PubMed · 69973
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E Simpson, D Thompson. 1977-08-13. Routine urinalysis.. https://doi.org/10.1016/s0140-6736(77)91529-x
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pKas for the acid dissociation of the carboxyl groups of bilirubin in water have been reported recently to be 8.1-8.4, or higher. These high values were attributed to intramolecular hydrogen bonding. They have led to suggestions that monoanions of bilirubin predominate at physiologic pH and are the species transported most readily into hepatocytes by carriers. Such high aqueous pKas are inconsistent with recent 13C nuclear magnetic resonance (NMR) measurements on mesobilirubin XIII alpha, done on aqueous solutions containing dimethyl sulfoxide. To investigate whether the presence of dimethyl sulfoxide leads to unreliable values when using 13C NMR spectroscopy to determine pKas of carboxylic acids that can undergo intramolecular hydrogen bonding, we measured the pKas of 13C-labeled fumaric, maleic, and phthalic acids in solutions containing up to 27 vol% dimethyl sulfoxide. In addition, we used 13C NMR to estimate the pKas of 2,2'-methylenebis[5-carbomethoxy-4-methylpyrrole-3-[1-13C] propanoic acid], a model for the two central rings of bilirubin. Our results show that 13C NMR of aqueous dimethyl sulfoxide solutions can be used with confidence to measure pKas of intramolecularly hydrogen-bonded carboxylic acids. They support our previous estimates for the pKas of bilirubin and confirm that intramolecular hydrogen bonding has little effect on the acidity of bilirubins in water. Together with previous studies and chemical arguments they strongly suggest that reported aqueous pKas of > 8, or even > 6, for the carboxyl groups of bilirubin are incorrect and that arguments used to rationalize them are questionable.
At 45 degrees C, phycocyanin 645 maximally undergoes a reversible and stable conformational change. The change is observed in the visible (chromophore) region of the absorption and circular dichroism (CD) spectra. In the absorption spectrum, the absorbance is lower at 45 degrees C but remains much closer to the normal spectrum than to a strongly denatured spectrum. In the CD, a similar situation exists except that a negative band on the blue edge of the spectrum is much more strongly affected at 45 degrees C than the other bands. On returning to 20 degrees C, all these changes are restored to the original states. The protein is an alpha 2 beta 2 dimer at both 20 and 45 degrees C, and CD in the far-UV shows the identical protein secondary structures at both 20 and 45 degrees C. Fluorescence studies show that energy transfer occurs at both temperatures. At 50 degrees C the results are saliently different as the secondary structure changes and the spectral changes are mostly irreversible. At 50 degrees C, some monomers (alpha beta) are produced, and these monomers are very unstable at that temperature, resulting in the formation of some fully denatured polypeptides. Stable monomers can be produced at 20 degrees C and have visible absorption and CD spectra identical to the dimer at 45 degrees C. Therefore, the chromophores are reporting a tertiary conformational change at 45 degrees C, in which the two halves of the dimer each assume a monomer-like conformation prior to dissociating. These results are compared with a hypothesis for the chromophore topography, and the CD change at the blue edge of the spectra may result from the separation at 45 degrees C of a chromophore pair.
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