pK_a and Aggregation of Bilirubin:  Titrimetric and Ultracentrifugation Studies on Water-Soluble Pegylated Conjugates of Bilirubin and Fatty Acids

Abstract

A water-soluble conjugate (1) with intact carboxyl groups was prepared by addition of poly(ethylene glycol) thiol (MPEG-SH) regiospecifically to the exo vinyl group of bilirubin. (1)H and (13)C NMR and absorbance spectroscopy in CDCl(3) and DMSO-d(6) confirmed the assigned structure and showed that pegylation did not disrupt the hydrogen-bonded ridge-tile conformation of the pigment moiety. Aqueous solutions of 1 were optically clear, but NMR signals were seen only from the MPEG portion and none from the tetrapyrrole, consistent with dissolved assemblies containing aggregated bilirubin cores within mobile polyether chains. On alkalinization (pH >12), signals from the pigment moiety reappeared. Titrimetric measurements on 1 in water showed the pK(a)'s of the two carboxyl groups to be similar (average 6.42). Control studies with pegylated half-esters of succinic, suberic, brassylic, thapsic, and 1,20-eicosanedioic acid showed that pegylation per se has little, if any, effect on carboxyl ionization. However, aggregation increases the apparent pK(a) by approximately 1-2 units. The molecularity of bilirubin in solution was further characterized by ultracentrifugation. Over the pH range 8.5-10 in buffer, bilirubin formed multimers with aggregation numbers ranging from approximately 2-7. Bilirubin is monomeric in DMSO or CHCl(3) at approximately 2 x 10(-)(5) M, but aggregation occurred when the CHCl(3) was contaminated with trace adventitious (perhaps lipoidal) impurities. These observations show that aggregation increases the pK(a)'s of aliphatic carboxylic acids relative to their monomer values in water. They are consistent with earlier (13)C NMR-based estimates of approximately 4.2 and approximately 4.9 for the aqueous pK(a)'s of bilirubin and similar studies of bilirubin in micellar bile-salt solutions. Together with earlier work, they confirm that the pK(a)'s of bilirubin are about normal for aliphatic carboxyls and suggest that the high (>7.5) values occasionally reported, including those based on CHCl(3) partitioning, are artifacts of aggregation or technique.

Imaging Endogenous Bilirubins with Two-Photon Fluorescence of Bilirubin Dimers

“…As a negative control, the red fluorescence disappeared when the bilirubins were dissolved in DMSO ( Figure 3A, red curve), in which the bilirubins were monomeric. 59 These evidence support that the bilirubin red fluorescence originates from its dimers. Although the single-photon fluorescence of bilirubin dimers was weak at pH 7.4 ( Figure 3A, black curve), the intensity was enhanced 3 times after association with HSA ( Figure 3B).…”

Revalidation and rationale for high pKa values of unconjugated bilirubin

“…[16] had reported the formation of UCB aggregates in some CHCl 3 solutions when pure distilled CHCl 3 , exposed to Parafilm ® or plastic caps, was used as solvent. They suggested that "adventitious lipid contaminants can cause aggregation in CHCl 3 " and that this may be relevant to our CHCl 3 -water partition studies [3].…”

Computational chemical analysis of unconjugated bilirubin anions and insights into pKa values clarification

“…43,46 The presence and persistence of folded, intramolecularly hydrogen-bonded conformation of bilirubin and its anions in solution is also experimentally supported by studies of Lightner and Nogales. 46 The hydrogen-carbon distances 11,47 account for the hydrogen bond effect (proton deshielding and a further downfield shift for these proton resonances) of the internal UCB hydrogen bond network.…”