Glycerol allows low-temperature phase separation of membrane proteins solubilized in Triton X-114: application to the purification of plant cytochromes P-450 and b5.
The potentiality of the Triton X-114 phase separation technique for the purification of proteins from plant microsomal membranes has been investigated. It was shown that glycerol significantly lowers the cloud point of Triton X-114 solutions in water and of Triton X-114 solubilizates from microsomal membranes. It was also established that solubilized membrane components decrease the temperature of Triton X-114 micellar aggregation. Solubilization of microsomal membrane using detergent to protein ratios lower than 3.5, however, resulted in complete inhibition of detergent phase separation. Phase partitioning of Triton X-114 microsomal solubilizates, performed at low temperature (4 degrees C), in the presence of glycerol, provided a very fast and efficient step for the purification of cytochromes P-450 and b5. Conditions allowing optimal recoveries of these cytochromes have been defined.
Werck-Reichhart
D
D
CNRS-UPR 406, Département d'Enzymologie Cellulaire et Moléculaire, Institut de Botanique, Strasbourg, France.
Benveniste
I
I
Teutsch
H
H
Durst
F
F
Gabriac
B
B
D003080:Cold Temperature; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification*; D015786:Cytochromes b5 / Q000302:isolation & purification*; D005990:Glycerol; D008565:Membrane Proteins / Q000302:isolation & purification; D008861:Microsomes / Q000737:chemistry; D017830:Octoxynol; D010940:Plant Proteins / Q000302:isolation & purification; D011092:Polyethylene Glycols
D008565:Membrane Proteins; D010940:Plant Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; C010615:Nonidet P-40; D005990:Glycerol