Characterization of heparin binding of human extracellular superoxide dismutase.

The C-terminal domain of human extracellular superoxide dismutase (hEC-SOD) plays a crucial role in the protein's interaction with heparin. Here we investigated this interaction in more detail by comparing the heparin-binding characteristics of two variants of hEC-SOD: the two fusion proteins containing the hEC-SOD C-terminal domain and a synthetic peptide homologous to the C-terminal. The interaction studies were performed using a surface plasmon resonance based technique on a BIAcore system. It should be emphasized that this is a model system. However, the kinetic constants, as measured, are valid in a comparative sense. Comparison of affinities for size-fractionated heparins revealed that octa- or decasaccharides are the smallest heparin fragments that can efficiently interact with the C-terminal domain of hEC-SOD. At physiological salt concentration, and pH 7.4, the hEC-SOD/heparin interaction was found to be of a high-affinity type, with an equilibrium dissociation constant, K(d), of 0.12 microM, which is 700 and 10-20 times lower than the K(d) values for the synthetic peptide and the fusion proteins, respectively. However, when an alpha-helical structure was induced in the synthetic peptide, by addition of 10% trifluoroethanol, the K(d) decreased to 0.64 microM. The differences in the K(d) values were mainly governed by differences in the association rate constants (k(ass)). The hEC-SOD/heparin interaction itself was found to have a fairly high dissociation rate constant (0.1 s(-)(1)), and a very high association rate constant (8 x 10(5) M(-)(1) s(-)(1)), suggesting that the interaction is mainly controlled by the association. These results together with circular dichroism spectra of the synthetic peptide suggest that an alpha-helical structure in the C-terminal is essential for optimal binding to heparin and that other parts of hEC-SOD moderate the affinity. Our data also demonstrate that the tetramerization itself does not substantially increase the affinity.