Chloroform-induced conformational changes in the bound pigment in bilirubin-albumin complexes

Chloroform-induced conformational changes of bilirubin (BR) bound to differ ent serum albumins were studied by circular dichroism (CD) and fluorescence spectroscopy. Addition of a small amount of chloroform (approximate to 20 mM) to a solution containing 20 mu M albumin and 15 mu M BR changed the sig n order and magnitude of the characteristic CD spectra of all BR-albumin co mplexes except BR-PSA complex which showed abnormal behavior. Monosignate n egative CD Cotton effects (CDCEs) of BR complexed with SSA, GSA and BuSA we re transformed into bisignate CDCEs in presence of chloroform akin to those exhibited by chloroform free solution of BR-HSA complex, indicating that t he pigment acquired right handed plus (P) chirality when chloroform was add ed to these complexes. Bisignate CD spectra of BR complexed with HSA and BS A showed complete inversion upon addition of chloroform corroborating earli er findings. On the other hand, changes observed with BR-RSA complex were s lightly different showing an additional CD band of weak intensity centered around 390 nm though inversion of CDCEs was similar to that of BR-HSA compl ex. Monosignate CD spectra of BR-PSA complex also showed three CD bands occ urring at 409, 470 and 514 nm after chloroform addition. These results indi cated significant but different effects of chloroform on the conformation o f bound BR in BR-albumin complexes which can be ascribed to the changes in the exciton chirality of bilirubin probably due to altered hydrophobic micr oenvironment induced by the binding of chloroform at or near the ligand bin ding site. Chloroform severely quenched the intrinsic tryptophan fluorescen ce of the protein and shifted the emission maxima towards blue region in al l the albumins except PSA. However, quantitative differences in both quench ing and blue shift were noted in different serum albumins. This suggests th at chloroform probably binds in the close vicinity of tryptophan residue(s) located in subdomain(s) IIA or IB and II both. The fluorescence of BR-albu min complexes was also found to be sensitive to the presence of a small amo unt of chloroform. But the changes observed in the fluorescence of the boun d pigment in presence of chloroform were less marked as compared to the cha nges in the intrinsic fluorescence of protein per se. Taken together, these results suggest that there is at least one conserved site for chloroform b inding in an these albumins which is at or near the BR binding site. (C) 20 00 Societe francaise de biochimie et biologie moleculaire / Editions scient ifiques et medicales Elsevier SAS.