In additional experiments the spatial structure of AAP10 was clarified using Fourier transformation NMR spectra (1H-NMR, 13C-NMR in D2O or DMSO at 25°C and 40°C). With these investigations it was possible to define the peaks identifying carbon atoms in the molecule and the various hydrogens. A chemical shift was found of the OH moiety of 4-Hyp to the lower field (from 3 to 4 ppm to 5.2 ppm) and of the NH2 moiety of alanine, both of which exhibited typical temperature dependence exceeding 5, calculated as follows:CS(25°) – CS(40° C) dT
where CS is chemical shift (ppm), T is temperature and dT=15°C. Additionally, two-dimensional 1HH-NMR and CH-NMR (COSY spectra) were performed for identification of neighbouring hydrogens by cross peaks. The NMR spectra are shown in Figure 6. From these data it was possible to define the preferred structure of AAP10, which is characterized by two hydrogen bonds stabilizing a semicyclic structure, as shown in Figure 7.
To evaluate whether the OH moiety of 4-Hyp is essential for the effect of AAP10,4-Hyp was replaced by proline. This peptide, AAP8TT, has no preferred structure because it is not stabilized by the hydrogen bond at 4-Hyp. After infusion of this peptide in cumulative concentrations in rabbit hearts, neither a decrease in dispersion nor an increase in the percentage of potential duration within the ±5 ms interval around the mean ARI was found (control 66.3±3.5%, 1 nmol/L AAP8TT 66.0±2.6%, 100 nmol/L AAP8TT 64.3±3.9%). Mean ARI was, however, also not affected by AAP8TT (AAP8TT control, 131.9±3.9; after AAP8TT, 134.1±5.3 ms).
Figure 6 Nuclear magnetic resonance (NMR) spectra of AAP10. Top left C-NMR; Bottom left H-NMR in dimethylsulphoxide; Top right CH-correlational spectroscopy (COSY) -NMR; Bottom right HH-COSY-NMR. All NMR were recorded at 25°C. Tetramethylsilan (0 ppm) served as a reference
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Figure 7 Strucure of AAP10 as determined by nuclear magnetic resonance spectra