(Received for publication, September 15, 1994; and in revised form, October 20, 1994)
From the
Select hydrogen-carbon distances have been determined from C{
H} heteronuclear Overhauser
effects observed in a 99% carbon-13 enriched
CO
H bilirubin analog,
[8
,12
-
C
]-mesobilirubin
XIII
. Analysis of the data confirms that the propionic acid
carbonyl lies within hydrogen bonding distance to the dipyrrinone
lactam and pyrrole N-H groups in chloroform and indicates,
surprisingly, that those distances are only slightly longer in dimethyl
sulfoxide solvent or when the carboxyl group is ionized in pH 7.4
aqueous buffered solutions of the pigment. The data supports the
presence and persistence of folded, intramolecularly hydrogen-bonded
bilirubin conformations in solution.
Bilirubin (Fig. 1),) the cytotoxic yellow-orange pigment
of jaundice is biosynthesized in normal metabolism of healthy adults at
the rate of about 300 mg per day. This water-insoluble and lipophilic
linear tetrapyrrole dicarboxylic acid is found characteristically in
mammals, where it is produced in copious quantities by catabolism of
heme, principally from the hemoglobin of red blood
cells(1, 2) . Bilirubin is essentially unexcretable in
its native form but is efficiently cleared by the body following
conversion in the liver to water-soluble glucuronides that are promptly
secreted into bile. Accumulation of bilirubin and its glucuronides in
the body is caused by certain pathologic conditions and sometimes
results in jaundice(3) . Although impaired excretion of the
glucuronides occurs in many types of hepatobiliary disease, retention
of native bilirubin is principally observed in newborn
babies(4) . What limits the facile excretability of bilirubin
is its poor water solubility (estimated K
3
10
M for bilirubin in water at 37
°C)(5) , high lipid/water partition
coefficient(6) , and proclivity to form association complexes
with serum albumin and other
proteins(1, 2, 3, 5, 6) ,
three interrelated properties that dominate the transport and
metabolism of the pigment in
vivo(1, 2, 3, 7) .
Figure 1:
A, porphyrin-like
and B, linear representations of bilirubin. C, linear
representation of mesobilirubin-IV.
Although
the structure of bilirubin has been well known since it was proved over
50 years ago(8) , the interesting biological and unusual
solubility properties of bilirubin (6) do not correlate well
with conventional linear representation (Fig. 1B)
favored by Fischer (8) or the porphyrin-like structure (Fig. 1A) that would follow logically from the
structure of the heme precursor. If bilirubin adopted such
conformations, which we now know are sterically disfavored, it would be
predictably polar, not lipophilic, and very likely excretable across
the liver into bile without resort to glucuronidation(9) . The
unusual properties of bilirubin are hardly changed in its analogs with
vinyl groups reduced to ethyls as in mesobilirubin IX, or even
when they are symmetrically arranged as in mesobilirubin III
or
XIII
. But major changes attend relocating the propionic acid
groups away from C-8 and C-12. For example, transposing the propionic
acid groups with the adjacent methyl groups at C-7 and C-13 yields a
pigment, mesobilirubin IV
(Fig. 1C), that is more
polar, and excretable without requiring glucuronidation or other
structural modification(9, 10) .
Correlation of
propionic acid group location and the chemical, biological, and
physical properties of bilirubin is, however, only one important
structural consideration. Other stereochemical determinants include
configuration and conformation of the tetrapyrrole skeleton, elements
of three-dimensional structure common to all bilirubin pigments.
Conformation plays a key role, especially the changes in shape brought
about by rotations of the dipyrrinone groups about the central CH groups at C-10. Such rotations bring about major stereochemical
changes, interconverting the linear and porphyrin-like structures of Fig. 1, A and B, inter alia, and
generating a vast array of pigment shapes, of which one type is
possessed of unique properties. In this conformation, the dipyrrinones
are rotated about the C-9 to C-10 and C-10 to C11 carbon-carbon single
bonds so as to create a ridge-tile shape where the propionic acid
CO
H groups and dipyrrinone NH and C=O groups are
brought into sufficiently close proximity to allow them to engage in
intramolecular hydrogen bonding. Such intramolecular hydrogen bonding
stabilizes the ridge-tile conformation (Fig. 2) to such an
extent that it is thought to persist and have a dominating influence on
conformation under a variety of solvation and ionization conditions. It
is also thought to explain many properties of the molecule, such as its
high oil-water partition coefficient(6) , its resistance to
hepatobiliary excretion(9) , and, unusual for a carboxylic
acid, its insolubility in aqueous sodium bicarbonate
solution(6) .
