Influence of succinylation of bovine serum albumin on its conformation and bilirubin binding.
In order to investigate the role of lysine residues in the interaction of bilirubin with bovine serum albumin, five succinylated preparations of albumin, namely: 23%, 39%, 49%, 55% and 87%, were prepared, and their conformational and bilirubin-binding properties were studied by the techniques of gel filtration, ultraviolet and visible spectroscopy, and fluorescence quenching. Gel filtration experiments performed at pH 7.0 and ionic strengths 0.15 and 1.0 suggested that the albumin molecule undergoes gradual disorganization with increase in succinylation. The Stokes radius and frictional ratio at ionic strength 0.15 increased from 3.7 nm and 1.36, respectively, for the native protein to 6.3 nm and 2.26 for maximally (87%) succinylated albumin. Interestingly, increase in ionic strength to 1.0 caused significant refolding in succinylated preparations as evidenced by a decrease in Stokes radius and frictional ratio (5.3 nm and 1.90 for 87% succinylated albumin). Progressive succinylation produced a steady decline in the intensity of bilirubin-induced fluorescence quenching, and in the visible spectral changes of the bilirubin-albumin complex at 480 nm. Both of these changes had a good correlation with increase in Stokes radius. Increase in ionic strength to 1.0 produced a significant reversal in these properties. From these results we conclude that probably none of the surface lysine residues is involved in bilirubin-albumin interaction, and that if lysine residues are involved in this interaction they must be buried in the protein interior.
D000418:Albumins; D001663:Bilirubin / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012709:Serum Albumin; D012710:Serum Albumin, Bovine; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D013386:Succinates; D014461:Ultracentrifugation
D000418:Albumins; D012709:Serum Albumin; D013386:Succinates; C034877:succinylalbumin; D012710:Serum Albumin, Bovine; D001663:Bilirubin