(Received for publication, August 15, 1995; and in revised form, November 7, 1995)
From the
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 values of the propionic acid groups of bilirubin in aqueous
solutions. Recently, pK
values of
6.7-9.3 have been reported for bilirubin-values much
greater than the value of
5 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
values of 99%
C-enriched (
CO
H)
[8
,12
-
C
]mesobilirubin-XIII
,
the corresponding biliverdin, and several monopropionic model compounds
by
C NMR spectroscopy. This technique allows direct
observation and quantitative measurement of the carboxylic acid and
carboxylate anion carbon signals. Analysis of the variation of carboxyl
C NMR chemical shift with pH gave rubin
pK
values of 4.2 and 4.9 and verdin
pK
values of 3.9 and 5.3 in aqueous
buffers containing only a very small quantity (0.086 mol fraction) of
dimethyl sulfoxide. When extrapolated to water, the
pK
values are essentially unchanged. The
data provide the first experimentally-determined
pK
values for a biliverdin. They indicate
that intramolecular hydrogen bonding has little effect on the acid
dissociation of bilirubin and suggest that the equilibrium acidity of
the bilirubin carboxylic acid groups is not abnormally high but similar
to the thermodynamic acidity found in other carboxylic acids, as
originally suggested by Overbeek et al. (Overbeek, J. T. G.,
Vink, C. L. J., and Deenstra, H. (1955) Recl. Trav. Chim. Pays-Bas 74, 81-84).
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, 2, 3) . The acidity constants of
bilirubin have been determined many times during the past 40 years (4, 5, 6, 7, 8, 9, 10, 11, 12, 13) ,
but those of heme and biliverdin have not been measured(3) . A
mean pK of
5.3 has been predicted
for porphyrin dicarboxylic acids(14) , and
pK
values of
5.0 and
7.2 have
been estimated for biliverdin(3) . A wide range of
pK
values has been reported for
bilirubin, with most investigators favoring a pK
of 6.2 to 6.5 (15) , but recent determinations
suggest that the true pK
values are even
higher (6.8 to
9.3) because of intramolecular hydrogen
bonding(11, 12) . Since most carboxylic acid
pK
values are
5 and values >6 are
seldom encountered(16, 17, 18) , these recent
determinations imply a remarkable and unprecedented effect of
intramolecular hydrogen bonding on proton dissociation. To investigate
this effect, we have used
C NMR to study the dissociation
of hydrogen-bonded and non-hydrogen-bonded pyrrolic acids and to
determine the pK
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 to have
little influence on the dissociation constants of the carboxyl groups.
[8,12
-
C
]Mesobilirubin-XIII
(1),
[8
,12
-
C
]mesobiliverdin-XIII
(2), [8
-
C]xanthobilirubic
acid (3, Table 1),
12-[8
-
C]despropionic
acid-12-ethylmesobiliverdin-XIII
(4),
12-[8
-
C]despropionic
acid-12-ethylmesobilirubin-XIII
(5), and
2-[1-
C]carbomethoxy-3,5-dimethyl-1H-pyrrole-4-propanoic
acid (6) (all 99%
C-enriched as
CO
H) were prepared as described previously (19, 20) or, for compounds 4 and 5, by
modification of reported methods(21) . Their
tetra-n-butylammonium salts were prepared as before (22) from the acid and tetra-n-butylammonium
hydroxide. NMR solutions ranging from 8
10
M to 2
10
M were
prepared in buffered deionized water or
water/(CD
)
SO mixtures. The
(CD
)
SO (99.9% d
) was
obtained from Cambridge Isotope Labs (Andover, MA). NMR samples were
prepared in NMR tubes by adding standard aliquots of a stock solution
of acid or its tetra-n-butylammonium salt to aqueous buffers.
The stock solutions were prepared as 6-8
10
M solutions in either H
O or in
(CD
)
SO and protected from light and oxygen.
Buffers were 0.1 M sodium acetate (for pH
3.2-6.8)
and 0.1 M Tris (for pH >
6.8). At pH < 3.2, either
0.1 M acetic acid, 0.1 M acetic acid-HCl, or 0.2 M HCl were used (nonbuffered). Phosphate buffers (0.1 M) were used to compare carboxyl
values derived from 0.1 M Tris buffer. No difference was detected at the same pH.
