The D/H fractionation factor is the extent to which a hydrogen at a particular site becomes enriched in 2H over 1H relative to the solvent. It is controversial whether or not this parameter correlates with hydrogen-bond strength. Loh et al. (1994) measured fractionation factors of amide protons in staphylococcal nuclease by a direct method. The protein was dissolved in varying percentages of D2O and the fractionation factor was determined by measuring the peakvolumes of the resonances in an H-N correlation spectrum (HSMQC) relative to a standard peak. The fractionation factor was obtained by linear least-squares analysis of the plot of the relative peak volume versus the mole fraction of H2O in solution. The drawback of this data is that water presaturation was employed. As water presaturation leads to attenuation of amide proton signals, the fractionation factors measured by Loh et al. (1994) might not be accurate and might have led to wrong conclusions. First, we are remeasuring the same fractionation factors, using the fast HSQC (FHSQC) detection scheme that avoids water saturation as well as dephasing of the water magnetization by gradients at the end of the experiment. The effects of increased solution viscosity in solutions with higher D2O concentrations, which might introduce significant error in the determined fractionation factors, will be addressed as well. Secondly, we are measuring the same fractionation factors with a triple resonance technique developed by LiWang & Bax (1996). An intraresidue H?-N correlation spectrum (H(CACO)N) of the protein dissolved in 50% H2O/ 50% D2O yields splittings in the nitrogen dimension that corresponds to the protonated and deuterated states of the amide. Measurement of the relative intensity of the two doublet components allows determination of the fractionation factors. The drawback of this technique is that corrections of the intensity ratio of the two doublet components are required to account for the different relaxation behavior in the N-1H and N-2H populations of the protein.
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