CHARACTERIZATION OF SPIN-LABELED FATTY-ACIDS AND HEMATOPORPHYRIN BINDING-SITES INTERACTIONS IN SERUM-ALBUMIN

Electron paramagnetic resonance (EPR) was used to investigate the spin-labelled fatty acid (SLFA) binding equilibrium to human (HSA) and bovine (BSA) serum albumin. The number of 5-doxyl stearate (5-DS) and 16-doxyl stearate (16-DS) binding sites on HSA and BSA were found to be equal, while the association constants, KA values (especially those of the primary binding site) were different. The applied EPR spectra analysis permitting a quantitative distinguishing between slow macromolecular rotation (τc) and fast anisotropic motion (steric restriction, S) of bound SLFA, allowed SLFA oxazolidinyl ring mobility to be estimated. The 5-DS nitroxide radical is completely immobilized within the HSA protein matrix (S ≈ 1.0, τc ≈ 56 ± 1 ns). The 5-DS when bound to BSA exhibited the presence of more extensive fluctuations (lower S and τc values) and its immersion depth with respect to BSA surface was calculated to be 4 ± 2 A ̊. The 16-DS oxazolidinyl radical bound to HSA was found to undergo moderated fluctuations (both S and τc are smaller with respect to 5-DS) and it is buried deeper within the protein core (rimm = 10 ± 2 A ̊ with respect to BSA surface). The tetrapyrrole ligands hematoporphyrin (Hp) and hematoporphyrin derivative (HpD) were found to induce well detectable changes in the SLFA binding patterns to serum albumin. The action mode was determined to be different for 16-DS (primary) and 5-DS (secondary) serum albumin binding sites: (i) 5-DS is extruded from several binding sites accompanied by an increase in KA in the remaining ones; (ii) simultaneous binding of 16-DS and Hp consists of cooperative and non-cooperative phases (both the number of the independent sites and the parameter of cooperativity, α, being dependent on Hp/HSA ratio); (iii) in principal the mobilities of 5-DS and 16-DS bound to HSA are changed, depending on the porphyrin/HSA ratio; and (iv) the effective immersion depth of the paramagnetic centres with respect to the protein surface is increased when Hp is present as a second ligand (rimm = 7 ± 2 and 16 ± 2 A ̊ for 5-DS and 16-DS, respectively). © 1990.