Studies With Staphylococcal Toxins: V. Possible Identification of Alpha Hemolysin With a Proteolytic Enzyme

Abstract

Alpha hernolysin has been separated from many of the other conlponents present in culture filtrates of four strains of Staphylococcr~s nureus using a column chromatographic procedure with carbosymethyl-cellulose as the selective adsorbent, and gradecl levels of phosphate bulfer a t pI-I 6.0 t o provide selective elution. The preparation of alpha hemolysin obtained in this manner hemolyzed rabbit and sheep erythrocytes, inducecl a dcrmonecrotic reaction in rabbits, was lethal to mice, ancl was protcolytic. The hemolytic activity of the preparation was stirnulatecl by ethylenediamine tetraacetic acid, presumably by removing toxic ious from solution. Divalcnt cations inhibited activity of the alpha hemolysin, but stimulated thc activity of a clisti~~ctive shecp hemolysin, which is shown to be a separate entity.Alpha hcmolysin, obtained by thc chromatographic procedure, and subjected further to resolution by zone clcctrophoresis retained its capacity to induce clcrmonecrosis, to hemolyze rabbit erythrocytes, and to hydrolyze casein. Apparent loss of the capac~ty of the preparation to hcmolyzc shccp crythrocytcs was discussed.The possibility that alpha hcmolysin is a specific proteolytic cnzylne is suggcstecl by thc observation that under no conclitions could a separation be clfectcd betwecn the hemolytic ancl proteolytic properties of the alpha hemolysin reco\;crcd from a number of dilfcrent cultures. 1;LIan~licript

Isolation and Composition of Staphylococcal Alpha Toxin

“…It has been suggested that alpha toxin is a proteolytic enzyme (Robinson et al 1960). By the casein digestion method of Kunitz ( 1947) the amount of trichloracetic acid-soluble 280 mp-absorbing material liberated from 10 mg. casein by 100 pg.…”

“…Purification of alpha toxin has been carried out by Wittler & Pillemer (1948), Turpin, Relyveld, Pillet & Raynaud (1954), Butler (1959), Robinson, Thatcher & Montford (1960), and Goshi & Cluff (1960), but in no instance have the products satisfied the usual tests of homogeneity. In order to define more clearly the nature and properties of the toxin, its purification and characterization were undertaken once more.…”

Isolation and Properties of a Staphylococcal Protease, Preferentially Cleaving Glutamoyl‐Peptide Bonds

“…These enzymes were free from a-hemolysin, which previously [2] was shown to be associated with proteolytic activity. Drapeau et al [3] purified a protease from strain V8 which later was studied in detail with respect to substrat,e specificity by Houmard and Drapeau [4].…”

Trypsin-Mediated Activation of the -Haemolysin of Staphylococcus Aureus

“…Ion-exchange chromatography was performed on a column of carboxymethylcellulose (CMC) according to the method of Robinson, Thatcher and Montford (1960). Haemolysin samples were dialysed against distilled water for 24 h, centrifuged and then dialysed against 0 .…”

“…A K50 x 100-cm column containing Sephadex G-75 (Pharmacia, Montreal, Canada) was set up according to the manufacturer's instructions and equilibrated with Hallander's buffer (Hallander, 1963). Haemolysin was applied to the column in 5-ml volumes and 15-ml fractions were collected by upward elution.Ion-exchange chromatography was performed on a column of carboxymethylcellulose (CMC) according to the method of Robinson, Thatcher and Montford (1960). Haemolysin samples were dialysed against distilled water for 24 h, centrifuged and then dialysed against 0 .…”