Chemical modification and labeling of glutamate residues at the stilbenedisulfonate site of human red blood cell band 3 protein.
A new method has been developed for the chemical modification and labeling of carboxyl groups in proteins. Carboxyl groups are activated with Woodward's reagent K (N-ethyl-5-phenylisoxazolium 3'-sulfonate), and the adducts are reduced with [3H]BH4. The method has been applied to the anion transport protein of the human red blood cell (band 3). Woodward's reagent K is a reasonably potent inhibitor of band 3-mediated anion transport; a 5-min exposure of intact cells to 2 mM reagent at pH 6.5 produces 80% inhibition of transport. The inhibition is a consequence of modification of residues that can be protected by 4,4'-dinitrostilbene-2,2'-disulfonate. Treatment of intact cells with Woodward's reagent K followed by B3H4 causes extensive labeling of band 3, with minimal labeling of intracellular proteins such as spectrin. Proteolytic digestion of the labeled protein reveals that both the 60- and the 35-kDa chymotryptic fragments are labeled and that the labeling of each is inhibitable by stilbenedisulfonate. If the reduction is performed at neutral pH the major labeled product is the primary alcohol corresponding to the original carboxylic acid. Liquid chromatography of acid hydrolysates of labeled affinity-purified band 3 shows that glutamate but not aspartate residues have been converted into the hydroxyl derivative. This is the first demonstration of the conversion of a glutamate carboxyl group to an alcohol in a protein. The labeling experiments reveal that there are two glutamate residues that are sufficiently close to the stilbenedisulfonate site for their labeling to be blocked by 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate and 4,4'-dinitrostilbene-2,2'-disulfonate.
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000031:analogs & derivatives; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D002918:Chymotrypsin / Q000378:metabolism; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D006801:Humans; D007555:Isoxazoles / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008745:Methylation; D008970:Molecular Weight; D013267:Stilbenes / Q000494:pharmacology
D001457:Anion Exchange Protein 1, Erythrocyte; D005971:Glutamates; D007555:Isoxazoles; D008565:Membrane Proteins; D013267:Stilbenes; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; C009686:N-ethyl-5-phenylisoxazolium-3'-sulfonate; D018698:Glutamic Acid; C018564:dihydro-DIDS; C022306:4,4'-dinitro-2,2'-stilbenedisulfonic acid; D002918:Chymotrypsin; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid