The locations of the three cysteine residues in the primary structure of the intrinsic segments of band 3 protein,and implications concerning the arrangement of band 3 protein in the bilayer

The intrinsic domains of band 3 protein contain three cysteine residues, one in a 17 kDa middle segment and two in a 35 kDa C-terminal segment. The latter are retained in an 8 kDa fragment produced by chymotrypsin treatment of ghosts. Cleavage of cysteine residues by 2-nitro-5-thiocyanobenzoic acid (NTCB) allows localization of this amino acid in the primary structure of the 8, 17, 35 and 52 (17 plus 35) kDa segments of band 3 protein. The mapping of these residues taken with other information concerning accessibility of various sites at the two sides of the membrane leads to the conclusion that band 3 protein crosses the membrane at least five times, or ten times in a dimer structure. The implications of this conclusion in terms of band 3 protein structure and function are briefly discussed.