Study of the specific interaction between L-methionine chromatography support and nucleotides

Abstract

The interaction of L-methionine-agarose with 5'-mononucleotide was investigated by saturation transfer difference (STD)-nuclear magnetic resonance (NMR) spectroscopy. Chromatographic experiments were also performed using homo-oligonucleotides of distinct molecular masses (1-30 nucleotides) to explore the effect of base hydrophobicity, temperature, pH and salt concentration on the retention of homo-oligonucleotides to L-methionine-agarose support. With STD-NMR, the results reveal that hydrophobic residues, such as the CH₃ of thymine and adenine, can preferentially recognise the L-methionine side chain of the support. Also, 5'-TMP led to more contacts with the support, while 5'-UMP presented fewer STD contacts. For 5'-UMP, 5'-CMP and 5'-GMP, the main interaction with the support was through the sugar-phosphate backbone. Similar binding profiles were obtained using chromatographic experiments. Indeed, 5'-TMP had the highest retention time, followed by 5'-GMP, 3'-AMP, 5'-UMP and 5'-CMP. In general, the retention factor of homo-oligonucleotides was higher for ammonium sulphate concentration 1.5 M. For the polyT₃-polyT₃₀ series, the retention time increased by about three-fold, indicating that larger homo-oligonucleotides have more hydrophobic bases, thus enhancing contact with the L-methionine support. The temperature (5, 20 and 35 °C) did not influence homo-oligonucleotide retention. However, the retention time slightly increased when the pH was lower than 9. The STD-NMR technique combined with chromatographic experiments was thus successfully used to screen amino acid-nucleotide interactions.