Solubilization of a membrane-bound diol dehydratase with retention of EPR g = 2.02 signal by using 2-(N-cyclohexylamino)ethanesulfonic acid buffer.

AUTOR(ES)

Hartmanis, M G

RESUMO

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.

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