Interaction between Heparin and Fibronectin: Using Quartz Crystal Microbalance with Dissipation, Immunochemistry and Isothermal Titration Calorimetry

The adsorption behavior of heparin and fibronectin was studied by quartz crystal microbalance with dissipation(QCM-D), and the interaction between heparin and fibronectin was evaluated using immunochemistry and isothermal titration calorimetry(ITC) measurement. The results showed that there was competitive adsorption between heparin and fibronectin, and the preadsorption of fibronectin could prevent subsequent heparin adsorption to some extent, and the adsorbed Hep/Fn complex on the surface was in a rigid form. The bioactivity of heparin and fibronectin could be affected by the bulk concentration of each, and both heparin and fibronectin in Hep/Fn complex formed under p H 4 condition displayed larger bioactivity than that formed under p H 7 condition. Moreover, the fibronectin showed more exposed cell-binding sites at the p H value lower than physiological condition. The results of ITC further suggested that the interaction between heparin and fibronectin under p H 4 was stronger than under p H 7, and the complex was also more stable. The study brings forth the detailed interaction between heparin and fibronectin, which will be helpful for better understanding the interaction mechanism of the two biomolecules. The results may be potentially useful for the development of new generation of cardiovascular biomaterials.

Thermodynamics of clayedrug complex dispersions: Isothermal titration calorimetry and high-performance liquid chromatography

An understanding of the thermodynamics of the complexation process utilized in sustaining drug release in clay matrices is of great importance.Several characterisation techniques as well as isothermal calorimetry were utilized in investigating the adsorption process of a model cationic drug(diltiazem hydrochloride,DIL)onto a pharmaceutical clay system(magnesium aluminium silicate,MAS).X-ray powder diffraction(XRPD),attenuated total reflectance Fourier transform infrared spectroscopy(ATRFTIR)and optical microscopy confirmed the successful formation of the DIL-MAS complexes.Drug quantification from the complexes demonstrated variable behaviour in the differing media used with DIL degrading to desacetyl diltiazem hydrochloride(DC-DIL)in the 2 M HCl media.Here also,the authors report for the first time two binding processes that occurred for DIL and MAS.A competitor binding model was thus proposed and the thermodynamics obtained suggested their binding processes to be enthalpy driven and entropically unfavourable.This information is of great importance for a formulator as care and consideration should be given with appropriate media selection as well as the nature of binding in complexes.

Exploring the taste presentation and receptor perception mechanism of salty peptides from Stropharia rugosoannulata based on molecular dynamics and thermodynamics simulation

The taste presentation and receptor perception mechanism of the salty peptide of Stropharia rugosoannulata were predicted and verified using peptide omics and molecular interaction techniques.The combination of aspartic acid(D)and glutamic acid(E),or peptide fragments composed of arginine(R),constitute the characteristic taste structural basis of salty peptides of S.rugosoannulata.The taste intensity of the salty peptide positively correlates with its concentration within a specific concentration range(0.25–1.0 mg/mL).The receptor more easily recognizes the first amino acid residue at the N-terminal of salty peptides and the aspartic acid residue in the peptides.GLU513,ASP707,and VAL508 are the critical amino acid residues for the receptor to recognize salty peptides.TRPV1 is specifically the receptor for recognizing salty peptides.Hydrogen bonds and electrostatic interactions are the main driving forces for the interactions between salty peptides and TRPV1 receptors.KSWDDFFTR has the most potent binding capacity with the receptor and has tremendous potential for application in sodium salt substitution.This study confirmed the taste receptor that specifically recognizes salty peptides,analyzed the receptor-peptide binding interaction,and provided a new idea for understanding the taste receptor perception of salty peptides.

Structural requirements and interaction mechanisms of ACE inhibitory peptides:molecular simulation and thermodynamics studies on LAPYK and its modifi ed peptides

The understanding of the structural requirements and the intermolecular-interaction mechanism are important for discovering potent angiotensin-converting enzyme(ACE)inhibitory peptides.In this study,we modifi ed an egg-white derived peptide,LAPYK,using the amino acids with different properties to produce the LAPYK-modified peptides.The ACE inhibitory activities of the modified peptides were determined to explore the structural requirements of ACE inhibitory peptides(ACEIPs).Molecular simulation and isothermal titration calorimetry analysis were used to investigate interactions between the peptides and ACE.We found that hydrophobicity and the amino acids with ring structures were benefi cial for the ACE inhibitory activities of the peptides.The results of the molecular mechanics poisson boltzmann surface area(MMPBSA)binding free energy calculations indicated that the polar solvation free energy(ΔG_(polar))of the charged peptides(LAPYK,LAPYE)were unfavorable for binding to ACE.On the other hand,the results of isothermal titration calorimetry analyses suggested that the enthalpy-driven ACE-peptide interactions were more favorable than the entropy-driven ACE-peptide interaction counterparts.

