Purification ofStaphylococcus aureusβ-Toxin: Comparison of Three Isoelectric Focusing Methods

Abstract

β-Toxin (β-hemolysin) is one of several extracellular proteins produced byStaphylococcus aureus.It is a sphingomyelinase which disrupts the membranes of erythrocytes and other mammalian cells. Despite its characterized mechanism of action, the role of β-toxin in human and animal disease remains unclear. In this report, we compare three gentle, rapid methods to purify enzymatically active β-toxin. Extracellular proteins inS. aureusstrain RN4220 cell supernatants, containing a high concentration of the toxin, were precipitated by ethanol, dialyzed, and separated by preparative isoelectric focusing (IEF). We compared the efficiency of three preparative IEF methods: a Sephadex flat-bed IEF using pH 3.5–10.0 Ampholine, a liquid IEF using pH 7.8–8.9 Rotolyte buffers, and a liquid IEF with two consecutive steps using pH 3.0–10.0 Bio-Lytes in the first separation followed by a second step using pH 6.0–8.0 Bio-Lytes. All three IEF methods purified milligram amounts of enzymatically and biologically active β-toxin. Typically, 2–5 mg of purified β-toxin was obtained from 1.2 liters of culture medium. The total enzymatic activity recovered and overall yield were similar for all three methods. However, the single-step liquid IEF separation using Rotolyte buffers was the most preferred method because it purified β-toxin to >95% purity, did not require dialysis to remove ampholytes, and was the most rapid of the three methods.