Involvement of tyrosine and lysine residues of retinol-binding protein in the interaction between retinol and retinol-binding protein and between retinol-binding protein and prealbumin. Acetylation with N-acetylimidazole and alkaline titration.
The behavior of holo-retinol-binding protein (RBP) from human plasma at alkaline pH was examined by absorption and circular dichroism measurements. Between pH 7.5 and 11.7 the ionization of the phenolic hydroxyl groups is reversible. However, there is a gradual irreversible loss of retinol as the pH is raised. After 4 hours at pH 11.7, 13 percent of retinol is lost from retinol-RBP. Alkaline titration of apo-RBP was time-independent and reversible between pH 7.5 and 11.7. The titration data of the phenolic hydroxyl groups in apo-RBP could be fitted with a single theoretical ionization curve of 8.6 phenolic groups having an apparent pK of 11. Acetylation of retinol-RBP with 10-fold molar excess of N-acetylimidazole over tyrosine resulted in the acetylation of all lysine residues and in the acetylation of 0.9 to 1.3 tyrosyl residues per molecule (out of eight). Acetylation of retinol-RBP, APO-RBP, and retinol-RBP-prealbumin complex with 50-fold molar excess of N-acetylimidazole resulted, again, with all of the lysine residues being acetylated and between 1.8 and 2.8 tyrosyl residues per molecule being acetylated. The acetylation did not affect the interaction between retinol and RBP. However, acetylation disrupted the normal binding between retinol-RBP and prealbumin. Deacetylation of tyrosyl residues with hydroxylamine failed to restore the normal binding of retinol-RBP to prealbumin. This excludes the acetylated tyrosyl-residues from being involved in the binding between the two proteins.
D000085:Acetates; D001059:Apoproteins; D001665:Binding Sites; D001798:Blood Proteins; D002942:Circular Dichroism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles; D007700:Kinetics; D008239:Lysine; D011485:Protein Binding; D011487:Protein Conformation; D011498:Protein Precursors / Q000097:blood*; D012709:Serum Albumin; D013056:Spectrophotometry, Ultraviolet; D014443:Tyrosine; D014801:Vitamin A / Q000031:analogs & derivatives* / Q000097:blood*
D000085:Acetates; D001059:Apoproteins; D001798:Blood Proteins; D007093:Imidazoles; D011498:Protein Precursors; D012709:Serum Albumin; D014801:Vitamin A; D014443:Tyrosine; D008239:Lysine