10-15 sample solutions were prepared at various pH values for use
in a complete titration curve. pH was determined using an Orion model
811 pH/mV microprocessor pH meter calibrated with standardized buffers.
Aqueous solutions were prepared by adding 50 µl of a 6-8
10
M stock solution of acid or its
salt dissolved in deionized H
O to 500 µl of buffer.
Aqueous dimethyl sulfoxide solutions were prepared by adding a
50-µl aliquot of a 6-8
10
M stock solution of acid or its salt in
(CD
)
SO to an aliquot of buffer. Final sample
concentrations typically ranged from 8
10
M to 2
10
M, with
the chemical shift (
) for the carboxyl group being
independent of concentration in this range. Only a single,
well-demarcated line appears in the carboxyl region of the
C NMR spectrum: that of the labeled carbon. Only trivial
UV-visible spectral changes, 0-2 nm bathochromic shifts, were
detected in the pigment solutions with added
(CD
)
SO, from 1.8 to 27 volume %, in pH 7.4 Tris
buffer, and solutions were usually optically clear. At 64 volume %
(CD
)
SO, the mesobilirubin absorption band
half-width decreases slightly, causing a 20% increase in molar
absorptivity and a 10-nm bathochromic shift of the absorption maximum.
Mesobilirubin-XIII
(1) and mesobiliverdin-XIII
(2) were less soluble than dipyrrole 3 and monopyrrole 6 at low pH. Their solutions were prepared and analyzed 1 or 2
orders of magnitude more dilute, and the
C NMR spectra
were run in 10-mm tubes with overnight scanning. Occasionally at low
pH, some turbidity developed; however, this did not interfere with
obtaining useful
values for the dissolved pigment.
NMR measurements of for carboxyl groups were
obtained with 60° pulse widths and duty cycles approximately 5
times T
on a Varian Unity Plus 500-MHz
spectrometer. Three separate determinations were carried out for each
acid; uncertainties in the measured pK
values were ± 0.05. The NMR instrument settings and
parameters specific to the Varian Unity Plus were as follows:
frequency, 125.706 MHz; spectral width, 28,368.8 Hz; acquisition time,
2.000 s; relaxation time, 0.000 s; pulse width, 5.0 µs; decouple,
H; high power, 40; decoupler continuously, on; Waltz 16
modulated software; double precision acquisition; line broadening, 1.8
Hz; and temperature, 25 °C. The number of acquisitions varied
depending on sample concentration. A sealed melting point capillary
insert filled with 50 µl of (CD
)
SO was used
as the lock and external reference to standardize all samples to an
independent of environment reference.
Carboxylic acid
pK values were determined either
graphically or by nonlinear regression analysis. In the first method,
titration curves were constructed by plotting
versus pH(19, 23) . The pK
values for monocarboxylic acids were read directly as the pH
when
= 0.5 (
C NMR titration curves were determined for the
following 99%
CO
H-enriched compounds:
mesobilirubin-XIII
(1) and mesobiliverdin-XIII
(2); their tetrapyrrole counterparts bearing only one propionic
acid (compounds 4 and 5, respectively); and di- and
monopyrrole calibration compounds, 3 and 6, which cannot
form intramolecular hydrogen bonds of the type seen in bilirubin. The
titration curves of all of these compounds showed two plateaux, at low
and high pH respectively, separated by
5 ppm. These plateaux
clearly represent the un-ionized and the fully ionized species,
respectively, and the chemical shift difference corresponds to the
titration shift,
=
-
Figure 1:
Upper panel, C NMR
titration curves showing the variation of carboxyl carbon
C NMR chemical shift (
) with changes in
pH for 99%
C-enriched
[8
-
C]xanthobilirubic acid (3) in
H
O-(CD
)
SO solutions, respectively.
The dashed lines connect the midpoint of the titration curve
at
= 0.5 (
As
noted previously for compound 6 and other carboxylic
acids(19) , the presence of (CD)
SO
cosolvent influences the titration curves by displacing them slightly
downward with increasing % (CD
)
SO. Yet their
shapes and the corresponding pK
values vary only
modestly over the range of
H
O/(CD
)
SO solvent mixtures used.
