4-acetamido-4--isothiocyanatostilbene-2-2--disulfonic-acid and eosine-5-isothiocyanate

Although eosin-5-maleimide (EM) covalently labels band 3 and has been thought to react at the external-facing anion transport site, EM reversibly inhibits Cl- exchange at 0 degrees C in a noncompetitive fashion, indicating that under these conditions it does not bind to the transport site [Knauf, P.A., N.M. Strong, J. Penikas, R.B. Wheeler, Jr., and S.J. Liu. Am. J. Physiol. 264 (Cell Physiol. 33): C1144-C1154 1993]. To see whether or not the covalent labeling by EM takes place at the same noncompetitive site as the reversible binding, we examined the dependence of reaction rate on EM concentration. The reaction rate saturates with increasing EM concentration, indicating that reversible binding precedes covalent reaction and that EM therefore acts as an affinity label. A more complex model in which reversible binding prevents a bimolecular reaction at a different site cannot, however, be ruled out. Cl- gradients across the membrane affect EM reversible binding in a manner suggesting that EM binds preferentially to the Eo form of band 3, with the transport site unloaded and facing outward. Thus EM binds to and probably reacts covalently with a site that is different from the transport site, but whose conformation is affected by the orientation of the transport site. Lysine-430, the amino acid residue which is covalently labeled by EM (4), may be near the transport site but does not seem to be directly involved in the binding of transported substrates such as chloride. EM binding to one band 3 monomer decreases the reactivity of the adjacent monomer but does not decrease the affinity constant of the reversible binding step that precedes covalent reaction. Although a small fraction (approximately 1%) of band 3 monomers fail to react with EM, EM nearly completely inhibits transport in those monomers with which it reacts.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amino Acid Sequence; Anion Exchange Protein 1, Erythrocyte; Binding Sites; Chlorides; Eosine Yellowish-(YS); Erythrocyte Membrane; Humans; Kinetics; Lysine; Mathematics; Models, Biological; Protein Conformation

The induced circular dichroism (CD) of erythrocyte ghosts with anion-transport inhibitors has been studied. A ghost-EITC (eosin 5-isothiocyanate) system shows an induced CD spectrum at the wavelength region corresponding to the absorption bands of EITC. Also a ghost-EMI (eosin 5-maleimide) system shows induced CD, but has bands of opposite sign to the EITC system. From the change of the CD intensity, the number of EITC molecules bound to one erythrocyte was estimated to be about 1.4 X 10(6), being close to the number of band 3 copies per ghost. The CD spectra of EITC and EMI systems show that a configurational structure of the moiety anchoring the EMI molecule is the reverse to that of EITC. The preferred conformation of bound EITC may be twisted in a right-handed sense. From the signs of the induced CD bands in ghost-stilbene disulfonate systems, the chirality of twisted stilbene derivatives seems to be a left-handed sense, as is the case for the EMI derivative. The CD spectra of EITC in the presence of DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonate) shows that the binding site of EITC may not be identical with that of DIDS. The results observed in this study reflect the ternary arrangement of the functional amino groups in anion recognition sites.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Anion Exchange Protein 1, Erythrocyte; Binding Sites; Biological Transport; Circular Dichroism; Eosine Yellowish-(YS); Erythrocyte Membrane; Humans; In Vitro Techniques; Ion Channels; Molecular Conformation; Structure-Activity Relationship