Isothermal titration calorimetry

Freire, Ernesto; Mayorga, Obdulio López; Straume, Martin

doi:10.1021/ac00217a002

Cited by 530 publications

(385 citation statements)

References 32 publications

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“…The fundamental principles of ITC are described elsewhere (22). All of the experiments were conducted under anaerobic conditions using 1 mM dithionite (Na 2 S 2 O 4 ) and an atmosphere of argon to prevent Fe 2+ oxidation from possible residual O 2 .…”

Section: Allmentioning

confidence: 99%

“…To fit the experimental data, it is necessary to estimate the heat associated with any injection in the calorimeter. To do that, the concentration of all species in equilibrium, free and bound, after each injection i, must be calculated from the total concentration of the macromolecule Solving eq 2 allows the calculation of the concentration of free ligand and, therefore, the concentration of ligand bound to the class j of binding sites Although an exact analytical solution is possible for other less complex binding equilibrium systems (22), in our case, which involves a quartic algebraic equation, a numerical solution is more convenient. To solve eq 2 numerically for any set of assumed values for n j and K j , the NewtonRaphson method was employed.…”

Section: Allmentioning

confidence: 99%

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Thermodynamic Analysis of Ferrous Ion Binding toEscherichia coliFerritin EcFtnA

et al. 2005

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Iron oxidation in the bacterial ferritin EcFtnA from Escherichia coli shows marked differences from its homologue human H-chain ferritin (HuHF). While the amino acid residues that constitute the dinuclear center in these proteins are highly conserved, EcFtnA has a third iron-binding site (C site) in close proximity to the dinuclear center that is seemingly responsible for these differences. Here, we describe the first thermodynamic study of Fe 2+ binding to EcFtnA and its variants to determine the location of the primary ferrous ion-binding sites on the protein and to better understand the role of the third C site in iron binding. Isothermal titration calorimetric analyses of the wild-type protein reveal the presence of two main classes of binding sites in the pH range of 6.5-7.5, ascribed to Fe 2+ binding, first at the A and then the B sites. Site-directed mutagenesis of ligands in the A, B, or C sites affects the apparent Fe 2+ -binding stoichiometries at the unaltered sites. The data imply some degree of inter-and intrasubunit negative cooperative interaction between sites. Unlike HuHF where only the A site initially binds Fe 2+ , both A and B sites in EcFtnA bind Fe 2+ , implying a role for the C site in influencing the binding of Fe 2+ at the B site of the di-iron center of EcFtnA. The ITC equations describing a binding model for three classes of independent binding sites are reported here for the first time.

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Section: Allmentioning

confidence: 99%

Section: Allmentioning

confidence: 99%

Thermodynamic Analysis of Ferrous Ion Binding toEscherichia coliFerritin EcFtnA

et al. 2005

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“…With the help of a non-linear curve fitting carried out by the Microcal Origin software, the binding thermodynamic parameters, K, n, and can be calculated from actual calorimetric data (Freire et al, 1990;Qu et al, 2002). The Gibbs free energy and entropy of the binding reaction can be obtained by Equations 1 and 2.…”

Section: Resultsmentioning

confidence: 99%

Interactions between rare-earth ions and DNA of Bashibai sheep

Aniwashi

Ha²,

Sulaiman³

et al. 2011

Genet. Mol. Res.

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ABSTRACT. The interaction between rare-earth ions and DNA from Bashibai sheep was studied by microcalorimetry and electrochemistry. The DNA chain was found to have four to five binding sites for rareearth ions. The binding affinity was about 10 -5 -10 -6 M. It was also found that smaller ions caused more heat to be released in the process of binding and bound more readily to the nucleic acid chain. This is attributed to the enhanced ability of polarization of smaller ions and reduced steric hindrance compared to larger ions. The electrochemistry results show that rare-earth ions could be inserted into the DNA helix, producing a new complex with electrochemically active groups. The rare-earth ions and DNA complex reached equilibrium after a 90-min incubation at room temperature.

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“…The only direct way to measure the heat change during complex formation at constant temperature is implemented by isothermal titration calorimetry (ITC) (Freire et al, 1990;Wiseman et al, 1989). In this method one binding partner is titrated into a solution containing the interacting partner, thereby generating or absorbing heat.…”

Section: Thermodynamics Of Protein-ligand Interactionmentioning

confidence: 99%