Iso-orotic acid was found to be very pH sensitive in water and deuterium oxide. Identical spectral curves were obtained in both solvent systems. A lowering of the extinction coefficient at pH 6, as compared with pH 1, could indicate an ionization effect rather than enolization since no appreciable wavelength shift was observed. However, enolization is predominant in the region of pH 12. A confirmation that pK2 in iso-orotic acid refers to proton dissociation from an enolic species was obtained by determining the pK2 of iso-orotic acid in water and in deuterium oxide. In the presence of Cu(II) ions at pH 3, there was noted a slight shift towards the visible in the region of the uracil absorption band of iso-orotic acid. This slight shift is attributed to complexing through the ketonic oxygen and carboxyl group.
Very little shift in the uracil lactam band occurred in going from the acidic to neutral region for orotic acid and a slight shift to longer wavelengths resulted when approaching the region of pH 12. Ultra-violet absorption spectra obtained in deuterium oxide indicated that even up to pH 12, orotic acid was still predominantly mono or diketonic. Deuterium iostope studies indicate at least one monoketonic form at pH 10. Spectral curves show that in the presence of Zn(II), Co(II) and Mn(II), orotic acid forms weak complexes. However, in the presence of Cu(II) and Ni(II) ions strong complexing is observed. A deuterium isotope effect was noted with orotic acid and Ni(II) ions in deuterium oxide resulting from the cleavage of the DN1 bond and verifying complexation through the carboxyl group and nitrogen in the 1-position.