Extraction and purification of a catalase from Candida albicans

Candida albicans yeast cells H317 were grown to mid-log phase, mechanically disrupted and the resulting crude extract clarified by centrifugation. This catalase rich fraction (1.26 x 10(-4) units/ml) was fractionated by liquid phase isoelectric focusing in a pH gradient ranging from 3 to 10 using the Rotofor Isoelectric Focusing Preparative Cell. After isoelectric separation, fractions containing catalase activity were focused between pH 6.7 and 9.3. Active fractions were pooled and re-focused. After the second fractionation, catalase activity increased to 1.52 x 10(-2) units/ml and was restricted to fractions ranging from pH 7.6 to 8.8. To this point a 121 fold purification was achieved. Native polyacrylamide gel electrophoresis analysis of active fractions revealed a band migrating between 272,000 and 132,000 daltons which showed catalase activity. Purification of C. albicans catalase will allow us to evaluate its potential role in protecting this opportunistic pathogen from products of the oxidative burst. Antibodies generated against the catalase provide means for the evaluation of neutralizing fungal defenses against products of the oxidative burst during phagocytosis.