AQUEOUS DISSOCIATION-CONSTANTS OF BILE-PIGMENTS AND SPARINGLY SOLUBLECARBOXYLIC-ACIDS BY C-13 NMR IN AQUEOUS DIMETHYL-SULFOXIDE - EFFECTS OF HYDROGEN-BONDING

pK(a)s for the acid dissociation of the carboxyl groups of bilirubin i n water have been reported recently to be 8.1-8.4, or higher. These hi gh values were attributed to intramolecular hydrogen bonding. They hav e led to suggestions that monoanions of bilirubin predominate at physi ologic pH and are the species transported most readily into hepatocyte s by carriers. Such high aqueous pK(a)s are inconsistent with recent C -13 nuclear magnetic resonance (NMR) measurements on mesobilirubin XII I alpha, done on aqueous solutions containing dimethyl sulfoxide. To i nvestigate whether the presence of dimethyl sulfoxide leads to unrelia ble values when using C-13 NMR spectroscopy to determine pK(a)s of car boxylic acids that can undergo intramolecular hydrogen bonding, we mea sured the pK(a)s of C-13-labeled fumaric, maleic, and phthalic acids i n solutions containing up to 27 vol % dimethyl sulfoxide. In addition, we used C-13 NMR to estimate the pK(a)s of 5-carbomethoxy-4-methylpyr role-3-[1-C-13]propanoic acid], a model for the two central rings of b ilirubin. Our results show that C-13 NMR of aqueous dimethyl sulfoxide solutions can be used with confidence to measure pK(a)s of intramolec ularly hydrogen-bonded carboxylic acids. They support our previous est imates for the pK(a)s of bilirubin and confirm that intramolecular hyd rogen bonding has little effect on the acidity of bilirubins in water. Together with previous studies and chemical arguments they strongly s uggest that reported aqueous pK(a)s of >8, or even >6, for the carboxy l groups of bilirubin are incorrect and that arguments used to rationa lize them are questionable.