Characterization of eosin 5-isothiocyanate binding site in band 3 protein of the human erythrocyte.
The characteristics of the anion transport system in human erythrocyte, which can be modified by eosin 5-isothiocyanate (EITC), were studied using the pH titration method and by measuring the sulfate efflux. Based on the pH dependence of EITC binding to the erythrocyte ghosts, it was found that the reaction rate was maximal at about pH 6.4, and that the pH profile of EITC binding was similar to that of divalent anion transport. The interaction between EITC and ghosts was interpreted by a two-step reaction, a fast ionic-binding reaction and a slow covalent-binding reaction. The induced CD spectrum of the EITC-ghost system was also dependent on pH. The intensity of the CD band at 530 nm was decreased in acidic pH region, and the inflection point was observed at about pH 6.3, indicating a participation of the histidine residue in the interaction of EITC with band 3. In order to characterize the EITC-binding site, the kinetics of sulfate efflux in intact and EITC-modified cells were examined at various pH values. The inhibitory effect of EITC was dependent on pH. From the experimental results, the followings are suggested. The rate of ionic interaction in the early stage is much slower than that in a general ionic reaction. A conformational change may participate in the reaction. The conformation of the EITC-binding site depends on pH, relating to the dissociation of the histidine residues. The EITC molecules act also as a competitive inhibitor to the sulfate efflux after binding covalently to band 3 protein.
D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D001665:Binding Sites; D002942:Circular Dichroism; D004801:Eosine Yellowish-(YS) / Q000031:analogs & derivatives* / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D013431:Sulfates / Q000378:metabolism
D001457:Anion Exchange Protein 1, Erythrocyte; D013431:Sulfates; D006639:Histidine; C012957:eosine-5-isothiocyanate; D004801:Eosine Yellowish-(YS)