A novel hemolysin from the lichen Parmelia pulla

A hitherto unknown hemolysin from the lichen Parmelia pulla was discovered and a method was developed for its purification to apparent homogeneity. Saline phosphate buffer pH 7.2 extracted the bulk of the hemolysin from the lichen thalli. From this extract the hemolysin was purified by ammonium sulfate precipitation and gel filtration with Sepharose 6-B column. The overall recovery was about 75% and the purified hemolysin appeared to be electrophoretically homogenous and had a native molecular weight of 32,600. The purified hemolysin had a pH optimum around 5.5, stable at room temperature and gradually loses its activity upon freezing and thawing. Polyacrylamide gel electrophoresis of the purified hemolysin in the presence of sodium dodecyl sulfate revealed the presence of two types of subunits with apparent molecular weight of 18,000 and 14,000 respectively, indicating a dimeric (alpha beta) type of structure. Immunoblotting analysis demonstrated the presence of this hemolysin in crude extract prepared from P tinictina but not in crude extract from P tiliacea and P acetabulum. The purified hemolysin lyses rabbit erythrocytes and the rate of hemolysis was linear dependence on protein concentration. Erythrocytes obtained from various species including human were also lysed by the purified hemolysin in a concentration dependent manner.