Natural-Abundance 13C Nuclear-Magnetic-Resonance Study of Toxin II from Anemonia sulcata

Abstract

Natural‐abundance 13C NMR spectra (at 15.04 MHz) of the polypeptide toxin II from the sea anemone Anemonia sulcata have been analysed and compared with corresponding spectra reported recently for a closely related polypeptide anthopleurin A. The spectra contain many resolved onecarbon and two‐carbon resonances from carbonyl, aromatic and methyl carbons, many of which have been assigned to individual carbons in the molecule on the basis of their chemical shifts, including their pH dependence, and by comparison with the 13C NMR spectrum of anthopleurin A. Analysis of the effects of pH on the spectrum yields estimates for the pKa values of a number of functional groups in the molecule, as follows: side‐chain carboxylates of the two aspartic acid residues 2 and 3.1; COOH‐terminal carboxylic acid, 3.5; imidazolium moieties of the two histidine residues, 6.7 and 7.6; NH2‐terminal ammonium, 8. The similarity between the pKa values of these functional groups in toxin II and those of corresponding groups in anthopleurin A, together with the close agreement between chemical shifts of conserved curbons, indicates that many local interactions are nearly identical in the two molecules, and thus supports the thesis that their overall conformations in solution are similar. However, the local interactions involving one of the aspartic acid residues are altered in toxin II. Together with other data, this leads to a proposal for the site in these two molecules which is responsible for their cardiac stimulatory activity.

Quantitative analysis of metabolite concentrations in human urine samples using 13C{1H} NMR spectroscopy

“…The uniqueness of 13 C NMR stems mainly from its increased chemical shift dispersion which makes it superior to 1 H NMR in analyzing of NMR spectra of polymers (Hatada and Kitayama 2004), polysaccharides (Gorin 1981), peptides (Norton et al 1980), and for the characterization of metabolic diseases (Wehrli et al 1997;Moolenaar et al 1999) where NMR spectra can be very complex and overcrowded, especially in the aliphatic region. For example, the analysis of the composition of high value edible oils is facilitated by this approach (Hidalgo and Zamora 2003).…”