Figure 2: Interconverting intramolecularly hydrogen-bonded enantiomeric conformers of bilirubin.
Stabilization of the bilirubin ridge-tile
conformation by a complementary network of intramolecular hydrogen
bonds was first detected by x-ray crystallography (11, 12) in the solid where bilirubin is folded into
either of two ridge-tile-shaped enantiomers (Fig. 2).
Intramolecular hydrogen bonding has also been detected by nuclear
magnetic resonance (NMR) ()in
solution(13, 14) , where the bilirubin conformers are
thought to persist as a pair of interconverting conformational
enantiomers (Fig. 2), and in organic solvents that do not
strongly perturb the matrix of intramolecular hydrogen bonds. The
conformational structure is less clear in solvents or other agents that
may disrupt the folded pigment's hydrogen bonding matrix. In the
strong hydrogen bond acceptor, (CD
)
SO,
C NMR analysis of segmental motion in the propionic acid
chains indicates that the -COOH residues are tethered to the
dipyrrinones via bound solvent molecules (15) , but the precise
conformation of the pigment in (CH
)
SO is
uncertain(16) . In metabolism, bilirubin is found bound to
albumin or other proteins, in membranes, micelles or to a lesser
extent, in aqueous solution. But here again, the conformation of
bilirubin is poorly understood. Yet, an intramolecularly hydrogen
bonded conformation is thought to be essential for hepatic
glucuronidation and excretion(9) . In order to understand
whether intramolecular hydrogen bonding persists in solutions of
bilirubin, we have prepared
[8
,12
-
C
]-mesobilirubin
XIII
, measured the NMR
C{
H}
heteronuclear nuclear Overhauser effects (NOE) between its dipyrrinone
N-H's and propionic acid
CO
H
groups, and calculated their non-bonded distances. These results are
important because they help define a comprehensive picture of
intramolecular hydrogen bonding and the probable shape and
stereochemistry of bilirubin in solution, and also illustrate a method
for determining the structure of bilirubin in biological tissues.
[8,12
-
C
]-Mesobilirubin
XIII
(99%
C-enriched), its dimethyl ester and methyl
[8
-
C]-xanthobilirubinate were
prepared as described previously(17) . The
tetra-n-butylammonium salt was prepared as described
previously (18) by suspending
[8
,12
-
C
]-mesobilirubin
XIII
(5.0 mg, 7.5
10
mmol) in
dichloromethane (2 ml) and adding excess methanolic
tetra-n-butylammonium hydroxide (Aldrich) (15.0 µl of 1 M, 1.5
10
mmol). The solution was
evaporated to dryness with a stream of dry N
, then dried
overnight under vacuum (0.1 torr) over P
O
. NMR
solutions ranging from 7.5
10
to 1.5
10
M were prepared in deuterated solvents,
CDCl
or (CD
)
SO, obtained from
Cambridge Isotope Labs. NMR solutions 3.5
10
M in aqueous medium were prepared by dissolving the
bis-tetra-n-butylammonium salt of
CO
H-labeled mesobilirubin XIII
in
(CD
)
SO, then diluting with pH 7.4 0.1 M phosphate buffer to give a final solution with 30% (volumes)
(CD
)
SO. The final pH is 7.4 and the
concentration of (CD
)
SO in the aqueous buffer
is 3.8 M (for a molar ratio of
15:1,
H
O:(CD
)
SO).
All experiments were
performed on a General Electric GN-300 NMR spectrometer. All samples
were degassed with argon and shielded from light. T measurements of the
C-labeled pigments were
performed by the inversion recovery method. All 75.58-MHz
C spectra were collected using a 90° pulse width of
18.0 s. The
C spectra was obtained coupled, with a minimum
of 256 scans and a pulse delay of 20 s (4 times T
). The
printed carbon resonances were digitized using Un-Plot-It and the areas
were determined by the Un-Plot-It software. No further data
manipulation was performed on the raw data.
