Apparent hydrogen bonding by strongly immobilized spin-labels.
The hyperfine separations of nitroxide spin-labels which are tightly bound within hemoglobin exhibit a substantial temperature dependence even when the hemoglobin is immobilized by freezing or precipitation. It is shown that NO.--HX hydrogen bond formation by the spin-label within its binding site is a good explanation for the observed temperature dependence. Comparative studies using different hemoglobin derivatives and two different spin-labels suggest that the HX group may be some element of the protein matrix and that this hydrogen bond may be a factor in the stabilization of the label within its binding site. The hyperfine separation of a fatty acid spin probe incorporated into aqueous bilayer dispersons of dipalmitoylphosphatidylcholine also exhibits a temperature dependence at low temperature which is qualitatively similar to that of the spin-labeled hemoglobin systems. Saturation transfer electron paramagnetic resonance measurements indicate that label motion is not the source of this temperature dependence. A hydrogen-bond equilibrium between water molecules and the nitroxide NO. group appears to be a plausible source of the temperature-dependent hyperfine separation in the lipid bilayer system. Small amplitude torsional oscillation or librational motion by the nitroxide may also produce additional changes in the hyperfine separation which are difficult to distinguish from hydrogen-bonding effects under some circumstances. The apparent hydrogen-bond equilibrium exhibits a strong thermal and environmental dependence which may be of importance in a number of biophysical spin-label measurements.
D004578:Electron Spin Resonance Spectroscopy; D006454:Hemoglobins; D006801:Humans; D006860:Hydrogen Bonding; D008433:Mathematics; D013113:Spin Labels; D013696:Temperature; D013816:Thermodynamics
D006454:Hemoglobins; D013113:Spin Labels