Partial purification and biochemical studies of hemolysin from Aeromonas hydrophila isolated from ulcerative disease snake-head fish

Abstract

Culture filtrate of Aeromonas hydrophila isolated from ulcerative-disease snake-head fish (Ophicephalus striatus) in Thailand was shown to be lethal to both snake-head and carp fishes under laboratory experimental condition. The filtrate contained a non-dialyzable, heat-labile substance with hemolytic activity. The substance, so-called hemolysin, was partially purified by acid precipitation followed by ion-exchange chromato-graphy on a column of QAE-Sephadex A-50 with about 300 folds of purification and 27.5% recovery yield. The apparent molecular weight of the native hemolysin was about 67K as detemined by Sephadex (G-100) gel filtration. The apparent molecular weight of purified hemolysin was about 48K as determined by SDS-PAGE in the presence of 2-mercaptoethanol. The purified hemolysin was protein in nature as shown to be sensitive to several proteolytic enzymes and was relatively unstable during purification. The activist was salt-dependent and optimum between pH 5-6. In addition, the hemolytic activity gradually declined during storage at -20 degree C but could be stabilized in 50% (v/v) glycerol. Many animal erythrocyte species (fish, sheep, human and mouse) were susceptible to the toxin at different degree. Hemolyic activity of the toxin was increased by adding sulfhydryl reducing agent but addition of sulfhydryl oxidizing agent significantly reduced the activity. The results indicated the relationship between free sulfhydryl group on the A.hydrophila hemolysin molecule and hemolytic activity. Proteolytic activity was not found in the present preparation of A.hydrophila hemolysin indicating that the purified hemolysin was attributable to be neither the protease associated substance nor the protease contaminated substance.