Abstract
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).
Highlights
To study the dissociation of hydrogen-bonded and non-hydrogen-bonded pyrrolic acids and to determine the pKa values of mesobilirubin-XIII␣ [1] and mesobiliverdin-XIII␣ [2], two synthetic surrogates that differ from bilirubin and biliverdin, respectively, only by trivial differences in the nature, i.e. vinyl groups reduced to ethyl, and sequence of alkyl side chains on the lactam rings
The titration curves of all of these compounds showed two plateaux, at low and high pH respectively, separated by ϳ5 ppm. These plateaux clearly represent the un-ionized and the fully ionized species, respectively, and the chemical shift difference corresponds to the titration shift, ⌬ ϭ ␦CO–2 Ϫ ␦CO2H, where ␦CO–2 is the chemical shift of the carboxylate anion and ␦ CO2H is the chemical shift of the carboxylic acid
The titration shifts observed (Table I) are consistent with those reported for a variety of carboxylic acids [20, 21] and were found to be independent of the number of carboxylic acid groups and almost unaffected by added dimethyl sulfoxide, up to about 30 volume %
Summary
[83,123-13C2]Mesobilirubin-XIII␣ [1], [83,123-13C2]mesobiliverdinXIII␣ [2], [83-13C]xanthobilirubic acid (3, Table I), 12-[83-13C]despropionic acid-12-ethylmesobiliverdin-XIII␣ [4], 12-[83-13C]despropionic acid-12-ethylmesobilirubin-XIII␣ [5], and 2-[1-13C]carbomethoxy-3,5dimethyl-1H-pyrrole-4-propanoic acid [6] (all 99% 13C-enriched as 13CO2H) were prepared as described previously [19, 20] or, for compounds 4 and 5, by modification of reported methods [21]. NMR samples were prepared in NMR tubes by adding standard aliquots of a stock solution of acid or its tetra-n-butylammonium salt to aqueous buffers. Aqueous solutions were prepared by adding 50 l of a 6 – 8 ϫ 10Ϫ3 M stock solution of acid or its salt dissolved in deionized H2O to 500 l of buffer. Aqueous dimethyl sulfoxide solutions were prepared by adding a 50-l aliquot of a 6 – 8 ϫ 10Ϫ3 M stock solution of acid or its salt in (CD3)2SO to an aliquot of buffer. Visible spectral changes, 0 –2 nm bathochromic shifts, were detected in the pigment solutions with added (CD3)2SO, from 1.8 to 27 volume %, in pH 7.4 Tris buffer, and solutions were usually optically clear. Carboxylic acid pKa values were determined either graphically or by nonlinear regression analysis. Ͻ n Ͻ 0.93 and pKa values within 0.03 units of those found by the graphical method
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