442. The chemistry of the bile pigments. Part IV. Spectro-photometric titration of the bile pigments

Gray, C. H.; Kulczycka, A.; Nicholson, D. C.

doi:10.1039/jr9610002276

Cited by 50 publications

(23 citation statements)

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“…(XV) (XVI) basic pyrrolenine nitrogen atom within the chromophore. The base strength decreases in the order shown, as found by spectrophotometric titration studies (Gray et al 1961). It appears therefore that the base strength decreases with increasing length of conjugation and the extent of this can, in turn, be assessed from the position of the visible absorption maximum.…”

Section: Vol 117 275supporting

confidence: 66%

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The oxidation products of crude mesobilirubinogen

Stoll

Gray

1970

Biochemical Journal

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Bile pigment esters were separated by ascending t.l.c. Apparently pure pigments, obtained by ferric chloride oxidation of crude mesobilirubinogen, derived from commercial bilirubin by reduction with sodium amalgam, were shown to be complex mixtures. Successive chromatography of their dimethyl esters on silica gel in methyl acetate-methyl propionate-dichloromethane-carbon tetrachloride (1:1:1:1, by vol.), ethyl methyl ketone-1,2-dichloroethane (1:2, v/v) and benzene-ethanol (100:3, v/v) revealed two major blue pigments (verdins), six major violet pigments (violins) and a red pigment (rhodin) together with numerous minor components. i-Urobilin dimethyl ester, prepared from mesobilirubinogen by dehydrogenation with aqueous iodine, was resolved into three major and at least four minor components on silica gel-kieselguhr (3:1, w/w) in benzene-ethanol (25:2, v/v). The chemical nature of these pigments was investigated by oxidation, by visible and u.v. spectroscopy, by mass spectrometry and by n.m.r. spectrometry. The evidence suggests unusual rearrangement of bilirubin during reduction leading to the formation of IIIalpha and XIIIalpha isomers. Isomeric forms of mesobiliviolin IXalpha and of i-urobilin IXalpha may also be formed.

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Section: Vol 117 275supporting

confidence: 66%

“…Preparation of crude me8obiliviolin and glaucobilin (Gray et al 1961). The petroleum extract of crude mesobilirubinogen from the reduction of bilirubin (100mg) was evaporated to dryness.…”

Section: Methodsmentioning

confidence: 99%

The oxidation products of crude mesobilirubinogen

Stoll

Gray

1970

Biochemical Journal

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“…However, precipitation of pigment, causing breaks or inflections in the titration curves, is a major problem with these methods, and spectrophotometric titrations are further complicated by autoxidation and aggregation of bilirubin and by the requirement for accurate extinction coefficients for the unionized and ionized forms of the pigment in water, which are not available. Using the spectrophotometric method, Gray et al (5) were unable to obtain accurate pK a values for bilirubin, but Moroi et al (11) found values of 6.1-6.5 for pK a1 and 7.3-7.6 for pK a2 , ascribing these remarkably high values, without explanation, to intramolecular hydrogen bonding (11). In contrast, using similar methods, Kolosov and Shapovalenko (9) estimated pK a1 and pK a2 for bilirubin to be 4.5 and 5.9, respectively.…”

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On the Acid Dissociation Constants of Bilirubin and Biliverdin

Lightner

Holmes

McDonagh

1996

Journal of Biological Chemistry

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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 K a values of the propionic acid groups of bilirubin in aqueous solutions. Recently, pK a values of 6.7-9.3 have been reported for bilirubin-values much greater than the value of ϳ5 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 pK a values of 99% Bilirubin and biliverdin are blue-green and yellow-orange pigments formed from heme during normal metabolism in humans and other animals (1, 2). Like heme, biliverdin and bilirubin have two propionic acid groups and low solubility in water (1-3). The acidity constants of bilirubin have been determined many times during the past 40 years (4 -13), but those of heme and biliverdin have not been measured (3). A mean pK a of ϳ5.3 has been predicted for porphyrin dicarboxylic acids (14), and pK a values of ϳ5.0 and ϳ7.2 have been estimated for biliverdin (3). A wide range of pK a values has been reported for bilirubin, with most investigators favoring a pK a of 6.2 to 6.5 (15), but recent determinations suggest that the true pK a values are even higher (6.8 to Ն 9.3) because of intramolecular hydrogen bonding (11,12). Since most carboxylic acid pK a values are ϳ5 and values Ͼ6 are seldom encountered (16 -18), these recent determinations imply a remarkable and unprecedented effect of intramolecular hydrogen bonding on proton dissociation. To investigate this effect, we have used 13 C NMR to study the dissociation of hydrogen-bonded and non-hydrogen-bonded pyrrolic acids and to determine the pK a 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. Being remote from the propionic acid side chains, the alkyl substituents on the lactam rings are expected

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“…Titration of bilirubin in aqueous solutions suggests a pK value of 7-8. 42 Since bilirubin tends to form insoluble aggregates below pH 8.0, determination of pK by titration of aqueous solutions of bilirubin can be misleading. 37 The pK values for bilirubin have been determined by 13 C nuclear magnetic resonance spectra, and potentiometric and spectrophotometric titrations in aqueous solutions.…”

Section: Chemistry Of Bilirubinmentioning

confidence: 99%