ISOLATION AND SOME PROPERTIES OF THE HEMOLYSINS FROM THE CRUDE VENOM EXTRACT OF SCORPION TELSON, HETEROMETRUS GRAVIMANUS

Abstract

The hemolytic and enzymic investigations of the crude venom from Heterometrus gravimanus scorpion have revealed the presence of both direct and indirect (phospholipase A) hemolysins. The direct hemolysins were isolated from the lyophilized crude venom extract by Sephadex G-100 gel filtration, SP-Sephadex C-25 column chromatography, gel filtration of Sepharose 4B and Sephacryl S-300. The two direct hemolysins having high molecular weights, P (A) (M.W. : nearly 200×10⁴) and P (B) (M.W. : 22×10⁴), were obtained with the activity recoveries of 8% and 39.4%, respectively. P (B) was further separated into three fractions by Sephadex G-200 gel filtration, namely F (A) (M.W. : 17×10⁴), F (B) (M.W. : 4.5×10⁴) and F (C) (M.W. : below 1×10⁴), with the significant losses of direct hemolytic activities. However, F (B) had phospholipase A activity and this activity was partially inhibited by p-bromophenacyl bromide. F (B) and F (C) were soluble, even after the treatment of dialysis and lyophilization, in the isotonic saline and distilled water, and were homogeneous in polyacrylamide gels after going through disc and vertical plate electrophoreses. Furthermore, the schlieren pattern of F (B) also showed homogeneity of the preparation. The protein parts of hemolysins were found to be acidic ones. Although ultraviolet spectra of them were somewhat varied in accordance with their protein contents in the samples, the spectra of exitation and emission were almost identical. The direct hemolysins are presumably glycoproteins, since they are positive in the sugar reactions, and the difference between P (A) and P (B) is supposed to consist in the sugar part rather than that of proteins.