Constante de dissociation | EPFL Graph Search

La constante de dissociation est la constante de réaction associée à la dissociation d'un composé chimique. Si un composé de formule AxBy se dissocie selon la réaction : AxBy ↔ xA + yB alors la constante de dissociation Kd est : K_{\mathrm d} = \frac{[\mathrm A]^x \times [\mathrm B]^y}{[\mathrm A_x \mathrm B_y]} où [X] est la concentration molaire de X. On définit aussi le pKd comme étant : pKd = −log(Kd). Cas de l'eau La constante de dissociation de l'eau, notée Ke (Kw en anglais), est la constante de réaction associée à la réaction chimique d'autoprotolyse de l'eau : : 2 = H3O+ + HO− Les produits de cette réaction sont les ions oxonium (anciennement appelés ions hydronium) et hydroxyde. À l'équilibre, et pour des solutions diluées, le K est égal au produit des concentrations en ions oxonium et hydroxyde. Dans les CNTP : : Ke = [H3O+]×[HO−] = 10−14 : et pKe = −log(Ke) = 14. La réaction de dissociation de l'eau est naturelle et spontanée ; l'eau dans sa

Exploring Ligand Affinities for Proteins by NMR of Long-Lived States

Roberto Buratto

The detection of molecules that can bind to active sites of protein targets and the measurement of their affinities is a promising application of NMR. Nowadays, the screening of drug candidates is routinely done by NMR in pharmaceutical industry. We have proposed to use the relaxation of Long-Lived States (LLS) for drug screening by NMR. Long-lived states are nuclear spin states whose decay time constant Tlls can be much longer than the longitudinal relaxation time T1. LLS can be used to screen and determine the dissociation constant KD of molecular fragments that bind weakly to protein targets. The use of LLS for fragment screening leads to a spectacular increase in contrast between free and bound ligands, and thus allows one to characterize binding of fragments with very weak affinities, with KD in the millimolar range, which is difficult to achieve by other methods such as ITC. By exploiting the LLS behavior of a spy molecule, we experimentally demonstrate that it is possible to measure dissociation constants KD as large as 12 mM, corresponding to very weak binding, where most other biophysical techniques fail, including other NMR methods based on the observation of ligands. Furthermore, we have combined LLS for screening for improved contrast with 1H dissolution-DNP to enhance the sensitivity. DNP-enhanced screening for measuring LLS signals of a weak ligand allows one to use very low concentrations of ligands and proteins. We observed dramatic differences between the spectra of the ligand in the presence or absence of a protein, or in the presence of the protein combined with a stronger ligand. Moreover, we have explored LLS involving pairs of 19F nuclei to study binding phenomena. Indeed, fluorine detection is quite interesting because it offers the possibility to perform screening experiments without any problems due to overlapping signals. In a customdesigned fluorinated ligand that binds trypsin, we have observed a promising ratio Tlls / T1 > 4. This fluorinated ligand has been used as spy molecule in competition experiments, which allowed us to rank the affinities and estimate dissociation constants of arbitrary ligands that do not contain any fluorine.

EPFL2015

On isotopically selective collisional energy transfer and infrared multiple photon absorption by vibrationally highly excited molecules

Richard Bossart

This study focuses on an interesting and important phenomenon that was employed in a new approach to laser isotope separation that has been recently proposed and developed in our laboratory for highly selective separation of carbon isotopes. This approach consists of pre-exciting CF3H molecules with the desired isotope to a low vibrational overtone of the CH-stretch with a subsequent selective infrared multiple photon dissociation (IRMPD) of only the pre-excited molecules by a CO2-laser pulse. Significant isotopic shifts of the employed CH overtone bands already allow high selectivity at the pre-excitation step. This selectivity, however, can be further greatly increased by increasing the pressure of the sample gas and/or the delay between the two laser pulses; that is, by increasing the number of molecular collisions during the process. At first glance, the observed effect contradicts the general expectations that the isotopic selectivity of such a process should drop with an increase in the number of collisions because of vibrational energy transfer between different isotopic species. In this work we have studied this phenomenon and found its physical origins. We propose the contribution of two different mechanisms to the observed enhancement of isotopic selectivity by collisions. First, the vibrational collisional relaxation itself is isotopically selective, that is vibrationally excited 12CF3H relax on the bath of cold, 12CF3H, molecules faster than the excited 13CF3H do on the same bath. The primary reason for such a selectivity could be a significant isotopic shift of the vibration (CF-stretch, we believe) that mediates the energy transfer. A second mechanism arises from the difference between the IRMPD probabilities of the two isotopic species by the CO2 laser tuned to a particular wavelength that enhances the dissociation yield of the targeted, carbon-13, species. As collisional deactivation of both species increases the number of photons they have to absorb to be dissociated, this difference in dissociation probability increases as well, yielding an additional isotopic selectivity of the process. We perform a set of experiments and numerical simulations to investigate these two mechanisms. Experimentally we find that, indeed, collisional vibrational deactivation of CF3H is isotopically selective. This is, perhaps, the first direct observation of isotopically selective collisional relaxation of highly excited medium-sized molecules. At low pressures and increased time-delay between the lasers both suggested mechanisms contribute equally to the enhancement of isotopic selectivity (with a slight dominance of the IRMPD step). We also perform numerical simulations of vibrational energy transfer (VET) between highly excited and cold CF3H of both isotopic species. The model we employ includes V-V energy transfer due to long-range dipole-dipole interactions and V-V',T,R energy transfer due to head-on collisions between two molecules. The results reproduce well the measured isotopic selectivity in vibrational energy transfer. At the next step, we propose a model for the simulation of the laser isotope separation process. We solve the master equations including vibrational energy transfer and absorption and stimulated emission of IR photons. The main improvement of our calculations with respect to existing models is that we introduce a dependence of the absorption/emission rates on the frequency of the laser, the internal energy of the molecule and its isotopic species. We are able to reproduce our experiments numerically and thus gain information on the laser isotope separation process. In particular, we find that at high sample pressures the mechanism of isotopically selective IRMPD rates prevails over the mechanism of different collisional relaxation rates.

EPFL2005