For example, for dipyrrinone acid 3 (Fig. 1), the
pK
values varied by only 0.13 pK units
over the range 9-64 volume % (2.5-31 mol %)
(CD
)
SO (Table 2). This surprisingly small
variation is probably due to the fact that in aqueous solutions
containing as much as 64 volume % dimethyl sulfoxide, water is still
the principle solvent (and base) on a molar scale. In fact, equimolar
solutions are not reached until the volume %
(CD
)
SO reaches
80%. Thus, when the solvent
is mainly water, especially when the volume % of
(CD
)
SO
27 (
91 mol % H
O),
aqueous dimethyl sulfoxide solutions can be used for estimating the
pK
values of water-insoluble carboxylic acids.
Previously, we noted that pK values determined
in aqueous dimethyl sulfoxide solutions could be extrapolated
accurately to known pK
values in
water(19) . For compound 6, the small correction to 100%
water was based on the excellent linear correlation between the
pK
values and the logarithm of the volume %
(CD
)
SO. Similar empirical correlations have
been noted previously(19) . The extrapolated pK
thus obtained (pK
= 4.7) is in
perfect agreement with that measured directly in water
(pK
= 4.7). For xanthobilirubic acid (3), the linear correlation (Fig. 1) led to a corrected
value of 4.6 for the apparent pK
in water.
Similarly, from the titration curves of the tetrapyrrole acids 4 and 5 (Fig. 2), which are monopropionic acid analogs
of biliverdin and bilirubin, the apparent pK
values were found to be 4.5 and 4.8, respectively, in aqueous
dimethyl sulfoxide (31 mol %), which extrapolated to 4.3 and 4.5,
respectively, for water (Table 2).
Figure 2:
Upper
panel, C NMR titration curves of tetrapyrrole
monopropionic acids 4 and 5 in aqueous (CD
)
SO
(0.31 mol fraction). The dashed lines connect the midpoint of
the titration curve at
= 0.5
(
The data do not
permit an accurate determination of pK and pK
for 1 and 2. However, the values can be estimated from the
titration curve of a calibration standard, adipic acid. From the known
acidity constants, pK
= 4.44
and pK
= 5.44(16) , and
the measured
versus pH titration curve, we
found that pK
for adipic acid
corresponds to the value of
when
=
Biliverdin and bilirubin are water-insoluble pigments that occur widely in nature(1) . Although similar in constitution, they have markedly different physicochemical properties largely because of their different three-dimensional structures and modes of hydrogen bonding. Biliverdins tend to assume helical, ``lock-washer,'' conformations in solution that are stabilized by intramolecular hydrogen bonding between NH groups and the unprotonated nitrogen atom(26, 27) ; whereas, bilirubin adopts enantiomeric conformations that are shaped like ridge tiles and are stabilized by intramolecular hydrogen bonds between the pyrrole/lactam functions and the propionic carboxyl (or carboxylate) groups (26, 28) (Fig. 3). In solution, enantiomeric conformers of bilirubin interconvert via nonplanar intermediates in which the hydrogen bonding network is never completely broken(28, 29) (not via flat non-hydrogen-bonded conformers as proposed recently by Ostrow et al.(30) ), and their chirality depends on their backbone shape (not on the unsymmetrical methyl-vinyl substitution pattern as erroneously stated by the same authors(30, 48) ). The ridge tile conformation is the only one that has been observed in crystals of bilirubin and its carboxylate salts (31, 32, 33) , and spectroscopic, particularly NMR, studies supported by energy calculations strongly indicate that similar conformers prevail in solution, even in the dipolar protophilic solvent dimethyl sulfoxide (22, 28, 34, 35) . The preferred conformation of bilirubin in protein-free aqueous solutions is not known, but calculations and the absorption spectra of freshly-made dilute solutions indicate that it is similar to that in dimethyl sulfoxide(29, 36) .
Figure 3: Upper left, ball and stick structure of biliverdin in its most stable porphyrin-like helical conformation; lower left, line drawings showing possible intramolecular hydrogen bonding between the propionic acid groups of biliverdin and its monoanion; upper right, ball and stick structure of bilirubin in its most stable intramolecularly hydrogen-bonded ridge-tile conformation; lower right, line drawings of bilirubin monoanion and dianion in their most stable intramolecularly hydrogen-bonded ridge-tile conformations. Atomic coordinates are from structures determined by x-ray crystallography (26, 27, 31, 33) and by molecular dynamics calculations(28) .
