Combined use of nuclear magnetic resonance and infrared spectroscopy for studying recognition processes between amphenicolic antibiotics and albumin

摘要

Biological reactions are mostly concerned with selective interactions between small ligands and\udmacromolecular receptors. The same ligands may activate responses of different intensities and/or effects\udin the presence of different receptors. Many approaches based on spectroscopic and non-spectroscopic\udmethods have been used to study interactions between small ligands and macromolecular receptors,\udincluding methods based on NMR and IR spectroscopic analysis of the solution behaviour of the\udligand in the presence of receptors. In this work, we investigated the interaction between ovine serum\udalbumin with two amphenicolic antibiotics [chloramphenicol (CAP) and thiamphenicol (TAP)], using a\udcombined approach based on NMR and IR methodologies, furnishing complementary information about\udthe recognition process occurring within the two systems. The two ligands, despite their similar structures,\udshowed different affinities towards albumin. NMR methodology is based on the comparison of selective and non-selective spin–lattice relaxation rates of the ligands in the presence and absence of\udmacromolecular receptors and RNS1\udand RSE1 temperature dependence analysis. From these studies, the\udligand–receptor binding strength was evaluated on the basis of the ‘affinity index.’ The derivation of\udthe affinity index from chemical equilibrium kinetics for both the CAP–albumin and TAP–albumin\udsystems allowed a comparison of the abilities of the two amphenicolic antibiotics to interact with the\udprotein. IR methodology is based on the comparison of the ligand–protein ‘complex’ spectra with those\udof the non-interacting systems. On the basis of the differences revealed, a more thorough IR analysis\udwas performed in order to understand the structural changes which occurred on both ligand and protein\udmolecules within the interacting system.