Tertiary structure of human α 1-acid glycoprotein (orosomucoid). Straightforward fluorescence experiments revealing the presence of a binding pocket

Abstract

Binding of hemin to α 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 α 1-acid glycoprotein with a dissociation constant equal to 60 μM. Addition of hemin to an α 1-acid glycoprotein–TNS complex induces the displacement of TNS from its binding site. At saturation (1 hemin for 1 protein) all the TNS has been displaced from its binding site. The dissociation constant of hemin–α 1-acid glycoprotein was found equal to 2 μM. Thus, TNS and hemin bind to the same hydrophobic site: the pocket of α 1-acid glycoprotein. Energy-transfer studies performed between the Trp residues of α 1-acid glycoprotein and hemin indicated that efficiency ( E) of Trp fluorescence quenching was equal to 80% and the Förster distance, R 0 at which the efficiency of energy transfer is 50% was calculated to be 26 Å, revealing a very high energy transfer.