Tertiary structure of human α1-acid glycoprotein (orosomucoid).: Straightforward fluorescence experiments revealing the presence of a binding pocket

Binding of hemin to alpha(1)-acid glycoprotein has been investigated. Hemin binds to the hydrophobic pocket of hemoproteins. The fluorescent probe 2-(p-toluidino)-6-naphthalenesulfonate (TNS) binds to a hydrophobic domain in a alpha(1)-acid glycoprotein with a dissociation constant equal to 60 muM. Addition of hemin to an alpha(1)-acid glycoprotein-TNS complex induces the displacement of TNS from its binding site. At saturation (I hemin for I protein) all the TNS has been displaced from its binding site. The dissociation constant of hemin-alpha(1)-acid glycoprotein was found equal to 2muM. Thus, TNS and hemin bind to the same hydrophobic site: the pocket of alpha(1)-acid glycoprotein. Energy-transfer studies performed between the Trp residues of alpha(1)-acid glycoprotein and hemin indicated that efficiency (E) of Trp fluorescence quenching was equal to 80% and the Forster distance, R-0 at which the efficiency of energy transfer is 50% was calculated to be 26Angstrom, revealing a very high energy transfer. (C) 2003 Elsevier Ltd. All rights reserved.