On the acid dissociation constants of bilirubin and biliverdin. pKa values from 13C NMR spectroscopy.

Biliverdin and bilirubin are naturally-occurring tetrapyrrolic bile pigments containing two propionic acid side chains. These side chains, and their propensity for ionization, are critical in the biological disposition of the pigments. Surprisingly, accurate dissociation constants for the propionic acid groups of biliverdin are unknown, and a wide range of values, extending over some 4 orders of magnitude, has been suggested for the Ka values of the propionic acid groups of bilirubin in aqueous solutions. Recently, pKa values of 6.7-9.3 have been reported for bilirubin--values much greater than the value of approximately 5 typical of propionic acid groups. These curiously high values, currently being used to explain the biological transport and metabolism of bilirubin and related compounds, have been attributed to intramolecular hydrogen bonding. We have determined the pKa values of 99% 13C-enriched (13CO2H) [8(3),12(3)-13C2]mesobilirubin-XIII, alpha, the corresponding biliverdin, and several monopropionic model compounds by 13C NMR spectroscopy. This technique allows direct observation and quantitative measurement of the carboxylic acid and carboxylate anion carbon signals. Analysis of the variation of carboxyl 13C NMR chemical shift with pH gave rubin pKa values of 4.2 and 4.9 and verdin pKa values of 3.9 and 5.3 in aqueous buffers containing only a very small quantity (0.086 mol fraction) of dimethyl sulfoxide. When extrapolated to water, the pKa values are essentially unchanged. The data provide the first experimentally-determined pKa values for a biliverdin. They indicate that intramolecular hydrogen bonding has little effect on the acid dissociation of bilirubin and suggest that the equilibrium acidity of the bilirubin carboxylic acid groups is not abnormally high but similar to the thermodynamic acidity found in other carboxylic acids, as originally suggested by Overbeek et al. (Overbeek, J. T. G., Vink, C. L. J., and Deenstra, H. (1955) Recl. Trav. Chim. Pays-Bas 74, 81-84).