C{
H} Heteronuclear NOEs were
determined by pulse techniques developed previously(19, 20, 21, 22, 23, 24) and
involve irradiating selected hydrogens (
H) while observing
signal enhancement of the
COOH
C NMR
resonance. The semiquantitative NOE values (an average of three to five
independent determinations and reported as average percent NOE) were
determined by subtracting integrated areas of the
COOH
off-resonance from the
COOH resonance observed during
selective
H saturation irradiation then divided by the
resonance peak area. For NOE determinations in aqueous buffer, a 1331
water suppression experiment was used in order to reduce the
H NMR signal due to H
O(25) . Normal
H saturation experiments could not be used due to the
exchange of the N-H's with solvent. The 1331 technique does not
reduce the magnitude of the H
O signal in the NMR; rather,
it applies a wave to the spectrum where no data points are taken in the
region of the H
O resonances. Data points are taken on each
side of the H
O resonance.
Approximate non-bonded H to
C interatomic distances were calculated
from the NOE results by calibration to a known
H to
C reference distance: in our work, the
C to
-H distance (2.12 Å) in the propionic acid segment,
-C(
)H
-C(
)H
-
COOH. The
formula (20) used for relating NOE to distance
is,
where r is the non-bonded distance between the COOH carbon and the proton of interest, (r
) (ref.) = 2.12 Å and
C{
H} (ref.) = 38% between the
-H to carboxylic acid carbon. The
-H to CO
H
carbon distance is constant for either
-H and is fixed by the
H-C
and C
-C(=O) bond distances
and the H-C
-C angle. These vary only insignificantly in
propionic acid. In CDCl
solvent, the two
-hydrogens
have distinctly different chemical shifts. Irradiation of either gives
exactly the same
C{
H} NOE (38%), and
that value was used for estimating the N-H
C(=O) non-bonded distances of this study according
to the equation above.
In NMR spectroscopy, the NOE occurs when the net intensities
of resonances change while a selected resonance is perturbed by
saturation or inversion. Its importance lies primarily in the fact that
the resonances which change their intensities are those from spin
systems that lie close to those directly affected by the
perturbation(19, 26) . This means that when the non-bonded distance between two hydrogens is small,
irradiation of one hydrogen can lead to an enhanced signal for the
other. With appropriate calibration non-bonded interatomic distances
may be calculated from NOE measurements(19, 27) . Such
homonuclear NOE's have been seen in bilirubin between the
dipyrrinone pyrrole and lactam hydrogens(13) , which lie some 2
Å apart and between the C-5 H and C-7 CH3, and between the C-15 H
and C-13 CH which lie some 3 Å apart. They have
provided evidence for the syn-Z configuration in the
dipyrrinones of bilirubin in solution (28) and for
conformational enantioselection forced by non-bonded steric effects in
bilirubin analogs with methyl groups on the
or
sites on the
propionic acid chains(29, 30) .
Although NOE
studies are most common in H{
H}
homo-NOE experiments, they are not limited to such and have been
expanded to include more complicated heteronuclear experiments,
especially those involving
H with
C resonances (21) or
H and
P
resonances(21) . Recently, heteronuclear
C{
H} NOE measurements have played a
major role in detecting hydrogen bonds (
H
O=
C) and
determining the secondary structure of peptides and
oligonucleotides(19, 20, 22) . In even
simpler systems, (E)- and (Z)-stereoisomers of
4(
-arylethylidene)-2-phenyl-5(4H)-oxazolones could be
distinguished by
C{
H} NOE
measurements (23) , while hydrogen bonding of water to
2-pyridones has been studied by
C{
H}
two-dimensional NOE spectroscopy (24) . In an especially
relevant example,
C{
H} heteronuclear
NOE measurements on valinomycin were used to identify the amide
carbonyl and NH partners involved in the intramolecular hydrogen
bonding that preserves its secondary structure(20) .
In our C{
H} NOE study of
CO
H-labeled mesobilirubin, in either
deuterated chloroform (CDCl
) or deuterated dimethyl
sulfoxide ((CD
)
SO) solvents, saturation
irradiation of either the pyrrole or lactam N-H's led to an
enhancement of the
COOH signal in the
C NMR.
