2,4-Dinitrophenyl [14C]cysteinyl disulfide allows selective radioactive labeling of protein thiols under spectrophotometric control.
2,4-Dinitrophenyl [1-14C]cysteinyl disulfide readily introduces by disulfide exchange [14C]cysteine as a label into proteins with exposed thiols. The release of an equivalent amount of colored 2,4-dinitrothiophenolate allows the labeling reaction to be followed spectrophotometrically. In reaction with two cysteine residues of rabbit skeletal muscle actin, the thiol selectivity of the reagent corresponded to that of 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman's reagent) and was superior to that of N-[14C]ethylmaleimide. Labeling of single SH groups of actin and papain proceeded faster than titration with Ellman's reagent under the same conditions. The [14C]cysteine label could be removed under mild conditions, e.g., with dithiothreitol, but proved to be stable during cyanogen bromide degradation of the protein and peptide purification. 2,4-Dinitrophenyl cysteinyl disulfide can be easily prepared within a few hours.
Drewes
G
G
Max-Planck-Institut für Medizinische Forschung, Heidelberg, Federal Republic of Germany.
Faulstich
H
H
D000199:Actins; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D004228:Dithionitrobenzoic Acid; D006868:Hydrolysis; D007202:Indicators and Reagents; D009132:Muscles / Q000737:chemistry; D010455:Peptides / Q000302:isolation & purification; D011506:Proteins / Q000032:analysis*; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000032:analysis*
D000199:Actins; D007202:Indicators and Reagents; D010455:Peptides; D011506:Proteins; D013438:Sulfhydryl Compounds; C065748:2,4-dinitrophenylcysteinyl disulfide; D004228:Dithionitrobenzoic Acid; D003545:Cysteine