Abstract
A first thiol-specific pH-sensitive nitroxide spin-label of the imidazolidine series, methanethiosulfonic acid S-(1-oxyl-2,2,3,5,5-pentamethylimidazolidin-4-ylmethyl) ester (IMTSL), has been synthesized and characterized. X-Band (9 GHz) and W-band (94 GHz) EPR spectral parameters of the new spin-label in its free form and covalently attached to an amino acid cysteine and a tripeptide glutathione were studied as a function of pH and solvent polarity. The pKa value of the protonatable tertiary amino group of the spin-label was found to be unaffected by other ionizable groups present in side chains of unstructured small peptides. The W-band EPR spectra were shown to allow for pKa determination from precise g-factor measurements. Is has been demonstrated that the high accuracy of pKa determination for pH-sensitive nitroxides could be achieved regardless of the frequency of measurements or the regime of spin exchange: fast at X-band and slow at W-band. IMTSL was found to react specifically with a model protein, iso-1-cytochrome c from the yeast Saccharomyces cerevisiae, giving EPR spectra very similar to those of the most commonly employed cysteine-specific label MTSL. CD data indicated no perturbations to the overall protein structure upon IMTSL labeling. It was found that for IMTSL, g iso correlates linearly with A iso, but the slopes are different for the neutral and charged forms of the nitroxide. This finding was attributed to the solvent effects on the spin density at the oxygen atom of the NO group and on the excitation energy of the oxygen lone-pair orbital.