Abstract
BackgroundUnconjugated bilirubin (UCB) is an unstable substance with very low aqueous solubility. Its aqueous pKa values affect many of its interactions, particularly their pH-dependence. A companion paper shows that only our prior solvent partition studies, leading to pKa values of 8.12 and 8.44, met all essential requirements for valid pKa determinations. Other published values, generally lower, some below 5.0, were shown to be invalid. The present work was designed to derive suitable models for interpreting published data on the pH-dependent binding of UCB with four agents, mentioned below, chosen because they are not, themselves, sensitive to changes in the pH range 4-10, and the data, mainly spectrometric, were of reasonable quality.ResultsThese analyses indicated that the high pKa values, dianion dimerization constant and solubilities of UCB at various pH values, derived from our partition studies, along with literature-derived pH- and time-dependent supersaturation effects, were essential for constructing useful models that showed good qualitative, and sometimes quantitative, fits with the data. In contrast, published pKa values below 5.0 were highly incompatible with the data for all systems considered. The primary species of bound UCB in our models were: undissociated diacid for phosphatidylcholine, dianion for dodecyl maltoside micelles and cyclodextrins, and both monoanions and dianion for sodium taurocholate. The resulting binding versus pH profiles differed strikingly from each other.ConclusionsThe insights derived from these analyses should be helpful to explore and interpret UCB binding to more complex, pH-sensitive, physiological moieties, such as proteins or membranes, in order to understand its functions.
Highlights
Unconjugated bilirubin (UCB) is an unstable substance with very low aqueous solubility
Criteria for Acceptability and Pitfalls in Interpretation Studies of UCB binding vs. pH should meet the same criteria of validity proposed in the companion paper that deals with UCB alone in simple systems [2]: 1) The UCB is pure; 2) There should be no significant degradation of UCB; 3) Measurements are made after a rapidly achieved equilibrium; 4) Unbound UCB concentrations are below to minimally above aqueous saturation; 5) The pH range studied should encompass all suggested pKa values of UCB, and sufficient data points should be available to permit mathematical modeling
Ionization and Binding Equilibria of Unbound UCB Species The equilibria governing the binding of unconjugated bilirubin (UCB) involve its three unbound species, the protonated diacid (H2B), the monoanions (HB-) and the dianion (B=), one or more of which binds independently to a larger host system [1], such as phospholipid vesicles, cyclodextrins, or micelles
Summary
Unconjugated bilirubin (UCB) is an unstable substance with very low aqueous solubility. Its aqueous pKa values affect many of its interactions, their pH-dependence. A companion paper shows that only our prior solvent partition studies, leading to pKa values of 8.12 and 8.44, met all essential requirements for valid pKa determinations. The true pKa values of unconjugated bilirubin (UCB) are an important determinant of the proportion of the three ionization species of UCB in present in solution, and of the overall aqueous solubility of UCB, at any given pH value [1]. In a companion paper [2] we re-examined many published studies that assessed pKa values for UCB in simple solutions, determined by a wide variety of methods. We summarized the deficiencies in the many reports which suggested that pKa values of UCB were below 7.0 and even below 5.0
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