Bilirubin Acidity. Titrimetric and 13C NMR Studies.

Abstract

Acidimetric titration of bilirubin IX-.alpha., dissolved in excess aqueous NaOH showed that 2 protons are dissociated with pK values well below 7 and that 1 or several additional acidic groups titrate with pK around 12.9. Precipitation of the nearly insoluble acid precluded determination of the 2 lower pK values by titration in aqueous solution. In dimethyl sulfoxide solution, 4 acidic protons were demonstrated, titrating 2 by 2 without precipitation. 13C NMR spectra of bilirubin IX-.alpha. were recorded and complete assignments were made by comparison with the spectra of bilirubin XIII-.alpha. and mesobilirubin etc. Such spectra, recorded after addition of 2 and 4 mol of base/mol of bilirubin IX-.alpha., showed that both carboxyl groups were titrated by the 1st 2 mol of base, and both lactams by the following 2 mol of base. Cotitrations of bilirubin IX-.alpha. with other acids, o- and m-hydroxybenzoic acid and 2-pyridone, were used to determine relative pK values in dimethyl sulfoxide solution,and pK values for the 4 acidic protons of bilirubin IX-.alpha. in aqueous solution were calculated from the Born equation. Both carboxyl groups exhibited pK = 4.4, and both lactams pK = 13.0, in good agreement with values expected from the chemical structure of the bilirubin molecule. Implications of these findings for understanding the mechanism of bilirubin neurotoxicity are discussed.