BIOMAT Database Logo BIOMAT Database Logo

Synthesis and characterization of a novel spin-labeled affinity probe of human erythrocyte band 3: characteristics of the stilbenedisulfonate binding site. 1996

D J Scothorn, and W E Wojcicki, and E J Hustedt, and A H Beth, and C E Cobb
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232-0615, USA.

A new spin-labeled maleimide derivative of the anion exchange inhibitor 4-4'-diaminodihydrostilbene-2,2'-disulfonate (H2DADS) has been synthesized as a site-specific molecular probe of the stilbenedisulfonate binding site of the anion exchange protein 1 (AE-1; band 3) in human erythrocytes. This probe, SL-H2DADS-maleimide, specifically and covalently labels the Mr 17 kDa integral membrane segment of band 3 with a 1:1 stoichiometry and inhibits essentially 100% of the band 3-mediated anion exchange. The linear V1 EPR spectrum of spin-labeled intact erythrocytes is indicative of a spatially isolated probe which is effectively immobilized on the submicrosecond time scale. Several independent lines of experimental evidence have shown that the nitroxide moiety of SL-H2DADS-maleimide-labeled band 3 is sequestered in a highly protected protein environment. These results are consistent with the observation that the spin-label is rigidly linked to band 3 in a fixed orientation with respect to the membrane normal axis [Hustedt, E. J., & Beth, A. H., (1996) Biochemistry 35, 6944-6954]. The nitroxide moieties of the SL-H2DADS-maleimide-labeled band 3 dimer are greater than 20 A from each other and are also more than 20 A from a monomer-monomer contact surface defined by cross-linking with the spin-labeled reagent BSSDA [bis(sulfo-N-succinimidyl)doxyl-2-spiro-5'-azelate]. These properties make SL-H2DADS-maleimide an extremely useful molecular probe for characterization of the physical properties of the band 3 stilbenedisulfonate binding site, determination of distances between the stilbenedisulfonate site and other segments of band 3, and investigation of the global rotational dynamics of human erythrocyte band 3.

UI MeSH Term Description Entries
D007202 Indicators and Reagents Substances used for the detection, identification, analysis, etc. of chemical, biological, or pathologic processes or conditions. Indicators are substances that change in physical appearance, e.g., color, at or approaching the endpoint of a chemical titration, e.g., on the passage between acidity and alkalinity. Reagents are substances used for the detection or determination of another substance by chemical or microscopical means, especially analysis. Types of reagents are precipitants, solvents, oxidizers, reducers, fluxes, and colorimetric reagents. (From Grant & Hackh's Chemical Dictionary, 5th ed, p301, p499) Indicator,Reagent,Reagents,Indicators,Reagents and Indicators
D004578 Electron Spin Resonance Spectroscopy A technique applicable to the wide variety of substances which exhibit paramagnetism because of the magnetic moments of unpaired electrons. The spectra are useful for detection and identification, for determination of electron structure, for study of interactions between molecules, and for measurement of nuclear spins and moments. (From McGraw-Hill Encyclopedia of Science and Technology, 7th edition) Electron nuclear double resonance (ENDOR) spectroscopy is a variant of the technique which can give enhanced resolution. Electron spin resonance analysis can now be used in vivo, including imaging applications such as MAGNETIC RESONANCE IMAGING. ENDOR,Electron Nuclear Double Resonance,Electron Paramagnetic Resonance,Paramagnetic Resonance,Electron Spin Resonance,Paramagnetic Resonance, Electron,Resonance, Electron Paramagnetic,Resonance, Electron Spin,Resonance, Paramagnetic
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
D000345 Affinity Labels Analogs of those substrates or compounds which bind naturally at the active sites of proteins, enzymes, antibodies, steroids, or physiological receptors. These analogs form a stable covalent bond at the binding site, thereby acting as inhibitors of the proteins or steroids. Affinity Labeling Reagents,Labeling Reagents, Affinity,Labels, Affinity,Reagents, Affinity Labeling
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
D001692 Biological Transport The movement of materials (including biochemical substances and drugs) through a biological system at the cellular level. The transport can be across cell membranes and epithelial layers. It also can occur within intracellular compartments and extracellular compartments. Transport, Biological,Biologic Transport,Transport, Biologic
D012856 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid A non-penetrating amino reagent (commonly called SITS) which acts as an inhibitor of anion transport in erythrocytes and other cells. 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid, Disodium Salt,SITS,SITS Disodium Salt,4 Acetamido 4' isothiocyanatostilbene 2,2' disulfonic Acid,Disodium Salt, SITS
D013113 Spin Labels Molecules which contain an atom or a group of atoms exhibiting an unpaired electron spin that can be detected by electron spin resonance spectroscopy and can be bonded to another molecule. (McGraw-Hill Dictionary of Chemical and Technical Terms, 4th ed) Spin Label,Label, Spin,Labels, Spin
D013431 Sulfates Inorganic salts of sulfuric acid. Sulfate,Sulfates, Inorganic,Inorganic Sulfates