The results are shown in Fig. 3and provide the first clear
evidence that the N-H and
CO
H groups of
bilirubin in solution lie very close to one another. The non-bonded H
to C (N-H to
COOH) distances calculated from the observed
NOE's in CDCl
are very nearly the same as those found
in crystals of bilirubin, where the pigment has been shown to adopt an
intramolecularly hydrogen bonded ridge-tile
conformation(11, 12) . They are also very nearly the
same as those found in the global minimum energy conformation of
bilirubin and mesobilirubin XIII
by molecular dynamics
calculations (31) (Fig. 3). The pigment in CDCl
solvent is thus shown by
C{
H}
NOE spectroscopy to adopt the hydrogen-bonded ridge-tile conformation.
Figure 3:
A, C{
H}
NOE enhancements of the
COOH resonance at 179.3
in
CDCl
and 174.0
in (CD
)
SO by
saturation irradiating the pyrrole and lactam hydrogens. B,
approximate non-bonded H to C distances (A) in the
N-H
O=
C hydrogen bond matrix
calculated from the observed
C{
H}
NOE enhancements are indicated on the structure. C, non-bonded
H to C distances (in the hydrogen bond matrix found by molecular
dynamics computations in the global energy minimum ridge-tile
conformation are indicated on the structure. In B, the upper
set of distances are for CDCl
solvent; the lower set are
for (CD
)
SO solvent. The H
O
distances in C found by molecular dynamics are
1.6, as
compared with
1.7 in the crystal ( (11) and (12) ).
The C{
H} NOE's seen in
(CD
)
SO solvent are somewhat smaller than those
found in CDCl
(Fig. 3A), indicating a
slightly longer distance (Table 1) between pyrrole and lactam
N-H's and the
COOH. The data suggest that dimethyl
sulfoxide does not completely break the matrix of intramolecular
hydrogen bonds but only slightly weakens the hydrogen-bonded ridge-tile
conformation, as suggested earlier by
C NMR T
relaxation times of the carbons in the propionic acid chains (13, 15, 32) and by circular dichoism
spectroscopy(33, 34) .
In strong contrast, no C{
H} NOE's (<0.1) are found
between the N-H's and
CO
CH
groups of
[8
,12
-
C
]-mesobilirubin
XIII
dimethyl ester. The data suggest large distances between the
dipyrrinone N-H groups and the propionic ester carbonyl carbon,
providing no support for an intramolecularly hydrogen-bonded
conformation in either chloroform or dimethyl sulfoxide. Although
bilirubin dimethyl ester is thought to be monomeric in the latter
solvent, in the former a dimer is favored, as shown by vapor phase
osmometry(35) .
Do intramolecularly hydrogen-bonded
conformations of bilirubin persist when the propionic acid groups are
ionized to carboxylate anions? The answer is apparently yes, as learned
from C{
H} NOE measurements on the
bis-tetra-n-butylammonium salts of
[8
,12
-
C
]-mesobilirubin
XIII
(Fig. 4). In CDCl
solvent, the NOE
enhancements found are comparably strong to those observed from the
free acid; whereas, in (CD
)
SO solvent the NOE
enhancements are somewhat larger than in the free acid. It would appear
that bilirubin carboxylate anions are just as tightly hydrogen-bonded
in chloroform solution as the free acids. And in dimethyl sulfoxide
solution the dicarboxylate anions are probably even more strongly
hydrogen bonded than the free acids. The data suggest that in the free
acid dimethyl sulfoxide solvent is more attracted to the carboxylic
acid OH group than to the dipyrrinone N-H's.
Figure 4:
A, C{
H}
NOE enhancements of the
CO
resonance at 179.5
in CDCl
and 177.6
in
(CD
)
SO by saturation irradiating the pyrrole
and lactam hydrogens. B, approximate H to C non-bonded
distances (Å) in the N-H
O=
C
hydrogen bonded pairs calculated from the observed
C{
H} NOE enhancements are indicated
on the structure. C, H to C non-bonded distances (Å) in
the hydrogen bonding matrix found by molecular dynamics computation in
the global energy minimum ridge-tile conformation are indicated on the
structure. In B, the upper set of distances are from
CDCl
solvent; the lower set are from
(CD
)
SO solvent.
