Decrease In Dissociation Rate Constant Research Articles

Affinity improvement by fine tuning of single-chain variable fragment against aflatoxin B1

Aflatoxin B1 (AFB1) produced in Aspergillus flavus is a major hepatocarcinogen found in foods and feed. For effective immunological detection of AFB1 at low concentrations, the development of high affinity antibody for AFB1 is required. Previously, an affinity-maturated single-chain variable fragment containing 6 mutations (scFv-M37) was isolated from an artificial mutagenic library, which showed a 9-fold higher affinity than its wild type scFv. In this study, the effect of the 6 mutated residues on the affinity improvement was characterized using surface plasmon resonance analysis, which identified a deleterious mutation (VH-A110T) located on a framework region of the scFv-M37. The back mutation of VH-A110T resulted in a 3.2-fold affinity improvement, which was attributed to decrease of dissociation rate constant (kd) in interaction between AFB1 and the back mutant scFv. The biophysical analyses using circular dichroism and gel filtration revealed that the back mutation of VH-A110T caused a subtle conformational change of the scFv toward tighter binding to AFB1.

The Mechanism by which TATA-Box Polymorphisms Associated with Human Hereditary Diseases Influence Interactions with the TATA-Binding Protein

SNPs in ТАТА boxes are the cause of monogenic diseases, contribute to a large number of complex diseases, and have implications for human sensitivity to external and internal environmental signals. The aim of this work was to explore the kinetic characteristics of the formation of human ТВР complexes with ТАТА boxes, in which the SNPs are associated with β-thalassemias of diverse severity, immunosuppression, neurological disorders, and so on. It has for the first time been demonstrated, using an electrophoretic mobility shift assay, that TBP interacts with SNP-containing ТАТА boxes with a significant (8-36-fold) decrease in TBP/ТАТА association rate constant (ka ) as compared with that in healthy people, a smaller decrease in dissociation rate constant (kd ) and changes in the half-lives of TBP/ТАТА complexes. Carriers of the -24G allele (rs 1800202T>G) in the TATA box of the triosephosphate isomerase gene promoter, associated with neurological and muscular disorders, were observed to have a 36-fold decrease in TBP/TATA association rate constant that are consistent with TPI deficiency shown for patients who carry this defective allele. The kinetic characteristics of TBP/ТАТА complexes obtained suggest that, at a molecular level, hereditary diseases are largely caused by changes in TBP/ТАТА association rates and these changes have a bearing on disease severity.

Enhanced thermal stability and mismatch discrimination of mutation-carrying DNA duplexes and their kinetic and thermodynamic properties in microchannel laminar flow

This article reports the enhancement of thermal stability involving normal duplex and mutation-carrying DNA duplexes in microchannel laminar flow. The application of an in-house temperature-controllable microchannel-type flow cell is demonstrated for improved discrimination of mismatch base pairs such as A–G and T–G that are difficult to distinguish due to the rather small thermal destabilizations. Enhancement in thermal stability is reflected by an increased thermal melting temperature achieved in microchannel laminar flow as compared with batch reactions. To examine the kinetics and thermodynamics of duplex–coil equilibrium of DNA oligomers, denaturation–renaturation hysteresis curves were measured. The influence of microchannel laminar flow on DNA base mismatch analysis was described from the kinetic and thermodynamic perspectives. An increasing trend was observed for association rate constant as flow rate increased. In contrast, an apparent decrease in dissociation rate constant was observed with increasing flow rate. The magnitudes of the activation energies of dissociation were nearly constant for both the batch and microchannel laminar flow systems at all flow rates. In contrast, the magnitudes of activation energies of association decreased as flow rate increased. These results clearly show how microchannel laminar flow induces change in reaction rate by effecting change in activation energy. We anticipate, therefore, that this approach based on microchannel laminar flow system holds great promise for improved mismatch discrimination in DNA analyses, particularly on single-base-pair mismatch, by pronouncedly enhancing thermal stability.

ChemInform Abstract: Kinetic Studies and Thermodynamic Results for Complex Formation and Solvation of Thallium(I) Cryptates in Acetonitrile and in Acetonitrile‐Water Mixtures.

Abstract Stability constants and dissociation rate constants of a range of thallium(I) cryptates in acetonitrile and of the cryptate Tl(2,2,2)+ in water-acetonitrile mixtures have been measured at 25°C. Solvation free energies of transfer for Tl+ from water to acetonitrile and to water-acetonitrile mixtures have been estimated from polarographic measurements using the ferrocene assumption. The results allow the calculation of transfer free energy data for the stable cryptate, TlCry+, and for the transition state (Tl+…Cry)≠. In mixtures of water and acetonitrile the stability constant of Tl(2,2,2)+ increases substantially with increasing acetonitrile content. This variation arises almost equally from an increase in the formation rate constant and a decrease in dissociation rate constant. Alternatively, the increase of stability constants for Tl(2,2,2)+ with increasing mole fraction of acetonitrile results from a strongly decreasing transfer free energy of the cryptate which surpasses in magnitude the increasing transfer free energies of the reactants.

Kinetic studies and thermodynamic results for complex formation and solvation of thallium(I) cryptates in acetonitrile and in acetonitrile-water mixtures

Stability constants and dissociation rate constants of a range of thallium(I) cryptates in acetonitrile and of the cryptate Tl(2,2,2) + in water-acetonitrile mixtures have been measured at 25°C. Solvation free energies of transfer for Tl + from water to acetonitrile and to water-acetonitrile mixtures have been estimated from polarographic measurements using the ferrocene assumption. The results allow the calculation of transfer free energy data for the stable cryptate, TlCry +, and for the transition state (Tl +…Cry) ≠. In mixtures of water and acetonitrile the stability constant of Tl(2,2,2) + increases substantially with increasing acetonitrile content. This variation arises almost equally from an increase in the formation rate constant and a decrease in dissociation rate constant. Alternatively, the increase of stability constants for Tl(2,2,2) + with increasing mole fraction of acetonitrile results from a strongly decreasing transfer free energy of the cryptate which surpasses in magnitude the increasing transfer free energies of the reactants.