A twin titration microcalorimeter for the study of biochemical reactions

McKinnon, Ian; Fall, Lana; Parody-Morreale, Antonio; Gill, Stanley J.

doi:10.1016/0003-2697(84)90398-1

Cited by 63 publications

(31 citation statements)

References 4 publications

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“…The characteristics of this instrument are similar to other calorimeters, such as those described by McKinnon et al (33) and Freire et al (34), but it is considerably less expensive to build. A full description of this calorimeter, together with a theoretical analysis of heat compensation technique in ITC, will also be published elsewhere.…”

Section: Methodssupporting

confidence: 55%

Thermodynamic Characterization of 5′-AMP Binding to Bovine Liver Glycogen Phosphorylase a

Garcı́a-Fuentes

Cámara-Artigas

López-Mayorga

et al. 1996

Journal of Biological Chemistry

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Section: Methodssupporting

confidence: 55%

Thermodynamic Characterization of 5′-AMP Binding to Bovine Liver Glycogen Phosphorylase a

Garcı́a-Fuentes

Cámara-Artigas

López-Mayorga

et al. 1996

Journal of Biological Chemistry

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“…Calorimetric measurements were performed using a high-precision twin titration isothermal microcalorimeter (16).…”

Section: Methodsmentioning

confidence: 99%

“…2 l) were added each time, and corrections were made for the heat effects due to stirring and dilution (16). Calibration experiments were carried out, employing HCl/NaOH titrations and electrical calibrations (16).…”

Section: Methodsmentioning

confidence: 99%

Thermodynamics of Inositol Hexakisphosphate Interaction with Human Oxyhemoglobin

Messana

Angeletti

Castagnola

et al. 1998

Journal of Biological Chemistry

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, which are located in the ␤ 1 ␤ 2 dyad axis, where they have been also proposed to interact with 2,3-diphosphoglycerate, whereas the third group does not appear easily identifiable. Calorimetric measurements of the heat associated with IHP binding at different pH values over the same range indicate that IHP binding is mostly enthalpy-driven at pH < 7 and mostly entropydriven at pH > 7.Human hemoglobin (Hb) 1 is functionally modulated by several non-heme ligands, such as organic phosphates (i.e. 2,3-diphosphoglycerate (2,3-DPG) and myo-inositol hexakisphosphate (IHP)), protons, and chloride ions (1-5), which bind at heterotropic interaction sites, topologically distinct from the heme at which homotropic ligands bind.The structure of this binding pocket has been determined for the interaction of 2,3-DPG (6), which has been shown to bind at the interface between the two ␤-chains, mainly involving three residues from either one of ␤-subunits (i.e. HisNA2(␤2), Lys-EF6(␤82), and HisH21(␤143), see Ref. 6).In more recent years, another organic phosphate, namely IHP (closely related to the inositol pentaphosphate, which is the physiological effector in avian erythrocytes; see Ref. 7), has often been employed to study the modulation of functional properties of human Hb (8, 9). It possesses additional negative charges with respect to 2,3-DPG, and it displays a much larger effect, which suggests the occurrence of additional electrostatic interactions with respect to 2,3-DPG, as from early model building studies on deoxy Hb (10). Therefore, the enhanced functional effect of IHP on the O 2 binding properties of human Hb with respect to 2,3-DPG could be related to a more widespread interaction surface, with the possibility of modulating ligand-linked conformational changes taking place over a larger portion of the whole tetramer.However, a comprehension of the origin for this enhanced effect starts from the characterization of the IHP interaction energy with deoxyHb and with oxyHb. Previous studies have shown that IHP binds HbO 2 , and its binding properties are pH-dependent (11,12). In this study, we have carried out a detailed analysis of the interaction of IHP with human HbO 2 , measuring the effect on (a) proton titration, (b) O 2 dissociation kinetics from fully liganded tetramer, and (c) heat associated to the reaction in order to give a quantitative description of the system and of the interplay between IHP and proton interaction with human HbO 2 . EXPERIMENTAL PROCEDURESHuman HbO 2 was obtained from the blood of healthy volunteers and stripped of anions according to the procedure reported by Riggs (13). Cells were washed three times with iso-osmotic NaCl solutions by centrifugation at 1000 ϫ g, and packed cells were lysed by adding 2 volumes of cold bidistilled water. Stroma were removed by centrifugation at 12,000 ϫ g for 30 min. Hemolysate was first filtered through a Sephadex G-25 column, equilibrated with 0.01 M Tris/HCl buffer, pH 8.0, and EDTA 10 Ϫ5 M, and afterward it was passed through a column of mixed bed ...

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“…In the case of biochemical reactions, to have the equilibrium constant for the process under a knowledge of these four quantities involves the exper-study determined in independent experiments. imental determination of two of them: the equilibrium In the last decade, several isothermal titration calorimeters to study biochemical reactions were developed in the laboratories of S. J. Gill (1,2), E. Freire (3), reproducible instruments, which allow a calorimetric used a Synchropack S300 column (100 1 4.6 mm i.d.) titration to be made by successive injections of one re-equilibrated in 50 mM tris [hydroxymetyl]aminoagent into a single solution of the other.…”

mentioning

confidence: 99%