Dissoziationsgleichgewichte von Glutathion

Abstract

The dependence of the 13 C‐NMR resonances on the pH of solutions is determined for reduced and oxidized glutathione and for the model substances glutamine, cysteine and acetylglycine by pulse Fourier transform (PFT) 13 C‐NMR spectroscopy. The 1 H‐noise decoupled 22.63 MHz PFT 13 C‐NMR spectra of 0.1–0.3 M aqueous solutions were measured on a Bruker HFX‐90 multinuclear spectrometer with an 18” magnet and equipped with a Fabritek 1074 and a PDP‐8‐I computer for accumulating the pulse interferograms and for performing their Fourier transformations. The pH induced variations of charge densities on the various carbon atoms of these molecules can be selectively observed. The pK and pI values obtained by these 13 C‐NMR investigations often agree very well with the electrometric and photometric determinations. However, large deviations from classical values are found for other dissociation equilibria. For glutamine we obtained the pK values 2.1 (COOH) and 9.1 (NH 3 + ) and an isoelectric point at 5.65. The pH induced shifts are particularly large for the ammonium dissociations. The titration of cysteine from pH 7 to pH 11 gives total shifts on the C α of 4.1 ppm, on the β of 6.10 ppm and on the carboxylic carbon of 10 ppm with two macroscopic pK ‐values at pH 8.25 and 10.25 corresponding to the ammonium and thiol dissociations. The curves show that cysteine exhibits mixed dissociation equilibria as reported previously. However a comparison of the results of the 13 C‐NMR measurements on reduced and oxidized glutathione indicates the thiol dissociation in oxidized glutathione to be largely independent of the ammonium dissociation at the glutamic acid residue and a pK ‐value of 9.2 for the thiol dissociation is found. Whereas the usual titrimetric methods register the macroscopic behaviour, e.g. an increase of the proton concentration. 13 C‐NMR spectroscopy allows investigations at each carbon atom concerning microscopic events resulting from — NH 3 + , — SH and — COOH dissociations.