The acid-base properties of
biliverdin and bilirubin are undoubtedly important determinants of
their transport, metabolism, and distribution within organisms, and the
pK values of the carboxyl groups of bilirubin are
thought to be a key factor in its hepatic transport and neurotoxicity
and in the formation of pigment gallstones (30, 37, 38) . In view of the extensive
literature on bile pigments and related compounds, it is surprising
that there are no reliable measurements of the pK
values of biliverdin and that the pK
values
of the carboxyl groups of bilirubin are controversial. There are
several probable reasons for this dearth of definitive data. First,
until recently there has been no persuasive reason to suppose that the
carboxyl pK
values are anomalous or substantially
different from the expected values of
4.5-5.5. Second,
commercial preparations of bilirubin and biliverdin are generally
impure, making them unsuitable for accurate pK
measurements, and difficult to purify(1) . Third,
bilirubin is prone to photoisomerization, even in dim light (39) , and unstable in alkaline aqueous solutions in the dark,
undergoing rapid radical reactions (1) that have often been
neglected in pK
studies. Fourth, ionization of the
carboxyl groups has little effect on the chromophores of biliverdin and
bilirubin, making spectrophotometric methods of pK
determination insensitive and unreliable. Last, but probably the
main reason, is the low solubility of the pigments in
water(1, 3) , particularly at pH < 7, which can
cause precipitation and phase separation during pK
determinations.
There appear to be only two reports in the
literature concerning the pK values of biliverdin.
Carey and Spivak (3) estimated pK
and pK
to be
5.0 and 7.2,
respectively, but precipitation of pigment at
pH 6.6 during
acidimetric titration banjaxed accurate measurements(3) . Gray et al.(5) were similarly unsuccessful. Many more
measurements of the pK
values of bilirubin have
been published (Table 3). Several groups have used potentiometric
or spectrophotometric back-titrations in water, methanol/water, or
water/detergent(5, 7, 9, 11) .
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 un-ionized 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
values for bilirubin, but Moroi et al.(11) found values of 6.1-6.5 for
pK
and 7.3-7.6 for
pK
, ascribing these remarkably high
values, without explanation, to intramolecular hydrogen
bonding(11) . In contrast, using similar methods, Kolosov and
Shapovalenko (9) estimated pK
and pK
for bilirubin to be 4.5
and 5.9, respectively.
Because of the problems in working with
aqueous solutions, Lee et al.(8) used
dimethylformamide, in which bilirubin is soluble, as cosolvent for
spectrophotometric titrations(8) . They estimated
pK and pK
values for bilirubin in water to be 4.3 and 5.3, respectively.
Hansen et al.(10) followed the ionization of
bilirubin in dimethyl sulfoxide by natural abundance
C NMR
and estimated the mean pK
of the two carboxyl
groups, by extrapolation, to be 4.4 in water(10) . These values
are close to those expected for aliphatic carboxyl groups (16) . More recently, Harman et al.(40) attempted to measure pK
values
for bilirubin by micellar electrokinetic capillary chromatography but
obtained inconclusive results.
Moroi et al.(11) and Ostrow et al.(12) used
solubility methods to determine the pK values for
bilirubin(11, 12) . In general, the solubility method
is less accurate than potentiometric, spectrophotometric, or
conductimetric methods and is particularly prone to error if the acid
contains impurities, especially impurities that are more soluble than
the acid itself. Moroi et al.(11) used analytically
pure bilirubin and estimated solubilities spectrophotometrically,
whereas Ostrow et al.(12) used poorly characterized
bilirubin preparations derived from unpurified commercial material and
estimated bilirubin solubilities spectrophotometrically and by the
nonspecific and rather insensitive diazo reaction. Both groups were
unable to accurately measure the intrinsic solubility of un-ionized
bilirubin and had to use estimates in calculating pK
values. Moroi et al.(11) estimated
pK
to be 6.0-6.5 and
pK
to be 7.3-7.7, whereas Ostrow et al.(12) found pK
to be 5.6-6.8 and pK
to be
>9.2. The latter incredibly high and widely separated
pK
values were attributed to retarded proton
dissociation caused by intramolecular hydrogen bonding. Recognizing the
methodological deficiencies (13, 30, 41) of
their earlier determinations, Ostrow and co-workers (13) subsequently remeasured dissociation constants for
bilirubin using a complicated solvent partitioning technique involving
extraction of chloroform solutions with aqueous buffer, back-extraction
of aqueous extracts with chloroform, evaporation of the extracts, and
diazo assay of the residues in dimethyl sulfoxide containing sodium
taurocholate and sodium EDTA(13) . They concluded that the two
pK
values of bilirubin are not widely separated,
as they had reported previously, but are similar, with values of
pK
= 8.12 and
pK
= 8.44. Derivation of these
values was based on questionable assumptions regarding the
solubilities, aggregation, and phase-transfer properties of bilirubin
species. Again intramolecular hydrogen bonding was invoked as the cause
of such remarkably high values.
