sepharose and Autolysis

Glutathione reductase was extracted from toluene-treated baker's yeast cells by a two-stage buffer autolysis method. The yeast cells were treated with toluene for 1 h at 40 degrees C. After removal of the toluene, the cells were then allowed to autolysis in buffer for 72 h at 4 degrees C. The cells were collected and resuspended in buffer. A second stage autolysis was carried out for another 96 h at 4 degrees C. The enzyme was purified to 786-fold from the second stage cell autolysate by using two steps of affinity chromatography with triazine dyes (Yellow H-E4G and Yellow H-E6G) coupled to Sepharose CL-4B. By using this simplified method, 1.44 mg (165 units/mg) of glutathione reductase was obtained from 65 g (wet weight) of yeast cells, equivalent to 80% enzyme recovery.

Topics: Autolysis; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Glutathione Reductase; Saccharomyces cerevisiae; Sepharose; Toluene; Triazines