy showed quantitatively good agreement with the volume MAE.

Unraveling the interaction of an opium poppy alkaloid noscapine ionic liquid with human hemoglobin: Biophysical and computational studies

Apart from a wide range of applications, nowadays, Ionic liquids (ILs) are showing their major importance in the field of pharmaceuticals for the development of effective therapeutics. Along with them, proteins are the major targets of a drug and a little alteration in their conformation can lead them to act differently. In the present study, a step has been taken towards the binding of a noscapine (Nos) based ionic liquid [Pip-Nos]OTf with human hemoglobin (Hb), as the blood plasma has a special role in the transport and targeting of a drug. Noscapine, is a phthalideisoquinoline alkaloid, derived from an opium poppy (Papaver Somniferum) having various biological properties. UV–Vis absorption spectroscopy, steady state fluorescence spectroscopy, circular dichroism (CD) and computational studies have been used to study the molecular interaction of [Pip-Nos]OTf with human hemoglobin. UV–Vis spectra showed an increase in absorption intensity with a considerable red (bathochromic) shift on continuous addition of [Pip-Nos]OTf. A static quenching was observed with the formation of ground state complex in fluorescence spectroscopy. CD analysis showed no significant changes in the secondary structure of Hb on the addition of [Pip-Nos]OTf. It was found to bind strongly with hemoglobin in its catalytic groove with a high molecular docking score of −265.47 kJ/mol. Besides, molecular dynamics simulation studies delineate the stable and prolonged binding of [Pip-Nos]OTf to hemoglobin with minimal deviations in deformations plot and covariance matrix statistics. These favorable outcomes of stable binding of [Pip-Nos]OTf with hemoglobin, also depicted certain common spectroscopic and in silico features to the previously reported interaction of this ionic liquid with BSA and conclusively, showed its promising candidature as cancer therapeutics.

Potential Foldamers Based on an ortho-Terphenyl Amino Acid.

We describe the synthesis and characterization of a new class of oligomers built from a terphenyl-based amino acid. These oligomeric amides are of interest because the adoption of specific conformations could potentially be driven by the coordinated formation of inter-residue hydrogen bonds and aromatic interactions. Although high-resolution structural data have proven inaccessible, circular dichroism and nuclear magnetic resonance studies suggest that the new oligomers fold concomitantly with discrete self-association in chloroform.

Purification and characterization of a novel acid-tolerant and heterodimeric β-glucosidase from pumpkin (Cucurbita moschata) seed

A novel β-glucosidase was purified from pumpkin (Cucurbitamoschata) seed by anion exchange chromatography and gel permeation chromatography, and its molecular mass was determined to be 42.8kDa by gel permeation chromatography. The heterodimeric structure consisting of two subunits, free from disulfide bonds, was determined by native-PAGE analysis followed by zymography. The enzyme was maximally active at pH 4.0 and 70°C, and Vmax, Km, and kcat values were 0.078 units mg-1 protein, 2.22mM, and 13.29min-1, respectively, employing p-nitrophenyl-β-d-glucopyranoside as the substrate. The high content of glycine determined by amino acid analysis implies that the enzyme possesses flexible conformations and interacts with cell membranes and walls in nature. Circular dichroism studies revealed that the high stability of the enzyme within the pH range of 2.0-10.0 is due to its reversible pH-responsive characteristics for α-helix-antiparallel β-sheet interconversion.

C-Term Faraday Rotation in Metallocene Containing Thin Films.

The Faraday effect is a magneto-optical (MO) phenomenon that causes the plane of linearly polarized light to rotate when passing through a medium subjected to a parallel magnetic field. Informed by the established quantum mechanical model developed by Buckingham and Stephens, we sought to identify molecules that would exhibit large MO responses. Magnetic circular dichroism studies of ferrocenium in the 1970s revealed its potential as an MO material; however, it has not been evaluated in the context of Faraday rotation and thin-film optical applications. Herein, we report near-infrared (NIR) Faraday rotation in thin films of decamethylferrocenium/poly(methyl methacrylate) composites with maximum Verdet constants of -3.45 × 104 deg T-1 m-1 at 810 nm (absorbance = 0.09) and -1.44 × 104 deg T-1 m-1 at 870 nm (absorbance = 0.01). These polymer-metallocene thin films deliver larger Verdet constants than commercially used NIR inorganic Faraday rotators and are facile and inexpensive to produce. The temperature dependence and distinct lineshape of the MO responses observed in decamethylferrocenium radical cations, decamethylmanganocene, and chromocene are in accordance with the quantum mechanical model. The observation of a strong C-term Faraday rotation in solid-state organometallic materials provides the groundwork for the development of high-performance metallocene-based Faraday rotators.

Spectroscopic and molecular docking studies for the binding and interaction aspects of curcumin-cysteine conjugate and rosmarinic acid with human telomeric G-quadruplex DNA

The binding and interaction aspects of potential anticancer ligands like: curcumin-cysteine (CC) and rosmarinic acid (RA) with human telomeric G-quadruplex DNA, a novel anticancer target, have been probed by spectroscopic and molecular docking approach. The circular dichroism study unravels the conformational switching from mixed hybrid to parallel structure for the short sequence of human telomeric G-quadruplex structure in the presence of both the ligands. Further a good correlation for binding affinity has been established from the emission and absorption binding spectrum analysis. Further our spectroscopic and molecular docking studies have suggested that the CC having better binding capability than RA to human telomeric G-quadruplex. The presence of L-cysteine moiety in CC ligand is responsible factor for its binding via both minor as well as major groove of human telomeric G-quadruplex DNA where-as RA binds only via minor groove of telomeric G-DNA.