The continuing disagreement over the
pK values of bilirubin(41, 42) ,
the increasing use of high values in the biomedical
literature(30) , and the unprecedented effects being ascribed
to intramolecular hydrogen bonding led us to reinvestigate the problem
using
C NMR. This method is rapid and sensitive, allows
direct observation of the carboxyl groups undergoing deprotonation (43) , and, unlike partitioning and solubility methods, is
insensitive to traces of polar impurities in the acid and does not
require extensive manipulation or extraction of solutions. Although the
technique has not been used extensively, its utility for measuring
dissociation constants accurately is well
established(44, 45) . It is particularly useful for
bile pigments, because it focuses specifically on the carboxyl group.
Ionization of pyrrole or lactam groups, which may occur at extreme pH
values and can complicate spectrophotometric titrations, is not
detected(21) . Natural abundance
C NMR (10) has already been used to estimate pK
values for bilirubin, but the results have been dismissed because
of the use of dimethyl sulfoxide as
solvent(12, 13, 30) . In our studies, we used
highly labeled compounds and high field NMR to increase sensitivity and
allow accurate measurements on dilute solutions. Since
COOH-labeled bilirubin and biliverdin are not easy to
synthesize, we used the corresponding available meso-XIII
analogs
as surrogates. The symmetrical side chain substitution pattern of these
compounds simplifies the NMR spectra and amplifies the
C
signals but is expected to have negligible effect on the intramolecular
hydrogen bonding or pK
values compared with the
natural analogs. We also measured the pK
values of
a dipyrrolic pigment that cannot undergo intramolecular hydrogen
bonding, of tetrapyrrole analogs with only one propionic acid side
chain, and of the dicarboxylic acid standard adipic acid. Use of this
interrelated set of compounds allowed us to examine specifically the
effects of intramolecular hydrogen bonding and electrostatic
interactions on pK
values. We used perdeuterated
(CD
)
SO as a cosolvent to facilitate the rapid
preparation of solutions and to ensure that solutions remained
homogeneous over a wide pH range, and we kept the concentration of
cosolvent in the final solutions to
31 mol %. Solutions were
optically clear and devoid of colloidal or particulate material, except
at low pH, where there was sometimes some turbidity. However, any
insoluble pigment present is not detected by the NMR under the
conditions used, nor does it seem to interfere with the determination
of the carboxyl chemical shift of the dissolved pigment. Although
pK
values determined by
C NMR
chemical shifts in (CD
)
SO may differ markedly
from those determined in water, pK
values of aryl
and pyrrolic carboxylic acids derived by NMR in aqueous solutions
containing up to 31 mol % (CD
)
SO differ little
from values determined in the absence of the organic cosolvent (28) . Thus, the caveat that pK
values
measured in aqueous organic solvents cannot be extrapolated to give
reliable pK
values for water (46) does not
apply to the present
C NMR method when aqueous dimethyl
sulfoxide solutions containing excess water are used.
As found
previously (21) (Table 2), values for the
pK of the monopyrrolic model compound 6 in
aqueous (CD
)
SO solutions were within about 0.1
pK
unit of the value measured in water, and
extrapolation from the aqueous (CD
)
SO data gave
a value for water that was identical within the experimental error to
the measured value of 4.7. Similarly, the water-insoluble dipyrrinone
xanthobilirubic acid 3 showed typical sigmoid pH versus chemical shift curves in aqueous (CD
)
SO
solutions (Fig. 1). Apparent pK
values,
determined from the midpoints of these curves varied by only 0.13
pK
units over the range 2.5-31 mol %
(CD
)
SO and were similar to the corresponding
values for monopyrrolic acid 6. In addition, the slopes of the
pK
versus log volume %
(CD
)
SO lines were similar for compounds 3 and 6, allowing an extrapolated value of 4.6 to be
determined for the apparent pK
of xanthobilirubic
acid in water.