Related Publications

D J Scothorn, and W E Wojcicki, and E J Hustedt, and A H Beth, and C E Cobb
Characterization of the stilbenedisulfonate binding site of the band 3 polypeptide of human erythrocyte membranes.
August 1983, Biochemistry,
D J Scothorn, and W E Wojcicki, and E J Hustedt, and A H Beth, and C E Cobb
Characterization of the stilbenedisulfonate binding site on band 3.
July 1995, Biochemistry,
D J Scothorn, and W E Wojcicki, and E J Hustedt, and A H Beth, and C E Cobb
Structural and binding properties of the stilbenedisulfonate sites on erythrocyte band 3: an electron paramagnetic resonance study using spin-labeled stilbenedisulfonates.
September 1993, Biochemistry,
D J Scothorn, and W E Wojcicki, and E J Hustedt, and A H Beth, and C E Cobb
Monoclonal antibodies against human erythrocyte band 3 protein. Localization of proteolytic cleavage sites and stilbenedisulfonate-binding lysine residues.
July 1986, The Journal of biological chemistry,
D J Scothorn, and W E Wojcicki, and E J Hustedt, and A H Beth, and C E Cobb
Characterization of eosin 5-isothiocyanate binding site in band 3 protein of the human erythrocyte.
February 1987, Biochimica et biophysica acta,
D J Scothorn, and W E Wojcicki, and E J Hustedt, and A H Beth, and C E Cobb
Characterization of the deoxyhemoglobin binding site on human erythrocyte band 3: implications for O2 regulation of erythrocyte properties.
January 2008, Blood,
D J Scothorn, and W E Wojcicki, and E J Hustedt, and A H Beth, and C E Cobb
Band 3 (AE1, SLC4A1)-mediated transport of stilbenedisulfonates. II: Evidence for transmembrane allosteric interactions between the "primary" stilbenedisulfonate binding site and the stilbenedisulfonate efflux site.
January 2006, Blood cells, molecules & diseases,
D J Scothorn, and W E Wojcicki, and E J Hustedt, and A H Beth, and C E Cobb
Allosteric effects in stilbenedisulfonate binding to band 3 protein (AE1).
November 1996, Cellular and molecular biology (Noisy-le-Grand, France),
D J Scothorn, and W E Wojcicki, and E J Hustedt, and A H Beth, and C E Cobb
Location of the stilbenedisulfonate binding site of the human erythrocyte anion-exchange system by resonance energy transfer.
October 1979, Biochemistry,
D J Scothorn, and W E Wojcicki, and E J Hustedt, and A H Beth, and C E Cobb
Stilbenedisulfonate binding kinetics to band 3 (AE 1): relationship between transport and stilbenedisulfonate binding sites and role of subunit interactions in transport.
January 2001, Blood cells, molecules & diseases,
Copied contents to your clipboard!