Biomaterial Database

Characterization of the stilbenedisulfonate binding site on band 3. 1995

L M Schopfer, and J M Salhany

Veterans Administration Medical Center, Omaha, Nebraska, USA.

Stilbenedisulfonates are potent inhibitors of Band 3 mediated anion exchange. They bind tightly to the protein and form a 1-to-1 reversible complex. Those stilbenedisulfonates which contain isothocyanato groups such as DIDS (4,4'-diisothiocyanato-2,2'-stilbenedisulfonate) and H2DIDS (4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate) can also react rapidly with lysine residues within the binding pocket to yield an irreversible covalent adduct. The reactive lysine residue is known as lysine-A, and is thought to have an unusually low pKa. In this report, we characterize the kinetics of DIDS adduct formation with respect to the effect of substrate anions, competitive inhibitory anions, and pH on the rate of covalent adduct formation. We investigate the following: (a) whether stilbenedisulfonates bind to or block access of substrate anions to the transport site; (b) whether the rapidity of the covalent reaction of DIDS at neutral pH is due to a low pKa for lysine-A within the binding pocket; and (c) whether once bound, DIDS and H2DIDS isothiocyanato groups are accessible to reagents. For this latter experiment, we have utilized a newly discovered reaction of the DIDS isothiocyanato groups with azide to test for accessibility. Our results show that substrate anions, DIDS, and Band 3 form a ternary complex. Significantly, the binding of large substrate anions, such as iodide, is not weakened by DIDS to any greater extent than is the binding of smaller substrates such as chloride or fluoride. These results are not consistent with a "partial blockade" hypothesis for the relationship between the stilbenedisulfonate and transport sites. Rather, they support an allosteric site-site interaction hypothesis. Our pH dependence results show that the apparent pKa for the DIDS/lysine-A reaction is greater than 9.26. This is consistent with typical lysine pKa values, and indicates that lysine-A does not have an unusually low pKa. Finally, we show that azide can react with the isothiocyanato groups of DIDS and H2DIDS within their Band 3 complexes, indicating that the stilbenedisulfonate binding site is accessible to solute. These results support a view which suggests that the stilbenedisulfonate site is a superficial inhibitory site on Band 3 which inhibits transport by allosteric interactions within the protein, rather than by either direct or partial blockade of the transport site.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D003432	Cross-Linking Reagents	Reagents with two reactive groups, usually at opposite ends of the molecule, that are capable of reacting with and thereby forming bridges between side chains of amino acids in proteins; the locations of naturally reactive areas within proteins can thereby be identified; may also be used for other macromolecules, like glycoproteins, nucleic acids, or other.	Bifunctional Reagent,Bifunctional Reagents,Cross Linking Reagent,Crosslinking Reagent,Cross Linking Reagents,Crosslinking Reagents,Linking Reagent, Cross,Linking Reagents, Cross,Reagent, Bifunctional,Reagent, Cross Linking,Reagent, Crosslinking,Reagents, Bifunctional,Reagents, Cross Linking,Reagents, Cross-Linking,Reagents, Crosslinking
D004910	Erythrocyte Membrane	The semi-permeable outer structure of a red blood cell. It is known as a red cell 'ghost' after HEMOLYSIS.	Erythrocyte Ghost,Red Cell Cytoskeleton,Red Cell Ghost,Erythrocyte Cytoskeleton,Cytoskeleton, Erythrocyte,Cytoskeleton, Red Cell,Erythrocyte Cytoskeletons,Erythrocyte Ghosts,Erythrocyte Membranes,Ghost, Erythrocyte,Ghost, Red Cell,Membrane, Erythrocyte,Red Cell Cytoskeletons,Red Cell Ghosts
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D006863	Hydrogen-Ion Concentration	The normality of a solution with respect to HYDROGEN ions; H+. It is related to acidity measurements in most cases by pH	pH,Concentration, Hydrogen-Ion,Concentrations, Hydrogen-Ion,Hydrogen Ion Concentration,Hydrogen-Ion Concentrations
D000838	Anions	Negatively charged atoms, radicals or groups of atoms which travel to the anode or positive pole during electrolysis.	Anion
D001457	Anion Exchange Protein 1, Erythrocyte	A major integral transmembrane protein of the ERYTHROCYTE MEMBRANE. It is the anion exchanger responsible for electroneutral transporting in CHLORIDE IONS in exchange of BICARBONATE IONS allowing CO2 uptake and transport from tissues to lungs by the red blood cells. Genetic mutations that result in a loss of the protein function have been associated with type 4 HEREDITARY SPHEROCYTOSIS.	Anion Transport Protein, Erythrocyte,Band 3 Protein,Erythrocyte Anion Transport Protein,Erythrocyte Membrane Band 3 Protein,AE1 Anion Exchanger,AE1 Chloride-Bicarbonate Exchanger,AE1 Cl- HCO3- Exchanger,AE1 Gene Product,Anion Exchanger 1,Antigens, CD233,Band 3 Anion Transport Protein,Band III Protein,CD233 Antigen,CD233 Antigens,Capnophorin,EPB3 Protein,Erythrocyte Anion Exchanger,Erythrocyte Membrane Anion Transport Protein,Erythrocyte Membrane Protein Band 3, Diego Blood Group,Protein Band 3,SLC4A1 Protein,Solute Carrier Family 4 Member 1,Solute Carrier Family 4, Anion Exchanger, Member 1,AE1 Chloride Bicarbonate Exchanger,AE1 Cl HCO3 Exchanger,Anion Exchanger, Erythrocyte,Antigen, CD233,Chloride-Bicarbonate Exchanger, AE1,Exchanger 1, Anion,Protein, EPB3
D001665	Binding Sites	The parts of a macromolecule that directly participate in its specific combination with another molecule.	Combining Site,Binding Site,Combining Sites,Site, Binding,Site, Combining,Sites, Binding,Sites, Combining
D017878	4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid	An inhibitor of anion conductance including band 3-mediated anion transport.	4,4'-Diisothiocyano-2,2'-Stilbene Disulfonic Acid,DIDS