Internal motions of band 3 of human erythrocytes

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Abstract
Band 3 was labeled with N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonate either exofacially in the intact washed erythrocytes or endofacially by treating inside-out vesicles. Exo labeling resulted in the labeling of several other proteins, besides band 3, which could not be removed from the membrane. The exo-labeled band 3 was extracted and purified by chromatography on DEAE-cellulose in Triton X-100. The endo labeling also resulted in the labeling of several other proteins. In this case, washing with NaOH removed all labeled material except band 3 from the vesicles. The lifetime of bound N-[(acetylamino)ethyl]-5-naphthylamine-1-sulfonate was heterogeneous, suggesting the positioning of the label in different environments either because different sites were labeled or because of positional freedom of the label at the same point of attachment. The main fraction of emission intensity had a lifetime near 20 ns, as expected for a hydrophobic environment. The rest showed a lifetime of .apprx. 3 ns in the exo-labeled band 3 and 9 ns in the endo-labeled band 3. Both lifetimes appeared to be independent of temperature between 5.degree. and 25.degree. C, suggesting shielding of the probe from the solvent. Quenching phenomena must be responsible for both the 3 and 9 ns lifetimes, not due to residual heme, as proven by the persistence of such quenching in the Triton X-100 extracted protein. The correlation times indicated the presence of a short component, between 2 and 4 ns in the different systems, probably due to the presence of a flexible portion in the structure of the protein. In the endo-labeled system, the anisotropy at long times failed to reach a 0 value, consistent with the hypothesis that the rotational motions of the membrane-embedded protein are constrained around a single axis, perpendicular to the plane of the lipid bilayer. Addition of 300 mM NaCl to the samples increased the value of the limiting anisotropy in the endo-labeled system and in the exo-labeled protein decreased the extent of depolarization produced by the motion corresponding to the short correlation time of the system. This suggests an increased rigidity of the system upon addition of NaCl.