Purification and Properties of Staphylococcal Beta Hemolysin II. Purification of Beta Hemolysin

Abstract

Staphylococcal beta hemolysin from the 681 strain of Staphylococcus aureus grown in a Heart Infusion dialysate semisolid medium under 10% carbon dioxide was obtained in an immunoelectrophoretically pure form by a combination of procedures of precipitation with 2 volumes of acetone followed by chromatography on diethylaminoethyl cellulose at pH 6.0. The acetone precipitation procedure did not show any deleterious effect on the hemolytic activity of the beta hemolysin unless the precipitate was left in contact with the acetone for at least 4 hr. The crude preparations contained two types of beta hemolysin. One of these represented the major portion of the total activity of beta hemolysin and behaved as a cation. The other represented a minor (1/5,000) portion of the total beta hemolysin activity and behaved as an anion. These active principles were designated as cationic and anionic beta hemolysins, respectively. An unexpected increase in the total beta hemolysin activity of the crude preparations was noted when these were concentrated by dialysis against polyethylene glycol (20 M). This effect was probably due to polyethylene glycol. A further unexpected increase in the titer of the acetoneprecipitated preparations occurred when these were lyophilized. The reason for this incremental increase is not known. It may be due to fragmentation of the beta hemolysin. Certain strains of Staphylococcus aureus are capable of producing a hemolytic macromolecule with characteristic ability to cause "hot-cold" lysis of sheep erythrocytes. This hemolysin, designated beta hemolysin by Glenny and Stevens (5), is most readily identified by the characteristic zone of darkening or discoloration that it produces on sheep blood-agar plates. Until recently, "hot-cold" lysis of sheep erythrocytes was the only activity attributed to beta hemolysin. Chesbro et al. (2) have now shown that the beta hemolysin which they obtained in partially purified form was leukocidal for guinea pig macrophages and was able to liberate mucopolysaccharides from the rabbit erythrocyte stroma, polysaccharides and mucopeptides from staphylococcal cell walls, and rhamnose, glucose, N-acetyl glucosamine, and at least two polysac-1 Portions of this study were presented at the 63rd Annual Meeting of the American Society for Microbiology, Cleveland, Ohio, May 1963. This paper is based on portions of a dissertation submitted by R. Haque to the Graduate School of The Ohio State University, Columbus, in partial fulfillment of the requirements for the Ph.D. degree.

Purification and Properties of Staphylococcal Delta Hemolysin

“…Proc., p. 78, 1970) isolated delta lysins with three different isoelectric points. Our findings support the idea that staphylococcal delta hemolysin is molecularly heterogeneous, i.e., it may, as staphylococcal alpha lysin (8,64) and beta lysin (13,30,48), exist in more than one active physicochemical form. Many other bacterial toxins are known to be physicochemically heterogeneous (2).…”

The hemolysins of Staphylococcus aureus.

“…Physicochemical characteristics. Numerous studies have shown that the hemolysin is a protein (83,92,133,208). Molecular weights range from 1.1 x 104 to 5.9 x 104 (26) to values of 1.6 x 104 to 3.8 x 104, depending upon the strain (see Table 9).…”

Beta Hemolysin: a Persistent Impurity in Preparations of Staphylococcal Nuclease and Enterotoxin

“…The beta-hemolytic activity eluted in two distinct peaks, the B2 peak overlapping the nuclease peak and the Bi peak overlapping the enterotoxin peak. Electrophoretically distinguishable forms of the beta hemolysin have been previously reported (11,24).…”

“…Their resolving power is diminished in the case of beta hemolysin, by the overlap of the electrophoretic and dimensional characteristics with those of the other two proteins. The problem thus posed in purifying these three proteins is compounded by the existence of a variety of electrophoretically and dimensionally distinguishable forms of each of the three (9,24,41). Thus, the successful employment of any combination of the foregoing techniques upon the exoproducts of a given strain does not bring with it any assurance of success when applied to a second strain.…”