Accessibility of the N-ethylmaleimide-unreactive sulfhydryl of human erythrocyte Band 3.
The human erythrocyte anion exchange protein, Band 3, was reacted with N-ethylmaleimide (NEM) in cells to a stoichiometry of 5.3 mol NEM per mol Band 3, indicating that all NEM-reactive cysteines in Band 3 were labeled. Quantitatively NEM-blocked Band 3 was still able to bind to and be eluted by reducing agents from a mercurial affinity resin, [p-(chloromercuribenzamido)ethylene]amino-Sepharose. Reaction of NEM-blocked Band 3 with p-chloromercuribenzoate (pCMB) did not prevent binding to the resin due to exchange of pCMB for the immobilized mercurial. pCMB has been reported to inhibit water and urea permeation across the red cell membrane, and this has been attributed to reaction with a NEM-reactive sulfhydryl in Band 3. The interaction of Band 3 with the immobilized ligand directly demonstrates the reaction of NEM-blocked Band 3 with a mercurial and indicates that the NEM-unreactive, pCMB-reactive sulfhydryl residue is accessible to within approximately equal to 12 A (the distance from the solid support to the Hg) of the surface of the solubilized Band 3 protein.
Werner
P K
PK
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Lieberman
D M
DM
Reithmeier
R A
RA
D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D002729:Chloromercuribenzoates / Q000378:metabolism; D002846:Chromatography, Affinity; D003545:Cysteine / Q000378:metabolism*; D005033:Ethylmaleimide / Q000378:metabolism*; D006801:Humans; D020245:p-Chloromercuribenzoic Acid
D001457:Anion Exchange Protein 1, Erythrocyte; D002729:Chloromercuribenzoates; D020245:p-Chloromercuribenzoic Acid; D003545:Cysteine; D005033:Ethylmaleimide