Specific fragmentation of human erythrocyte spectrin by chemical cleavage at cysteine residues.
Spectrin, isolated from human erythrocyte membrane, was specifically cleaved at the amino side of its cysteine residues by reacting it with 2-nitro-5-thiocyanobenzoic acid at pH 8.0 and incubating the product at pH 9.0. Conditions were developed to obtain quantitative cleavage, with virtually no side reactions due to exposure to the alkaline pH. The solubility and aggregation state of the spectrin fragments in 0.2 M sodium chloride, in 7 M guanidine hydrochloride or in 10 M urea, at pH 8.0, allow separation and partial purification of the fragments by gel filtration or by ion-exchange chromatography. Our results strongly suggest that various parts of the spectrin molecules have similar amino acid compositions. Due to the relatively limited number of fragments, this cleavage method is a promising tool for further elucidation of the structure of spectrin and for understanding its role in the erythrocyte membrane.
D000596:Amino Acids / Q000032:analysis; D055598:Chemical Phenomena; D002621:Chemistry; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003545:Cysteine / Q000032:analysis*; D004910:Erythrocyte Membrane / Q000032:analysis*; D004912:Erythrocytes / Q000032:analysis*; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis*; D008722:Methods; D010446:Peptide Fragments / Q000032:analysis; D013049:Spectrin / Q000032:analysis*
D000596:Amino Acids; D008565:Membrane Proteins; D010446:Peptide Fragments; D013049:Spectrin; D003545:Cysteine