CONFORMATIONAL-ANALYSIS OF SYMMETRICAL BILIRUBIN ANALOGS WITH VARYINGLENGTH ALKANOIC ACIDS - ENANTIOSELECTIVITY BY HUMAN SERUM-ALBUMIN

Symmetric analogues of mesobilirubin-XIII alpha, with propionic acid g roups shortened to acetic and lengthened to undecanoic, exhibit induce d circular dichroism (ICD) in pH 7.5 buffered aqueous [1-5% dimethyl s ulfoxide (DMSO) co-solvent] solution in the presence of human serum al bumin (HSA). The CD spectra exhibit bisignate Cotton effects with Delt a epsilon(434)(max) = +87, Delta epsilon(389)(max) = -54 (acetic), Del ta epsilon(436)(max) = +37, Delta epsilon(388)(max) = -42 (propionic), Delta epsilon(434)(max) = -15, Delta epsilon(370)(max) = +8 (butyric) , [Delta epsilon(433)(max) = -97, Delta epsilon(388)(max) = +89 in 30% aqueous DMSO], Delta epsilon(449)(max) = +6, Delta epsilon(397)(max) = -46 (valeric), Delta epsilon(440)(max) = +57, Delta epsilon(392)(max ) = -96 (caproic), Delta epsilon(440)(max) = +15, Delta epsilon(393)(m ax) = -21 (caprylic) and Delta epsilon(448)(max) = +18, Delta epsilon( 385)(max) = -31 (undecanoic). These values result from chromophore con formation (i.e. exciton coupling) and enantioselectivity by the protei n (i.e. preference for a given bilirubin enantiomer). The UV-VIS spect ra of the acetic to butyric, caprylic and undecanoic complexes are sim ilar in shape, with a shoulder in addition to the main band, and remin iscent of that of the bilirubin-IX alpha HSA complex, indicating an an alogous, folded conformation for all. The spectra of the valeric and c aproic complexes, in turn, are more symmetric and red-shifted, suggest ing a more extended conformation. Experimental CD values in each of th ese two series have been interpreted in terms of the different enantio selectivity by the protein, with the right handed acetic and caproic e nantiomers fitting best the protein binding site (Delta Delta epsilon ca. 150) and the protein showing a lower preference; for the right han ded propionic enantiomer (Delta Delta epsilon ca. 80) and even lower f or the right handed valeric, caprylic and undecanoic enantiomer (Delta Delta epsilon ca. 50), but left handed butyric enantiomer (Delta Delt a epsilon ca. 24). The differences observed in the UV-VIS spectra of e ach 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 (reduc tion from two to one transition for valeric and caproic-but not for th e rest) reflect the changes in pigment conformation induced by the pro tein. These changes are especially noticeable for the caproic and capr ylic analogues. Taken collectively the present results indicate that t he length of the alkanoic acid chains at C8 and C12 is essential for d etermining not only the pigment conformation, but also the enantiosele ctivity by the protein (through specific pigment-protein interactions) and agree with previous suggestions that these interactions may invol ve (at least) one salt linkage and hydrogen bonding. The effect upon t he ICD of each rubin-HSA complex of other parameters such as the perce ntage of DMSO used as carrier in the solution and the nature of the bu ffer has also been investigated. Surprisingly, an increase in the amou nt of DMSO from 3-30% results in dramatic changes in the observed CD o f the butyric and (to a lesser extent) propionic, undecanoic complexes . These have been interpreted in terms of selective changes in the ter tiary structure of the protein.