Fluorescence studies of beta-adrenergic ligand binding to alpha 1-acid glycoprotein with 1-anilino-8-naphthalene sulfonate, isoprenaline, adrenaline and propranolol.
The present study shows that ANS (1-anilino-8-naphthalene sulfonate), propranolol, isoprenaline, adrenaline and dopamine have common binding sites on AAG (alpha 1-acid glycoprotein). A fluorescence technique was employed to characterize the interaction between the ligands and AAG at 20-22 degrees. The binding of ANS to AAG caused increased fluorescence intensity at emission and excitation wavelengths of 400 and 470 nm. In this situation, propranolol displaced ANS in a concentration-dependent mode with an apparent dissociation constant of 6.2 +/- 0.01 microM, whereas isoprenaline did not reduce the ANS-AAG fluorescence. However, in the presence of AAG, catecholamines caused a marked increase of fluorescence at excitation and emission wavelengths of 250 and 325 nm, respectively. These wavelengths were employed to characterize the binding of isoprenaline, adrenaline and propranolol to AAG. Two subsets of binding sites were demonstrated. The Kd values were 0.87 +/- 0.03 and 25.1 +/- 10.7 microM for ANS, 0.76 +/- 0.09 and 133 +/- 30.4 microM for propranolol, 140 +/- 14 and 2.18 +/- 0.58 mM for isoprenaline, 137 +/- 24 and 14.8 +/- 0.1 mM for adrenaline, respectively. AAG had identical high affinity binding capacity for these ligands (n approximately 1). However, the second class of binding sites showed ligand-dependent binding capacity: n = 1 for ANS, n approximately 10 for propranolol, n approximately 15 for adrenaline, n approximately 20 for isoprenaline, respectively. ANS, propranolol, dopamine and adrenaline caused concentration-dependent inhibition of isoprenaline binding to AAG with apparent dissociation constants of 5.1 +/- 1.8 microM, 6.4 +/- 1.1 microM, 0.57 +/- 0.13 mM and 1.5 +/- 0.46 mM, respectively.
Johansen
A K
AK
Department of Pharmacology, University of Tromsø, Norway.
Willassen
N P
NP
Sager
G
G
D000817:Anilino Naphthalenesulfonates; D001205:Ascorbic Acid / Q000737:chemistry; D001665:Binding Sites; D004298:Dopamine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004837:Epinephrine / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007545:Isoproterenol / Q000737:chemistry / Q000378:metabolism*; D009961:Orosomucoid / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011433:Propranolol / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D000817:Anilino Naphthalenesulfonates; D009961:Orosomucoid; D011433:Propranolol; D007545:Isoproterenol; D001205:Ascorbic Acid; D004298:Dopamine; D004837:Epinephrine