J. Membrane Biol. 81, 139-147 (1984) The Journal of MembraneBiology 9 Springer-Verlag 1984' Functional Evidence for Distinct Interaction of Hydrophobic Arylisothiocyanates with the Erythrocyte Anion Transport Protein Santa O. Cacciola*, Hans Sigrist, Markus Reist, Z. Ioav Cabantchik, and Peter Zahler Institute of Biochemistry, University of Berne, Switzerland, and Department of Biological Chemistry, Hebrew University of Jerusalem, Jerusalem, Israel Summary. Human erythrocytes were treated with various hydro- phobic arylisothiocyanates under conditions which favor modifi- cation of distinct proteinaceous nucleophiles. The morphological appearance of phenylisothiocyanate-treated cells was discoid and membrane-bound hydrolases (human acetylcholinesterase, sheep phospholipase A2) were fully active following membrane modification. Noncharged hydrophobic arylisothiocyanates, in- cluding phenylisothiocyanate, ~-naphthylisothiocyanate and het- erobifunctional azidoarylisothiocyanates inhibited [35S]-sulfate efflux irreversibly. Protection against modification-induced inhibition of sulfate transport was attained by the simultaneous presence of the specific reversible anion transport inhibitor 4,4'-dinitrostilbene-2,2'-disulfonate. Selective protection of a functionally relevant domain of band 3 is concluded to occur based on the above-derived information. Key Words erythmcyte membrane - chemical modification 9 arylisothiocyanates 9 band 3 protein 9 anion transport Introduction Anion exchange in erythrocytes is mediated by a 95,000-dalton polypeptide, known as band 3 pro- tein. Under physiological conditions the protein ef- fects transmembrane exchange of C1- and HCO~- anions. The protein-mediated process is inhibited in vitro by inorganic and organic anions, structurally related to physiological substrates (for reviews on the subject see Deuticke, 1977; Cabantchik, Knauf & Rothstein, 1978; Knauf, 1979). Investigations which included reversibly and irreversibly binding inhibitors have indicated that the minimum require- ment for anion transport inhibition is the availability of at least one of differing molecular characteristics. The efficient anion transport inhibitor bears a nega- tive charge (anion), most obviously to interact non- covalently with a positively charged group allocated * Present address: Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218. in the functional domain of the protein (Cabantchik et al., 1978; Knauf, 1979). Furthermore, inhibition is significantly enhanced by the reagent's lipophilic- ity (Barzilay, Ship & Cabantchik, 1979; Cousin & Motais, 1982) and by the electron-attracting capac- ity of substituents within the inhibitor molecule (Barzilay et al., 1979; Kitagawa, Terada & Kame- tami, 1982). Reagents which provide either of the characteristics mentioned in conjunction with a co- valently reactive function are appropriate for selec- tive modification of the anion transport protein. Hydrophobic arylisothiocyanates fulfill the sec- ond and the third requirement for selective covalent inhibition of anion transport. Polar negatively charged groups which may direct the inhibitors to the anion binding site of band 3 are absent. The reagents are therefore of particular use for the ex- ploration of otherwise inaccessible hydrophobic do- mains of band 3. Apolar arylisothiocyanates favor- ably partition into lipophilic media and are thus capable of covalent membrane protein modification at sites where crucial transmembrane functions may occur. The covalent modification reaction ef- fects thiocarbamylation of reactive nucleophilic groups (R--S-; R--O-: ~N-; R--NH2) which, in an apolar environment, is most favorable for lysine e-amino groups (Sigrist & Zahler, 1982). In this study the effects of hydrophobic aryl- isothiocyanates on erythrocyte morphology, mem- brane-bound hydrolase activities and the process of anion exchange are investigated. With respect to anion transport, the inhibitory potency of various arylisothiocyanates including hetero-bifunctional azidoarylisothiocyanates are compared by analyz- ing [35S]-sulfate equilibrium exchange in labeled erythrocytes. Distinct interaction of hydrophobic arylisothiocyanates with the erythrocyte membrane is concluded from experiments which document that arylisothiocyanate-induced anion transport in-