The biliverdin analog 4 with one propionic
acid showed a typical sigmoid titration curve in 31 mol %
(CD)
SO (Fig. 2), qualitatively similar
to those obtained with the mono and dipyrrolic acids 3 and 6. This curve undoubtedly corresponds to ionization of the lone
carboxyl group. The apparent pK
for 4 in 31
mol % (CD
)
SO was 4.5-close to, but
slightly lower than the corresponding values for 3 and 6.
Assuming that the slope of the pK
versus log volume % (CD
)
SO plot for 4 would be similar to that for xanthobilirubic acid, we estimated the
pK
for 4 in water to be
4.3. Thus, the
measured and extrapolated pK
values for the three
monocarboxylic model compounds, 3, 4, and 6, for
which intramolecular hydrogen bonding of the type seen in bilirubin is
impossible, were similar and within the range expected for propionic
acid groups. These results indicate that the presence of mono-, di-, or
tetrapyrrole components has no unusual effect on propanoic side chain
acidity, and they lend further support to the reliability and utility
of
C NMR for determining the pK
values of bile pigments.
In contrast to the propionic acid
group in verdin 4, the single propionic acid group in rubin 5 can participate in intramolecular hydrogen bonding of the type seen
in the ridge tile conformation of bilirubin. Nevertheless, in 31 mol %
(CD)
SO, 5 displayed a sigmoid titration
curve (Fig. 2) that resembled the titration curve for the
corresponding verdin 4 and yielded an apparent pK
of 4.8. Extrapolation to zero mol %
(CD
)
SO, as for the verdin 4, gave an
estimated pK
of 4.5 for 5 in water.
Comparison of the pK
values of 4 and 5 indicates that intramolecular hydrogen bonding within a ridge-tile
conformation has little effect, if any, on the pK
.
Hydrogen bonding may retard the dissociation of the COOH proton
slightly, but it does not reduce the dissociation constant by orders of
magnitude, as suggested
previously(11, 12, 13, 30, 41) .
The titration curves of the biliverdin analog 2 and the
bilirubin analog 1 in aqueous (CD)
SO
solutions were qualitatively similar and showed unambiguously that the
pK
values for both compounds are below 5.5. The
pK
values for the first and second ionizations,
estimated using adipic acid as a standard and extrapolated to zero mol
% (CD
)
SO, were 3.9 and 5.3 for the verdin 2 and 4.2 and 4.9 for the rubin 1. From the measured
pK
of the monopropionic verdin 5,
pK
and pK
for the biliverdin analog 2 would be expected to be 4.0
and 4.6, respectively, taking into account the statistical factor but
assuming negligible electrostatic repulsion between the carboxylate
groups in the dianion. Thus, the measured pK
was close to the calculated value, but the measured
pK
was greater than expected. The
difference between the measured and calculated
pK
values and the rather large
difference of 1.4 pK
units between
pK
and pK
suggests that the monoanion of verdin 2 may be stabilized
by intramolecular hydrogen bonding (Fig. 3) and that there may
be significant electrostatic repulsion between the propionate groups in 2, which is reasonable for a helical conformation. In contrast,
the experimental values for both pK
and pK
for the rubin 1 were close to the values (4.2 and 4.0) calculated from the measured
pK
of the monopropionic rubin 4. This
excellent agreement indicates that electrostatic interactions between
the carboxylate groups in bilirubins are unimportant with respect to
pK
values, as expected for ridge-tile conformers.
Again, comparison of the pK
values of the verdin 2 with those of the rubin 1 indicates that intramolecular
hydrogen bonding of the carboxyl groups has no significant effect on
either pK
or
pK
.
These studies provide the first
dissociation constants for the carboxyl groups of a synthetic
biliverdin closely resembling the naturally occurring biliverdin
IX, and they strongly suggest that the pK
values for the two carboxyl groups of monomeric bilirubin in
water are close to 4.2 and 4.9, respectively. These values are
consistent with the observed solubility properties of bilirubin (4) . Our results are in good agreement with previous
measurements by Lucassen(6) , Hansen et
al.(10) , and by Lee et al.(8) , and with
the values originally assumed by Overbeek et al.(4) some 40 years ago. The much higher values that have
been reported recently have been rationalized by invoking
intramolecular hydrogen
bonding(11, 12, 13, 30, 41) .