Exploring the binding mechanism of β-resorcylic acid with calf thymus DNA: Insights from multi-spectroscopic, thermodynamic and bioinformatics approaches

β-resorcylic acid (BR) is a phytochemical which is widely used in the food industry as a flavouring agent and preservative. It has also been found to exhibit antibacterial action against several types of food-borne bacteria. DNA is the main molecular target for many small molecules of therapeutic importance. Hence, the interest is rapidly growing among the researchers to elucidate the interaction between small molecules and DNA. Thus, paving the way to design novel DNA-specific drugs. In this study, an attempt was made to examine the mechanism of binding of BR with calf thymus DNA (ctDNA) with the help of various experiments based on spectroscopy and in silico studies. The spectroscopic studies like UV absorption and fluorescence affirmed the complex formation between BR and ctDNA. The observed binding constant was in the order of 103 M−1 which is indicative of the groove binding mechanism. These findings were further verified by dye-displacement assay, potassium iodide quenching, urea denaturation assay, the study of the effect of ssDNA, circular dichroism and DNA thermal denaturing studies. Different temperature-based fluorescence and isothermal titration calorimetry (ITC) experiments were employed to evaluate thermodynamic parameters. The analysis of thermodynamic parameters supports the enthalpically driven, exothermic and spontaneous nature of the reaction between BR and ctDNA. The forces involved in the binding process were mainly found to be hydrogen bonding, van der Waals and hydrophobic interactions. The results obtained from the molecular docking and molecular dynamics (MD) simulation were consistent with the in vitro experiments, which support the groove binding mode of BR with ctDNA.

Conformational evaluation of fusidic acid derivatives by 750 MHz 1H NMR and VCD

Fusidic acid (1a) is a commercial antibiotic widely used in therapy for methicillin-resistant Staphylococcus aureus infections. Although the history of spectroscopic and conformational characteristics of 1a is long and old, the complete 1H NMR parameters set assignment remains incomplete due to the large signal superposition in most conventionally used NMR spectrometers. Thus, in the present study the 750 MHz 1H NMR spectra of the two methyl acetylfusidates 1b and 1c were completely assigned by means of total-line-shape-fitting calculations using the PERCH iterative spectra analysis. The task was facilitated taking advantage of the data for 3β-acetoxypregna-Δ5,16‑dien-20-one and cholesteryl benzoate, thereby allowing to obtain excellent correlations between experimental vicinal coupling constants with values calculated by the Altona program. It turned out that the vicinal coupling constant values were not compatible with the published boat conformation of B-ring. Thus, a vibrational circular dichroism study of 1b and 1c, using density functional theory calculated spectra to compare with experimental spectra, revealed that ring B in these trans-syn-trans 29-nor-protostane triterpenoids exists in equilibrium between two twisted boat conformers. This is very useful for future docking calculations to test the antibiotic in front of proteins to find further specific pharmacological applications.

Acid‐Mediated Dissociation of Haemophilus ducreyi Cytolethal Distending Toxin Subunits Upon Cell Entry

Haemophilus ducreyi is a Gram-negative bacterium responsible for the sexually transmitted disease chancroid. It releases a heterotrimeric cytolethal distending toxin (CDT) that contains a catalytic subunit (CdtB) which has a DNAse activity and two binding components (CdtA and CdtC) responsible for entry into the cell. Our hypothesis predicts that the endocytosed holotoxin disassembles through two steps, where CdtA is dissociated from CdtB/CdtC subunits in the late endosomes due to the low pH of this compartment. Afterward, CdtC is released in the endoplasmic reticulum. This allows CdtB to enter the nucleus, causing DNA damage and cell cycle arrest. To study the mechanism of CdtA dissociation, each Cdt subunit was expressed in bacteria transformed with Cdt expression plasmids. Subunit purification was confirmed using SDS-PAGE with Coomassie stain. Afterward, circular dichroism studies tested the structural stabilities of each Cdt subunit at extracellular pH (7.4), early endosome pH (6.3), and late endosome pH (5.2). These structural studies showed that CdtB and CdtC do not undergo significant secondary structure changes at acidic pH. Acid-induces changes were observed with the CdtA subunit, causing its unfolding, aggregation and precipitation at pH 5.2. Additional studies assembled the holotoxin from the individual subunits and introduced it to different pH levels to evaluate the association and dissociation of the subunits using size exclusion chromatography. The results showed that the active holotoxin disassembles at a pH of 5.2, suggesting that the toxin dissociates in the acidified endosomal compartments due to the unfolding of CdtA. Knowledge of the CDT dissociation mechanism could be used to find treatments for chancroid that would prevent toxin function by blocking its disassembly.

Characterization of a Bowman\u2013Birk type trypsin inhibitor purified from seeds of Solanum surattense

A Bowman–Birk type trypsin inhibitor protein (SSTI) from seeds of the medicinal plant Solanum surattense was isolated, purified and characterized. SSTI showed a single band on SDS-PAGE corresponding to 11.4 kDa molecular weight. It is a glycoprotein (2.8% glycosylation) that differentially interacted with trypsin and chymotrypsin in a concentration-dependent manner. Its peptide sequence is similar to other Bowman–Birk type protease inhibitors found in Glycine max and Phaseolus acutifolius. The inhibitory activity was stable over a wide range of pH (1–10) and temperatures (10–100° C). Far-UV Circular Dichroism (CD) studies showed that SSTI contains β sheets (~ 23%) and α helix (~ 6%) and demonstrated structural stability at wide pH and high temperature. The kinetic analysis revealed a noncompetitive (mixed) type nature of SSTI and low inhibitor constant (Ki) values (16.6 × 10−8 M) suggested strong inhibitory activity. Isothermal titration calorimetric analysis revealed its high affinity towards trypsin with dissociation constant (Kd) 2.28 µM.