Low solubility of unconjugated bilirubin in dimethylsulfoxide – water systems: implications for pKa determinations
BackgroundAqueous pKa values of unconjugated bilirubin are important determinants of its solubility and transport. Published pKa data on an analog, mesobilirubin-XIIIα, studied by 13C-NMR in buffered solutions containing 27 and 64 vol% (C2H3)2SO because of low aqueous solubility of mesobilirubin, were extrapolated to obtain pKa values in water of 4.2 and 4.9. Previous chloroform-water partition data on bilirubin diacid led to higher estimates of its pKa, 8.12 and 8.44, and its aqueous solubility. A thermodynamic analysis, using this solubility and a published solubility in DMSO, suggested that the systems used to measure 13C-NMR shifts were highly supersaturated. This expectation was assessed by measuring the residual concentrations of bilirubin in the supernatants of comparable DMSO-buffer systems, after mild centrifugation to remove microprecipitates.ResultsExtensive sedimentation was observed from numerous systems, many of which appeared optically clear. The very low supernatant concentrations at the lowest pH values (4.1-5.9) were compatible with the above thermodynamic analysis. Extensive sedimentation and low supernatant concentrations occurred also at pH as high as 7.2.ConclusionsThe present study strongly supports the validity of the aqueous solubility of bilirubin diacid derived from partition data, and, therefore, the corresponding high pKa values. Many of the mesobilirubin systems in the 13C-NMR studies were probably supersaturated, contained microsuspensions, and were not true solutions. This, and previously documented errors in pH determinations that caused serious errors in pKa values of the many soluble reference acids and mesobilirubin, raise doubts regarding the low pKa estimates for mesobilirubin from the 13C-NMR studies.
Review: Bilirubin pKa studies; new models and theories indicate high pKa values in water, dimethylformamide and DMSO
BackgroundCorrect aqueous pKa values of unconjugated bilirubin (UCB), a poorly-soluble, unstable substance, are essential for understanding its functions. Our prior solvent partition studies, of unlabeled and [14C] UCB, indicated pKa values above 8.0. These high values were attributed to effects of internal H-bonding in UCB. Many earlier and subsequent studies have reported lower pKa values, some even below 5.0, which are often used to describe the behavior of UCB. We here review 18 published studies that assessed aqueous pKa values of UCB, critically evaluating their methodologies in relation to essential preconditions for valid pKa measurements (short-duration experiments with purified UCB below saturation and accounting for self-association of UCB).ResultsThese re-assessments identified major deficiencies that invalidate the results of all but our partition studies. New theoretical modeling of UCB titrations shows remarkable, unexpected effects of self-association, yielding falsely low pKa estimates, and provides some rationalization of the titration anomalies. The titration behavior reported for a soluble thioether conjugate of UCB at high aqueous concentrations is shown to be highly anomalous. Theoretical re-interpretations of data in DMSO and dimethylformamide show that those indirectly-derived aqueous pKa values are unacceptable, and indicate new, high average pKa values for UCB in non-aqueous media (>11 in DMSO and, probably, >10 in dimethylformamide).ConclusionsNo reliable aqueous pKa values of UCB are available for comparison with our partition-derived results. A companion paper shows that only the high pKa values can explain the pH-dependence of UCB binding to phospholipids, cyclodextrins, and alkyl-glycoside and bile salt micelles.
New Trends in Fungal Biooxidation
Biooxidations using isolated biocatalysts for the oxidative conversion of organic compounds and materials are becoming more and more important in the industrial sector. Research in this direction is part of the rapidly developing field of White Biotechnology and is carried out not least against the background of sustainable development and the need to replace environmentally risky technologies by eco-friendly processes. Whereas whole cells are already widely used as oxidative biocatalysts, the application of isolated enzymes is still limited to a few examples. One reason for that is the cost-intensive production and laborious purification of enzymes which are mostly intracellular and sometimes membrane-bound proteins with low stability and complex co-factor requirements. The use of secreted oxidoreductases offers several advantages: they are easier to separate and purify, need only cheap co-substrates such as dioxygen or peroxides and are far more stable than intracellular enzymes. Filamentous fungi secrete a broad spectrum of oxidative biocatalysts which are involved, amongst others, in the degradation of recalcitrant biopolymers, the synthesis of melanins as well as the detoxification of plant ingredients, microbial metabolites and organopollutants. We focus here on novel secreted enzymes - peroxygenases and DyP-type peroxidases - with remarkable catalytic properties. Furthermore selected features of “classic” fungal oxidoreductases, such as laccase, tyrosinase and chloroperoxidase, are discussed against the background of innovative recent developments in the field of enzyme application.