Chromatographic separation of extruded iron-sulfur cores from the apoproteins of Clostridium pasteurianum and spinach ferredoxins in aqueous Triton X-100/urea.
Iron-sulfur core extrusions from spinach [( 2Fe-2S]) and Clostridium pasteurianum (2[4Fe-4S]) ferredoxins in aqueous Triton X-100/urea containing excess benzenethiol yield quantitatively [FenSn(SPh)4]2- with n = 2 and n = 4, respectively. The iron-sulfur cluster can be separated from the corresponding apoprotein by rapid passage of the extrusion mixture over a small anaerobic column of Whatman DE-52 anion-exchange cellulose. Essentially quantitative recovery of [FenSn (SPh)4]2- is achieved in the eluate. The apoprotein remaining on the column can be eluted with 0.5 M NaCl. Most of the residual Triton X-100 and benzenethiol can be removed by passage of the apoprotein eluate over a small column of Bio-Beads SM-2, a hydrophobic polystyrene adsorbent. Apoprotein recovery is comparable to that obtained by other chromatographic methods. At least with spinach ferredoxin, the apoprotein prepared in this fashion can be reconstituted. The procedures developed in this work are potentially most applicable to selective removal of [2Fe-2S] and [4Fe-4S] centers from a multicenter enzyme without irreversible denaturation.
D001059:Apoproteins / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000302:isolation & purification*; D055598:Chemical Phenomena; D002621:Chemistry; D002852:Chromatography, Ion Exchange; D003013:Clostridium / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005288:Ferredoxins / Q000302:isolation & purification*; D007506:Iron-Sulfur Proteins / Q000302:isolation & purification*; D008667:Metalloproteins / Q000302:isolation & purification*; D017830:Octoxynol; D010940:Plant Proteins / Q000302:isolation & purification*; D011092:Polyethylene Glycols; D012996:Solutions; D013053:Spectrophotometry; D014508:Urea
D001059:Apoproteins; D001426:Bacterial Proteins; D005288:Ferredoxins; D007506:Iron-Sulfur Proteins; D008667:Metalloproteins; D010940:Plant Proteins; D012996:Solutions; D011092:Polyethylene Glycols; D014508:Urea; D017830:Octoxynol