However, these rationalizations appear to be based on misconceptions
concerning the effects of ionization and solvent on the structure of
bilirubin in solution and erroneous ideas concerning the effects of
hydrogen bonding on acidity. Some misconceptions are that the
hydrogen-bonded structure of bilirubin in solution is
rigid(30, 47) , that intramolecular hydrogen bonding
is not maintained in dimethyl
sulfoxide(12, 13, 30, 48) , that
flat (non-hydrogen-bonded) conformations of bilirubin occur in
solution(12, 30) , that intramolecular hydrogen
bonding in the free acid suppresses ionization of the carboxyl
groups(11, 12, 13, 30, 41, 49, 50) ,
and that the carboxyl groups no longer undergo intramolecular hydrogen
bonding after they are ionized(12, 49) . Such
misconceptions have fueled the view that the acid dissociation
constants of bilirubin are abnormally low because of hydrogen bonding.
In addition, the focus on deprotonation or dissociation has neglected
the potential stabilization of the carboxylate anion by intramolecular
hydrogen bonding. In general, the effect of intramolecular hydrogen
bonding on the pK
values of dicarboxylic acids is
small except for instances in which intramolecular hydrogen bonding
between carboxyl and carboxylate groups occurs in the
monoanion(17, 51, 52) , which is sterically
unlikely for bilirubin, though possible for biliverdin. In such acids,
for example maleic or phthalic acid, hydrogen bonding invariably lowers
pK
rather than increases it, as
claimed for bilirubin(30) . In bilirubin, proton dissociation
might be facilitated somewhat by stabilization of the resulting
carboxylate anion by intramolecular hydrogen bonding to a dipyrrinone,
but this stabilization would be offset by electrostatic repulsion
between carboxylate and lactam oxygens. When large effects of
intramolecular hydrogen bonding occur in dicarboxylic acids, they are
manifested by abnormally large values of K
/K
(17, 51, 52) , which is clearly not the
case for bilirubin.
The biochemical relevance of the earlier
measurements by Hansen et al.(10) and by Lee et
al.(8) has been cursorily disregarded on the unfounded
basis that intramolecular hydrogen bonding within the ridge-tile
structure of bilirubin is not maintained in dimethylformamide and
dimethyl sulfoxide(12, 13, 30) , the solvents
in which the measurements were made. For dimethylformamide, there is
little evidence to show whether this is true or not, but for dimethyl
sulfoxide there is extensive NMR and spectroscopic evidence that
intramolecular hydrogen bonding is maintained, albeit slightly
altered(34, 35, 53) . This evidence indicates
that the ridge tile structure prevails, although somewhat flattened,
and that, at high sulfoxide concentrations in organic solvents or in
pure anhydrous dimethyl sulfoxide, a sulfoxide group intercalates
between each propionic carboxyl and its apposite dipyrrinone NH
groups(35, 54, 55) . Although such solvation
might influence proton dissociation, it is not likely to increase it by
several orders of magnitude. In the present studies, where all
measurements were done in solutions containing excess water, solvation
or putative hydrogen bond breaking by dimethyl sulfoxide is evidently
irrelevant(56) , as shown by the weak effects of dimethyl
sulfoxide concentration on the carboxyl chemical shifts and the
measured pK values. This conclusion is supported
by measurements of the second ionization constants for adipic, malonic,
and maleic acids by the same technique, which gave values identical to
those measured in water. (
)Thus, there is no plausible
reason to expect that intramolecular hydrogen bonding would decrease
the acidity of bilirubin, and arguments invoking hydrogen bonding to
explain improbably large measured pK
values are
specious.
We conclude that the acid dissociation constants of
natural bilirubin and biliverdin are likely to be almost the same and
within the normal range for aliphatic propionic acids. Consequently,
when unbound bilirubin and biliverdin occur in the aqueous phase of
living tissues at physiologic pH, the predominant species present will
be the dianions, not the monoanions or un-ionized acids, and current
theories of bilirubin distribution, toxicity, and gallstone formation
that assume otherwise are implausible and need reevaluating. We
detected no major effects of intramolecular hydrogen bonding on acid
dissociation of the bilirubin analog mesobilirubin-XIII and only a
weak effect on the second ionization of the biliverdin analog
mesobiliverdin-XIII
, confounding the prevalent notion that such
bonding inhibits proton dissociation from bilirubin. Intramolecular
hydrogen bonding is undoubtedly important in the biological chemistry
of bilirubin-not because it retards carboxyl dissociation, but
because it engenders lipophilicity(57) .