Thermal stability and ligand-binding properties of human plasma alpha 1-acid glycoprotein (orosomucoid) as determined with fluorescent probes.
The fluorescence of 1,8-anilinonaphthalene sulfonate is enhanced and blue-shifted upon binding to alpha 1-acid glycoprotein, a human plasma protein of uncertain function. Fluorescence titrations of delipidated protein indicate at least two classes of binding sites having dissociation constants of 0.33 microM and 12 microM at 25 degrees C in 0.02 M potassium phosphate/0.15 M NaCl, pH 7.4. Exclusion chromatography measurements indicate only 1 binding site per mol protein, suggesting that the heterogeneity is due to differences between protein molecules, the origin of which remains unclear. The fluorescence of a mixture of dye and protein is progressively diminished upon addition of ethanol and other organic solvents whose presence could be detected at concentrations as low as 100 mM. Addition of the adrenergic drug propranolol to a mixture of alpha 1-acid glycoprotein (2.5 microM) and 1,8-anilinonaphthalene sulfonate (4 microM) caused a hyperbolic decrease in dye fluorescence to 30% of the initial value, with half-maximal response near 1 microM propranolol. When the protein-dye mixture was heated, the fluorescence of the dye exhibited a reversible downward transition with midpoint near 65 degrees C, compared to a midpoint of 58.5 degrees C obtained by intrinsic fluorescence in the absence of dye. This stabilization was confirmed with fluorescein-labeled protein, whose fluorescence polarization revealed a melting transition at 58.8 degrees C in the absence of ligands which increased by 5-6 Cdeg in the presence of 1,8-anilinonaphthalene sulfonate or propranolol. The sensitivity of 1,8-anilinonaphthalene sulfonate fluorescence to changes in the conformation and ligand environment of alpha 1-acid glycoprotein should facilitate efforts to understand the structure and function of this acute-phase reactant.
Busby
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Ingham
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D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D003016:Clostridium perfringens / Q000201:enzymology; D004355:Drug Stability; D000431:Ethanol / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D008024:Ligands; D009439:Neuraminidase; D009961:Orosomucoid / Q000302:isolation & purification / Q000378:metabolism*; D011433:Propranolol / Q000378:metabolism; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D000817:Anilino Naphthalenesulfonates; D008024:Ligands; D009961:Orosomucoid; D000431:Ethanol; C039256:6-anilino-1-naphthalenesulfonic acid; D011433:Propranolol; D009439:Neuraminidase