Thermodynamic study on the interaction between anti-tumor drug tegafur and human serum albumin

The changes of thermodynamic properties of the system on interaction between tegafur and human serum albumin （HSA） and the changes of secondary structure units of HSA in the system at 298.15 K have been investigated by the Nano-Watt-Scale isothermal titration calorimetry （ITC）, the Langmuirs binding model and the circular dichroism （CD） spectrometry.

The Streptococcus agalactiae Ribose Binding Protein B (RbsB) Mediates Quorum Sensing Signal Uptake via Interaction with Autoinducer-2 Signals

Understanding aquatic pathogen in sediments or aquacultural water is crucial to protect public health from soilborne and waterborne diseases.Quorum sensing(QS)was increasingly reported in biological wastewater treatment processes because of their inherent roles in biofilm development,bacterial aggregation and so on.The widely QS signals was Antoinducer-2(AI-2),primarily involved to allow the possibility of interspecies communication.However,the cellular components that mediate the response of Streptococcus agalactiae to AI-2 have not been fully characterized.Analysis of the complete genome sequence of S.agalactiae indi-cated that its RbsB protein has similarity to Escherichia coli LsrB and Aggregatibacter actinomycetemcomitans RbsB proteins that bind AI-2.We hypothesized that RbsB protein mediates quorum sensing signal uptake via interaction with AI-2.To evaluate the regulatory effect of RbsB on QS system,the recombinant plasmid pGEX-6p-1-RbsB was constructed and RbsB protein was purified with GST-tag.To further elucidate the role of RbsB protein binding to DPD(AI-2 precursor dihydroxypentanedione),the systemati-cally throughput circular dichroism(CD)spectroscopy,isothermal titration calorimetry200(ITC200)and molecular docking methods were employed.The high expression of soluble RbsB protein with molecular weight of 33 kDa was obtained.The thermodynamics results(ΔH<0,ΔS<0,ΔG<0)with ITC determination indicated that the binding process between DPD and RbsB was exothermic and spontaneous,with hydrogen bonds and van der Waals forces as the main binding forces.Obviously,DPD can be more easily combined with RbsB in a dose-dependent manner,suggesting that RbsB was changed in the microenvironment of DPD when the DPD concentration was between 0.8-1.0mmolL−1 and reaching the maximum binding amount.According to molecular docking,3 hydrophobic residues involved in DPD and RbsB protein stable binding were be found,and also hydrogen bonding plays a key role in the formation of the new complex.RbsB efficiently inhibited V.harveyi bioluminescence induced by both S.agalactiae AI-2 and V.harveyi AI-2 in a dose-dependent manner.However,our results suggest that RbsB may play a role in the response of S.agalactiace to AI-2.

Application of a simple calorimetric data analysis on the binding study of cyanide ions by Jack bean urease

Cyanide ion was studied as an effector of Jack bean urease(JBU) at 300 K in 30 mmol/LTris buffer,pH 7 by isothermal titration calorimetry(ITC).The simple novel model was used for CN^- + JBU interaction over the whole range of CN^- concentrations.The binding parameters recovered from the simple novel model were attributed to the cyanide ion interaction.It was found that cyanide ion acted as a noncooperative inhibitor of JBU,and there is a set of 12 identical and independent binding sites for CN^- ions.The di...

Thermodynamic study of CN^- ion inhibition of Jack bean urease using the extended solvation theory

Cyanide ion was studied as an inhibitor of Jack bean urease at 300 K in 30 mmol/L tris buffer, pH 7. The inhibition was investigated by isothermal titration calorimetry （ITC）. The extended solvation model was used for CN^- ＋ JBU interaction over the whole range of CN^- concentrations. The binding parameters recovered from the solvation model were attributed to the cyanide ion interaction. It was found that cyanide ion acted as a non-cooperative inhibitor ofurease, and there is a set of 12 ± 0.12 identical and independent binding sites for CN- ions. The dissociation equilibrium constant is 749.99 umol/L. The molar enthalpy of binding is AH= -13.60 kJ mol^-1.

