Arsenical-based affinity chromatography of vicinal dithiol-containing proteins: purification of L1210 leukemia cytoplasmic proteins and the recombinant rat c-erb A beta 1 T3 receptor.
Proteins containing vicinal dithiols were purified by affinity chromatography using Sepharose 4B linked to aminohexanoyl-4-aminophenylarsineoxide (As-Sepharose). The protein vicinal dithiols form stable dithioarsine derivatives with the arsine oxide moieties of the gel. The adsorbed proteins were eluted, at physiological pH, by buffers containing beta-mercaptoethanol or dithiothreitol. The dithiol proteins were identified by their specific labeling with N-iodoacetyl-3-[125I]-iodotyrosine. Cytoplasmic thiol proteins of L1210 murine leukemia lymphoblasts were separated into three classes by interaction with As-Sepharose. Proteins that did not bind to the gel consisted of monothiol proteins; proteins eluted by beta-mercaptoethanol include vicinal dithiol-containing proteins with low affinity for the arsine oxide. DL-Dithiothreitol (DTT) elutes a large group of vicinal dithiol-containing proteins with high affinity for the arsine groups. Gradient elution allowed characterization of the relative affinities of dithiol proteins for the As-Sepharose. A one-step purification of the L-triiodothyronine recombinant rat c-erb A beta 1 T3 receptor synthesized in yeast required pretreatment with DTT for binding to As-Sepharose and resulted in a 62-fold increase in specific activity. The procedure allows purification of proteins inhibited by phenylarsine oxide such as phosphotyrosine phosphatases, proteins that are subject to redox regulation, and dithiol proteins that are targets of oxidative stress.
Kalef
E
E
Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel.
Walfish
P G
PG
Gitler
C
C
D000327:Adsorption; D000818:Animals; D001152:Arsenicals; D002846:Chromatography, Affinity / Q000379:methods; D003593:Cytoplasm / Q000737:chemistry; D004229:Dithiothreitol; D007939:Leukemia L1210 / Q000378:metabolism*; D008214:Lymphocytes / Q000737:chemistry; D051379:Mice; D009363:Neoplasm Proteins / Q000302:isolation & purification*; D051381:Rats; D011988:Receptors, Thyroid Hormone / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000302:isolation & purification; D012685:Sepharose; D013438:Sulfhydryl Compounds / Q000302:isolation & purification*
D001152:Arsenicals; D009363:Neoplasm Proteins; D011988:Receptors, Thyroid Hormone; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D012685:Sepharose; D004229:Dithiothreitol