Interactions of bilirubin and other ligands with ligandin.
Circular dichroism methods were used to study the structure of rat ligandin and the binding of organic anions to the protein. Ligandin has a highly ordered secondary structure with about 40%alpha helix, 15% beta structure, and 45% random coil. Bilirubin binding occurred primarily at a single high affinity site on the protein. The binding constant for bilirubin (5 X 10-7 Mminus 1) was the highest among the ligands studied. The bilirubin-ligandin complex exhibited a well-defined circular dichroic spectrum with two major overlapping ellipticity bands of opposite sign in the bilirubin absorption region. This spectrum was virtually a mirror image of that of human or rat serum albumin-bilirubin complexes. Studies on the direct transfer of bilirubin from ligandin to rat serum albumin showed that sasociation constants of bilirubin-ligandin complexes were approximately tenfold less than those of the bilirubin-albumin system. Ligandin exhibited a broad specificity with respect to the typeof ligand bond. A series of organic anions inclucing dyes used clinically for liver function tests, fatty acids, hormones, heme derivatives, bile acids, and other ligands that were considered likely to interact with ligandin, were examined. Most induced ellipticity changes consistent with competitive displacement of bilirubin from ligandin and relative affinities of these compounds for ligandin were determined based on their effectiveness in desplacing the bilirubin. Some substances such as glutathione, conjugated sulfobromophthaleins and lithocholic acid bound to ligandin but induced anomalous spectral shifts, when added to ligandin-bilirubin complexes. Other compounds, including some that act as substrates for the glutathione transferase activity exhibited by ligandin, revealed no apparent competitive effects with respect to the bilitubin binding site.
D000818:Animals; D001663:Bilirubin; D001665:Binding Sites; D002352:Carrier Proteins; D002942:Circular Dichroism; D005978:Glutathione; D005982:Glutathione Transferase; D007208:Indocyanine Green; D007700:Kinetics; D008024:Ligands; D008099:Liver / Q000032:analysis; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet
D002352:Carrier Proteins; D008024:Ligands; D005982:Glutathione Transferase; D005978:Glutathione; D007208:Indocyanine Green; D001663:Bilirubin