Appendix I ¹⁵N Isotope Effects on the Raman Spectra of 5′IMP and 5′UMP-d₃

Abstract

Baker’s yeast was grown in (¹⁵NH₄)₂SO₄ as the sole nitrogen source, and RNA was extracted from it with 1.5% sodium dodecyl sulfate, 2% ethanol, and phosphate buffer pH 7.2. After centrifugation, the supernatant was dialyzed against pH 7.2 phosphate buffer. The ¹⁵N-RNA was digested with nuclease Pl, and a mixture solution of nucleotides was obtained. Four nucleotides, 5′AMP, 5′UMP, 5′GMP, and 5′CMP were separated from one another by Dowex I-X2 column chromatography. The final product, purified 5′ UMP- ¹⁵N₂, gave only one spot in a thin-layer chromatography. A mass-spectrometric analysis of ammonia produced by micro-Kjeldahl method from this sample indicated that 90% of the whole nitrogen atoms in it are ¹⁵N.