Is bilirubin in
pH 7.4 aqueous buffer intramolecularly hydrogen bonded? When
[8,12
-
C
]-mesobilirubin
XIII
is dissolved in a small quantity of
(CD
)
SO and diluted with 0.1 M pH 7.4
phosphate buffer, the
C NMR resonance for the propionic
carboxyl (
181.9 ppm downfield from (CH
)
Si
standard) is observed at the same position (
181.9 ppm) as in a
solution where the bis-tetra-n-butylammonium salt is similarly
dissolved. The carboxyl resonance is thus apparently insensitive to the
cation used (tetra-n-butylammonium versus sodium or
potassium) for the carboxylate anion. When the
bis-tetra-n-butylammonium salt is dissolved in H
O,
10% D
O (no (CD
)
SO) at pH 8.0 or
11.0, the carboxyl resonance appears at essentially the same shielding,
182.5 and 182.4, respectively. Since the carboxyl
C
NMR resonances of carboxylic acids are known to be sensitive to
ionization (
181.5 for acetate ion versus 176.8 for
acetic acid)(36) , the observed resonances for our
bilirubin analog are consistent with predominantly ionized (carboxylate
ion) species at pH 8, 11, and even 7.4.
The C{
H} NOE's observed for
[8
,12
-
C
]-mesobilirubin
XIII
in pH 7.4 aqueous phosphate buffer containing 3.8 M (CD
)
SO (Fig. 5) are significant and
meaningful. Although smaller than those seen for the anion in either
CDCl
or pure (CD
)
SO, they are
comparable to those seen for the acid in
(CD
)
SO. The weaker NOE's are consistent
with a somewhat more open or flatter ridge-tile conformation, and the
computed N-H to
C=O distance is still within the
range found for long hydrogen bonds (
H
O=
C). The data are
summarized in Table 1.
Figure 5:
A, C{
H}
NOE enhancements of the
CO
resonance at 181.9
by saturation irradiating the pyrrole
and lactam hydrogens. B, approximate H to C non-bonded
distances (Å) in the N-H
O=
C
hydrogen bonded pairs calculated from the observed
C{
H} NOE enhancements are indicated
on the structure. C, H to C non-bonded distances (Å)
found in the hydrogen bond matrix by molecular dynamics computations in
the global energy minimum ridge-tile
conformation.
The results of the C{
H}
NOE study on
[8
,12
-
C
]-mesobilirubin
XIII
and its bispropionate anion indicate that the carboxylic acid
and carboxylate anion carbons are sufficiently close to the dipyrrinone
pyrrole and lactam N-H groups to produce an NOE, but the carboxylic
ester carbon is not. The larger NOEs are found in the more lipophilic
solvent (CDCl
) as might be expected, where the calculated
pyrrole/lactam N-H to
COOH non-bonded H to C distances are
essentially the same as those computed by molecular dynamics or found
in crystals of bilirubin. As might also be expected, the smaller NOEs
are found in the more polar solvents, (CD
)
SO
and H
O, where solvent is thought to interfere with the
intramolecular hydrogen bonds. What is surprising, perhaps, is that the
NOEs are found in all of these solvents. The data clearly indicate,
however, that in the acid, and even in the propionate anion,
the carboxyl group lies close to the dipyrrinone NH groups. In fact,
the computed distance between the carboxyl carbon and dipyrrinone
N-H's lies within the distance of a long hydrogen bond, either
N-H
O=
C(-OH) or
N-H
O=
C(-O
)
N-H
O-
C(=O).
These results are important because they reveal the persistence of
intramolecular hydrogen bonding in bilirubin and its anions, in organic
(CDCl, (CD
)
SO) solvents, and in
water. They suggest a way to learn the conformation of bilirubin in
lipids, on proteins, or in intact cells or other biological tissues
using
C{
H} heteronuclear NOE
measurements. They indicate that key steps in bilirubin metabolism,
such as glucuronidation, might be followed by
C NMR
spectroscopy. They also suggest that bilirubin pK
values in microenvironments might be determined from the
CO
H-labeled pigment.