pH dependence of 13C-15N coupling constants of highly 14N-enriched amino acids isolated from mass cultivation of algae.

The alga Ankistrodesmus braunii was grown with [14N]nitrate under optimized conditions of a large-scale mass cultivation. 19.7% of the dried algae were isolated as a mixture of amino acids. The 15N-labelled amino acids (15N content up to 98%) were separated by ion exchange chromatography using pyridine acetate gradients. The 15N cotent of the analytically pure amino acid was determined by combined gas-liquid chromatography-mass spectrometry of the trifluoroacetylated methylesters and by emission spectroscopy in the 15N analysator. Using pulse Fourier transform 13C nuclear magnetic resonance, the pH dependence of the 13C-15N coupling constants of Asp, Pro, Ser, Glu, Gly, Ala, Val, Ile and Leu was determined in aqueous solutions. Increasing coupling constants were found with pH and decreasing electron density, respectively. The relation of Binsch et al. (Binsch, G., Lambert, J.B., Roberts, B.W. and Roberts, J.D. (1964) J.Am. Chem. Soc. 86,5564-5570) between the coupling constant and the product of the S-part of the 13C and 15N hybridization SC - SN = 80 - J (13C-45X) fits best in acidic medium. The magnitude of coupling constants correlates well with the electron densities calculated by Del Re et al. (Del Re, G., Pullman, B. and Yonezawa, T. (1963) Biochim. Biophys. Acta 75, 153-182). The recording of 13C nuclear magnetic resonance spectra over the entire pH range revealed no change in the sign of the 13C-15N coupling constants of the amino acids.