DETERMINATION OF THE ORIENTATION OF A BAND-3 AFFINITY SPIN-LABEL RELATIVE TO THE MEMBRANE NORMAL AXIS OF THE HUMAN ERYTHROCYTE

The orientation of the nitroxide moiety of an isotopically substituted spin-labeled derivative of dihydrostilbenedisulfonate ([N-15,H-2(13)] -SL-H(2)DADS-maleimide) covalently coupled at the extracellular stilbe nedisulfonate binding site of the human erythrocyte anion exchange pro tein, band 3, has been determined relative to the membrane normal axis of intact cells. The X-band linear electron paramagnetic resonance (E PR) spectra of [N-15,H-2(13)]-SL-H(2)DADS-maleimide-labeled band 3 in intact erythrocytes oriented by flow through an EPR flat cell have bee n obtained for two orthogonal orientations of the sample in the DC mag netic field. Two different methods of analysis have provided very simi lar values for the angles alpha(1) and beta(1) which uniquely define t he orientation of the nitroxide axis frame relative to the membrane no rmal axis. In the first approach, a variable fraction of the cells, f, were taken to be biconcave disks perfectly oriented relative to the f lat cell surface with the remainder, 1 - f, isotropically oriented. Si multaneous nonlinear least squares analysis of the spectra obtained at the two sample orientations yielded best fit values of f = 0.60, alph a(1) = 58 degrees, and beta(1) = 36 degrees. In the second approach, t he EPR spectra of flow-oriented intact erythrocytes labeled with the f atty acid spin-label, [N-15,H-2(12)]-5-nitroxyl Stearate, have been ob tained at the two sample orientations. These two spectra have been use d to determine a model-independent distribution of membrane normal ori entations in the sample. Using this experimentally determined membrane normal orientation distribution, the EPR spectra of [N-15,H-2(13)]-SL -H(2)DADS-maleimide-labeled erythrocytes were then reanalyzed to obtai n a second determination of the nitroxide orientation, alpha(1) = 61 d egrees and beta(1) = 37 degrees. The orientation of the nitroxide with respect to the membrane normal axis determined in the present study i s nearly identical to the orientation of the nitroxide with respect to the uniaxial rotational diffusion axis, alpha = 66 degrees and beta = 34 degrees, as determined from saturation transfer EPR (ST-EPR) studi es [Hustedt, E. H., & Beth, A. H. (1995) Biophys. J. 69, 1409-1423]. T his result supports the conclusion that the motion observed using ST-E PR spectroscopy is, in fact, the uniaxial rotational diffusion of band 3 about the membrane normal.