Synthesis and characterization of a novel spin-labeled affinity probe of human erythrocyte band 3: characteristics of the stilbenedisulfonate binding site.
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.
Scothorn
D J
DJ
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232-0615, USA.
Wojcicki
W E
WE
Hustedt
E J
EJ
Beth
A H
AH
Cobb
C E
CE
D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry; D000345:Affinity Labels / Q000138:chemical synthesis*; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D001692:Biological Transport; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004910:Erythrocyte Membrane / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D007202:Indicators and Reagents; D013113:Spin Labels / Q000138:chemical synthesis*; D013431:Sulfates / Q000097:blood
C097423:4'-maleimido-4-((1-oxyl-2,2,5,5-tetramethyl-3-maleimidyl)carbonylamino)dihydrostilbene-2,2'-disulfonate; D000345:Affinity Labels; D001457:Anion Exchange Protein 1, Erythrocyte; D007202:Indicators and Reagents; D013113:Spin Labels; D013431:Sulfates; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid