Characterization of Heparin Binding of Human Extracellular Superoxide Dismutase

• Published in: Biochemistry (Easton) Vol. 39; no. 1; pp. 230 - 236
• Main Authors: Lookene, Aivar, Stenlund, Peter, Tibell, Lena A. E
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 11.01.2000
• Subjects: Amino Acid Sequence; Binding, Competitive; Biosensing Techniques; Carbonic Anhydrases - genetics; Carbonic Anhydrases - metabolism; Extracellular Space - enzymology; Heparin - chemistry; Heparin - genetics; Heparin - metabolism; Humans; Kinetics; MEDICIN; MEDICINE; Molecular Sequence Data; Oligosaccharides - chemistry; Oligosaccharides - metabolism; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - metabolism; Protein Binding - genetics; Protein Structure, Secondary; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; Static Electricity; Superoxide Dismutase - chemistry; Superoxide Dismutase - genetics; Superoxide Dismutase - metabolism
• ISSN: 0006-2960; 1520-4995; 1520-4995
• DOI: 10.1021/bi991512x

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 μM, 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 α-helical structure was induced in the synthetic peptide, by addition of 10% trifluoroethanol, the K d decreased to 0.64 μM. 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 × 105 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 α-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.