Separation of microsomal cytochrome b5 via phase separation in a mixed solution of Triton X-114 and charged dextran.
The successful introduction of a charged dextran into the Triton X-114 phase separation system for the selective extraction of cytochrome b5 (cyt. b5) in liver microsomes is described. In the absence of charged dextran, 55% of total microsomal proteins and 84% of cyt. b5 were extracted into the surfactant-rich phase. In the presence of anionic dextran sulfate, the extractability of total microsomal proteins was greatly reduced while that of cyt. b5 was increased. After triplicate extraction, cyt. b5 was purified more than 10-fold from microsomes with a recovery of 91% in the surfactant-rich phase. In view of its operational simplicity, this method provides a good means for the partial purification of cyt. b5 prior to chromatographic separations.
Tani
H
H
Laboratory of Bioanalytical Chemistry, Graduate School of Engineering, Hokkaido University, Sapporo, Japan.
Ooura
T
T
Kamidate
T
T
Kamataki
T
T
Watanabe
H
H
D000818:Animals; D002498:Centrifugation; D015786:Cytochromes b5 / Q000302:isolation & purification*; D003902:Detergents; D003911:Dextrans / Q000737:chemistry; D004563:Electrochemistry; D004983:Ethanolamines; D008565:Membrane Proteins / Q000302:isolation & purification; D008862:Microsomes, Liver / Q000737:chemistry; D017830:Octoxynol; D011092:Polyethylene Glycols; D012996:Solutions; D013552:Swine; D013696:Temperature
D003902:Detergents; D003911:Dextrans; D004983:Ethanolamines; D008565:Membrane Proteins; D012996:Solutions; D011092:Polyethylene Glycols; D017830:Octoxynol; D015786:Cytochromes b5; C010615:Nonidet P-40; C007369:2-diethylaminoethanol