Conformational analysis of symmetric bilirubin analogues with varying
length alkanoic acids. Enantioselectivity by human serum albumin
Abstract
Symmetric analogues of mesobilirubin-XIIIα, with
propionic acid groups shortened to acetic and lengthened to undecanoic,
exhibit induced circular dichroism (ICD) in pH 7.5 buffered aqueous
[1–5% dimethyl sulfoxide (DMSO) co-solvent] solution in the
presence of human serum albumin (HSA). The CD spectra exhibit bisignate
Cotton effects with
ωεmax434
= +87,
ωεmax389
= -54 (acetic),
ωεmax436
= +37,
ωεmax388
= -42 (propionic),
ωεmax420
= -15,
ωεmax370
= +8 (butyric),
[ωεmax433
= -97,
ωεmax388
= +89 in 30% aqueous DMSO],
ωεmax449
= +6,
ωεmax397
= -46 (valeric),
ωεmax440
= +57,
ωεmax392
= -96 (caproic),
ωεmax440
= +15,
ωεmax393
= -21 (caprylic) and
ωεmax448
= +18,
ωεmax385
= -31 (undecanoic). These values result
from chromophore conformation (i.e. exciton coupling) and
enantioselectivity by the protein (i.e. preference for a given
bilirubin enantiomer). The UV–VIS spectra of the acetic to
butyric, caprylic and undecanoic complexes are similar in shape, with a
shoulder in addition to the main band, and reminiscent of that of the
bilirubin-IXα HSA complex, indicating an analogous,
folded conformation for all. The spectra of the valeric and caproic
complexes, in turn, are more symmetric and red-shifted, suggesting a
more extended conformation. Experimental CD values in each of these two
series have been interpreted in terms of the different
enantioselectivity by the protein, with the right handed acetic and
caproic enantiomers fitting best the protein binding site
(ωωε
ca. 150) and the protein showing a lower preference for the
right handed propionic enantiomer
(ωωε
ca. 80) and even lower for the right handed valeric, caprylic
and undecanoic enantiomer
(ωωε
ca. 50), but left handed butyric enantiomer
(ωωε
ca. 24).The differences observed in the UV–VIS spectra of each
complexed (in aqueous buffer) vs. uncomplexed pigment
(in MeOH), i.e. spectral shifts (7–11 nm for
acetic to butyric and undecanoic, 12 nm for valeric and 16–18 nm
for caproic and caprylic) and shape (reduction from two to one
transition for valeric and caproic—but not for the rest) reflect
the changes in pigment conformation induced by the protein. These
changes are especially noticeable for the caproic and caprylic
analogues.Taken collectively, the present results indicate that the length of
the alkanoic acid chains at C8 and C12 is essential for determining not
only the pigment conformation, but also the enantioselectivity by the
protein (through specific pigment–protein interactions) and agree
with previous suggestions that these interactions may involve (at least)
one salt linkage and hydrogen bonding.The effect upon the ICD of each rubin-HSA complex of other
parameters such as the percentage of DMSO used as carrier in the
solution and the nature of the buffer has also been investigated.
Surprisingly, an increase in the amount of DMSO from 3–30% results
in dramatic changes in the observed CD of the butyric and (to a lesser
extent) propionic, undecanoic complexes. These have been interpreted in
terms of selective changes in the tertiary structure of the
protein.
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