Caracterización energética de la unión de 1, 3, 6 - naftalén trisulfonato al factor de crecimiento para fibroblastos ácido humano: implicaciones para su uso como agente anti-angiogénico / Energetic characterization of 1, 3, 6 - naphtalene trisulfonate binding to human acidic fi broblast growth factor: implications for its anti-angiogenic use

Se ha estudiado, mediante calorimetría isotérmica de reacción, la interacción del agente anticancerígeno 1,3,6-naftalén trisulfonato con el factor de crecimiento para fi broblastos ácido humano. La afi nidad decrece con el aumento de la fuerza iónica. A pH 7,0 y NaCl 0,15 M, la constante de unión de la proteína con el ligando se encuentra en el rango 102 – 103 M-1, una afi nidad dos órdenes de magnitud menor que la del FGFa por heparina. El cambio de entalpía favorece la interacción, siendo el cambio de entropía desfavorable. De la dependencia del cambio de entalpía con la temperatura se calculó un pequeño cambio en la capacidad calorífi ca del proceso, con un valor excepcionalmente positivo de 90 cal K-1mol-1. A partir de los datos termodinámicos medidos y de ecuaciones paramétricas establecidas en la literatura, se calcularon cambios en la superfi cie accesible al disolvente, tanto polar como apolar, que acompañan a la interacción. Los resultados se compararon con los medidos mediante resonancia magnética nuclear. El estudio incluye consideraciones de bioenergética estructural sobre el posible uso de 1,3,6-naftalén trisulfonato como agente antiangiogénico o como molécula líder para el desarrollo de fármacos anti-angiogénicos

The equilibrium interaction of anti-cancer agent 1,3,6-naphatalene trisulfonate with human acidic fi broblast growth factor has been studied by calorimetry. The affi nity decreases with increasing ionic strength. At pH 7.0 and 0.15 M NaCl concentration, a binding constant of the protein with the ligand was estimated in the 102 – 103 M-1 range, an affi nity two orders of magnitude lower than that of aFGF with heparin. The interaction is enthalpically driven, and the entropy change is unfavorable. A small heat capacity change with an unusual positive value of 90 cal K-1mol-1 was determined from the temperature dependence of the enthalpies. Changes in accessible apolar and polar surface areas in the interaction were calculated from the thermodynamic data obtained and parametric equations in the literature. The results were compared with those measured from NMR data. The study includes structural bioenergetic considerations about the possible use of 1,3,6-naphatalene trisulfonate as an anti-angiogenic agent itself, or as a lead for the development of anti-angiogenic drugs

Myo-inositol hexasulphate and low molecular weight heparin binding to human acidic fibroblast growth factor: a calorimetric and FTIR study.

The interaction of an amino-terminal-truncated 139 amino-acids form of human acidic fibroblast growth factor with myo-inositol hexasulphate and low molecular weight (3500 g mol(-1)) heparin has been studied by isothermal titration calorimetry, differential scanning calorimetry and Fourier transform infrared spectroscopy. A slightly higher affinity for the monosaccharide has been measured. The binding of the ligands causes an increase of 13--15 degrees C in the melting temperature of the free protein (45 degrees C). From measured enthalpy and heat capacity changes, calculations of changes in accessible surface areas have been made. These calculations, together with infrared spectroscopy data, indicate that a small conformational change is induced by the binding of both ligands. This conformational change would affect the tertiary structure, not the secondary one.

Energetics of myo-inositol hexasulfate binding to human acidic fibroblast growth factor effect of ionic strength and temperature.

The binding of myo-inositol hexasulfate to an N-terminal truncated 132-amino-acid human acidic fibroblast growth factor form was studied by isothermal titration calorimetry. The technique yields values for the enthalpy change and equilibrium constant, from which the Gibbs energy and entropy change can also be calculated. Experiments in different buffers and pH values show that the proton balance in the reaction is negligible. Experiments at pH 7.0 in the presence of 0.2-0.6 M NaCl showed that the enthalpy and Gibbs energy changes parallel behaviour with ionic strength change, with values in the -21 to -11 kJ x mol(-1) range in the first case and in the -31 to -22 kJ x mol(-1) range in the second. No dependence of entropy on ionic strength was found, with a constant value of approximately 35 J x K(-1) x mol(-1) at all ionic strengths studied. The results can be interpreted in molecular terms by a model in which competitive binding of 3-4 chloride ions to the myo-inositol-binding site is assumed. Isothermal titration calorimetry was also performed at different temperatures and yielded a value of -142+/-13 J x K(-1) x mol(-1) for the heat-capacity change at pH 7.0 and 0.4 M NaCl. Using different parametric equations in the literature, changes on ligand binding in the range -100 to -200 A2 in solvent-accessible surface areas, both polar and apolar, were calculated from thermodynamic data. These values suggest a negligible overall conformational change in the protein when the ligand binds and agree closely with calculations performed with NMR structural data, in which it is shown that the most important negative change in total solvent-accessible surface area occurs in the amino acids Ile56, Gln57, Leu58 and Leu149, in the high-affinity receptor-binding region of the protein.

Differential scanning calorimetry of lobster haemocyanin.

Differential scanning calorimetry has been performed with Palinurus vulgaris haemocyanin monomers and hexamers. The denaturation of the protein is irreversible. Both the temperature of the transition maximum and the enthalpy are lower for the monomer than for the hexamer. A scan rate dependence of the temperature of the maxima is found for both the monomer and the hexamer; for the hexamer at least, this can be explained in terms of a two-state kinetic model. Some comments are made as to the use of equilibrium thermodynamics in the analysis of irreversible scanning calorimetric traces.