Simplified purification and biophysicochemical characteristics of Kanagawa phenomenon-associated hemolysin of Vibrio parahaemolyticus

Abstract

Kanagawa phenomenon-associated hemolysin (K-hemolysin) was purified by Sephadex gel and ion-exchange column chromatography, after the culture supernatant had been adsorbed on and eluted from diethylaminoethyl-Sepharose CL-6B, and acid precipitated. K-hemolysin was a heat-stable and trypsin-susceptible protein with an apparent molecular weight of 44,000, the subunit of which was 22,000. The isoelectric point was 4.9. The minimum hemolytic dose was 0.1 mug/ml. The fifty percent lethal dose by intravenous injection was 1.4 mug. Electron microscopy of the small intestine of suckling mice orally challenged with the highest dose (50 mug) not only showed disappearance of epithelial cell microvilli, but also structural disturbances of the endoplasmic reticulum and mitochondrial swelling. One blueing dose representing permeability factor activity was 0.3 mug, and positive reaction in the rabbit ileal loop appeared at above 125 mug. Besides these data in experimental models, we discovered the appearance of an antibody in patients which neutralizes K-hemolysin during the course of the disease. This finding reinforces our view that K-hemolysin plays a most significant role in the pathogenesis of this enteric human disease.