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 ωIµmax434 = +87, ωIµmax389 = -54 (acetic), ωIµmax436 = +37, ωIµmax388 = -42 (propionic), ωIµmax420 = -15, ωIµmax370 = +8 (butyric), [ωIµmax433 = -97, ωIµmax388 = +89 in 30% aqueous DMSO], ωIµmax449 = +6, ωIµmax397 = -46 (valeric), ωIµmax440 = +57, ωIµmax392 = -96 (caproic), ωIµmax440 = +15, ωIµmax393 = -21 (caprylic) and ωIµmax448 = +18, ωIµmax385 = -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 (ωωIµ ca. 150) and the protein showing a lower preference for the right handed propionic enantiomer (ωωIµ ca. 80) and even lower for the right handed valeric, caprylic and undecanoic enantiomer (ωωIµ ca. 50), but left handed butyric enantiomer (ωωIµ 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.