Solubilization of a membrane-bound diol dehydratase with retention of EPR g = 2.02 signal by using 2-(N-cyclohexylamino)ethanesulfonic acid buffer.
A procedure for solubilization of the oxygen-labile, membrane-bound diol dehydratase from Clostridium glycolicum with retention of enzymatic activity is described. The procedure involves sonication of crude membrane preparations anaerobically in 0.1 M 2-(N-cyclohexylamino)ethane-sulfonic acid (CHES) buffer (pH 8.6-9.0) containing 2 mM dithiothreitol. The addition of dimethylsulfoxide (30%) and lysophosphatidylcholine (0.15 mg/ml) to the solubilization buffer resulted in a 10-fold increase in recovery of solubilized diol dehydratase activity. After ultracentrifugation, an overall recovery of 50% of the activity initially present in the crude membrane preparations was achieved. Active membrane preparations and the solubilized enzyme exhibited an EPR signal at g = 2.02. Both enzyme activity and EPR signal were sensitive to oxygen and the radical scavengers, NH2OH and hydroxy-urea.
D002021:Buffers; D002462:Cell Membrane / Q000201:enzymology; D003013:Clostridium / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D006836:Hydro-Lyases / Q000302:isolation & purification*; D007700:Kinetics; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D010713:Phosphatidylcholines / Q000494:pharmacology; D011408:Propanediol Dehydratase / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D013654:Taurine / Q000031:analogs & derivatives
D002021:Buffers; D008244:Lysophosphatidylcholines; D010713:Phosphatidylcholines; C050927:2-(N-cyclohexylamino)ethanesulfonic acid; D013654:Taurine; D006836:Hydro-Lyases; D011408:Propanediol Dehydratase