SYNTHESIS AND CHARACTERIZATION OF A NOVEL SPIN-LABELED AFFINITY PROBEOF HUMAN ERYTHROCYTE BAND-3 - CHARACTERISTICS OF THE STILBENEDISULFONATE BINDING-SITE

A new spin-labeled maleimide derivative of the anion exchange inhibito r 4,4'-diaminodihy drostilbene-2,2'-disulfonate (H(2)DADS) has been sy nthesized as a site-specific molecular probe of the stilbenedisulfonat e binding site of the anion exchange protein 1 (AE-1; band 3) in human erythrocytes. This probe, SL-H(2)DADS-maleimide, specifically and cov alently labels the M(r) 17 kDa integral membrane segment of band 3 wit h a 1:1 stoichiometry and inhibits essentially 100% of the band 3-medi ated anion exchange. The linear V-1 EPR spectrum of spin-labeled intac t erythrocytes is indicative of a spatially isolated probe which is ef fectively immobilized on the submicrosecond time scale. Several indepe ndent lines of experimental evidence have shown that the nitroxide moi ety of SL-H(2)DADS-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 moie ties of the SL-H(2)DADS-maleimide-labeled band 3 dimer are greater tha n 20 Angstrom from each other and are also more than 20 Angstrom from a monomer-monomer contact surface defined by cross-linking with the sp in-labeled reagent BSSDA is(sulfo-N-succinimidyl)doxyl-2-spiro-5'-azel ate]. These properties make SL-H(2)DADS-mmaleimide an extremely useful molecular probe for characterization of the physical properties of th e 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.