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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

1991; Elsevier BV; Volume: 197; Issue: 1 Linguagem: Inglês

10.1016/0003-2697(91)90367-3

1096-0309

Danièle Werck‐Reichhart, Irène Benveniste, H. Teutsch, Francis Durst, Brigitte Gabriac,

Microbial Metabolites in Food Biotechnology

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°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.