Affinity chromatography of cysteine-containing histone.
Affinity chromatography based on the reaction between SH groups in protein and +HgC6H4CO groups in the p-mercuribenzoylaminoethyl derivative of Sepharose 4B was examined with a crude preparation of calf thymus cysteine-containing histone.  Adsorption of the histone onto the column by specific coupling was found to be optimal in 0.1 M citrate buffer, pH 5.5, containing 5M urea to prevent any aggregation of histones and their non-specific adsorption onto the column, and elution from the column was successfully performed by cleavage of the resulting S-Hg bond with urea-buffer solution containing 0.05 M 2-mercaptoethanol.  Under these conditions both the adsorption and elution were quantitative; no adsorption was observed when either SH-blocked histone or unsubstituted Sepharose was used.  The cysteine-containing histone thus recovered, after further purification by Bio-Gel P-60 chromatography to remove some cysteine-containing nonhistone proteins contaminating the starting material, showed a single band on polyacrylamide gel electrophoresis and an amino acid composition agreeing with the known sequence of this histone.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002729:Chloromercuribenzoates; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003545:Cysteine / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D006657:Histones / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008623:Mercaptoethanol; D008626:Mercuribenzoates; D011485:Protein Binding; D013950:Thymus Gland
D000596:Amino Acids; D002729:Chloromercuribenzoates; D006657:Histones; D008626:Mercuribenzoates; D008623:Mercaptoethanol; D003545:Cysteine