Calorimetric Studies of the Synergistic Effect: The Reaction of RE(TTA)_3 with N503 in Toluene

Titration calorimetry has been used to study the thermodynamics of reaction in toluene solution of 2-thenoyltriflueroacetone(TTA)complexes of rare earths(RE=Y,Nd,Eu,Yb)with N,N dimethylheptyl-acetamide(N503)at 298.15 K.The heat of the reaction was determined with a TRONAC Model 1250 automatic isoperibol calorimeter.The data and figures of titration curve were recorded by the com- puter automatically.An iterative technique of least-square analysis was used to calculate the equilibrium con- stants and the thermodynamic functions including the entropy and free energy changes,minimizing the error square sum by a computer program.

A predictive,simple and rapid method for thermodynamic study on the binding of copper ion with Alzheimer's amyloid β peptide

The interaction of CuCl2 to the first 16 residues of the Alzheimer＇s amyliod β peptide, Aβ（1-16） was studied by isothermal titration calorimetry at pH 7.2 and 37℃ in aqueous solution.

Microcalorimetric Study of Acetylcholine and Acetylthiocholine Hydrolysis by Acetylcholinesterase

Acetylcholinesterase (AChE) is an important enzyme responsible for the cleavage of acetylcholine. Studies of the activity of this enzyme use an artificial substrate, acetylthiocholine, because a product of its catalysis, thiocholine, readily generates a light absorbing product upon reaction with Elman’s reagent 5,5’-dithiobis-(2-nitrobenzoic acid (DTNB). The hydrolysis of acetylcholine cannot be assayed with this method. The isothermal titration calorimetry can assay the hydrolysis of both substrates, without requiring additional reagents other than the enzyme and the substrate. To compare kinetic values obtained in the hydrolysis of acetylcholine (ACh) and acetylthiocholine (ATCh), with carbaryl acting as inhibitor, a calorimetric technique was used to evaluate kinetic properties of the two reactions. This method can show the hydrolysis of both substrates by the heat exchange that occurs during catalysis. In addition, it allowed the assessment of the AChE inhibition by carbaryl, a common insecticide. The results show a similarity between values obtained with both substrates, which are slightly higher for acetylcholine, the enzyme natural substrate. Enzymatic parameters values from ATCh and ACh were similar to each other and inhibitory constants using carbaryl were also similar, displaying that any approach to ACh is feasible using ATCh. The results obtained from ITC show the precision achieved by the calorimetric method.

Crystal structure of E. synthetase and ligand revealed key residues coil arglnyl-tRNA binding studies in arginine recognition

The arginyl-tRNA synthetase （ArgRS） catalyzes the esterification reaction between L-arginine and its cog- nate tRNAArg. Previously reported structures of ArgRS shed considerable light on the tRNA recognition mech- anism, while the aspect of amino acid binding in ArgRS remains largely unexplored. Here we report the first crystal structure of E. coli ArgRS （eArgRS） complexed with L-arginine, and a series of mutational studies using isothermal titration calorimetry （ITC）. Combined with previously reported work on ArgRS, our results eluci- dated the structural and functional roles of a series of important residues in the active site, which furthered our understanding of this unique enzyme.

A Calorimetric Study on the Interaction of Zinc and Cadmium Ions with Jack Bean Urease

A thermodynamic study on the interaction of Jack bean urease, JBU, with Zn2＋ and Cd2＋ ions was studied by isothermal titration calorimetry （ITC） at 290, 300 and 310 K in 30 mmol/L Tris buffer solution, pH 7.0. The heats of JBU＋Zn2＋ and JBU＋Cd2＋ interactions are reported and analyzed in terms of the extended solvation theory. It was indicated that there is a set of 12 identical and non-interacting binding sites for Zn2＋ and Cd2＋ ions. The interactions of Zn2＋ and Cd2＋ ions with JBU are exothermic and both enthalpy and entropy driven. The association equilibrium constants for JBU＋Zn2＋ complexes are 4118.20, 3354.70 and 2790.62 Lomol 1 at 290, 300 and 310 K respectively. The association equilibrium constants for JBU＋Cd2＋ interactions are 2831.6 and 2386.28 Lomol 1 at 300 and 310 K, respectively.