title: Equilibrium studies of a fluorescent tacrolimus binding to surfactant protein A.
abstract: Tacrolimus (FK506) is a hydrophobic immunosuppressive agent used in kidney, liver, and lung transplantation. The objective of this study was to characterize the binding of FK506 to surfactant protein A (SP-A), an abundant lipoprotein found in the alveolar fluid that functions as part of the innate immune system in the lung. We have synthesized a novel derivative of FK506 in which a dansyl moiety was covalently bound via cadaverine to the C22 position of the FK506 molecule (DNS-FK). Using the fluorescence and anisotropy properties of DNS-FK, we demonstrated that tacrolimus avidly binds to SP-A with an apparent equilibrium association constant (K(app)) of 10(7)M(-1) and a Gibbs binding free energy of -40 kJ mol(-1)K(-1). Derivatization of FK506 at the C22 position did not block FK506 binding to the cytosolic immunophilin FK506-binding protein (FK-BP) or human serum albumin (HSA), both used as controls of tacrolimus-binding proteins. K(app) for FK-BP/DNS-FK and HSA/DNS-FK complexes were 1.5 x 10(7) and 10(7)M(-1), respectively. The high sensitivity of this analytical technique makes it suitable for binding analysis of FK506 to proteins.
authors.affiliation: Department of Biochemistry and Molecular Biology I, Complutense University of Madrid, 28040 Madrid, Spain.
authors.forename: Olga
authors.initials: O
authors.lastname: Cañadas
authors.forename: Alejandra
authors.initials: A
authors.lastname: Sáenz
authors.forename: Guillermo
authors.initials: G
authors.lastname: Orellana
authors.forename: Cristina
authors.initials: C
authors.lastname: Casals
mesh_terms: D005454:Fluorescence Polarization; D006801:Humans; D015394:Molecular Structure; D011485:Protein Binding; D037662:Pulmonary Surfactant-Associated Protein A / Q000378:metabolism*; D016559:Tacrolimus / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D037662:Pulmonary Surfactant-Associated Protein A; D016559:Tacrolimus