Effects of carrier ampholyte contamination on the biological and biochemical properties of streptococcal pyrogenic exotoxin type C.

Three streptococcal pyrogenic exotoxins (SPEs), designated as SPE A, B, and C, have been purified and characterized. Routine purification of the SPEs includes the technique of isoelectric focusing. An earlier study showed that the removal of commercial carrier ampholytes (Ampholines) from SPE was difficult. The physiochemical properties of SPE C were previously reported; however, the SPE C preparation used in those experiments was contaminated with Ampholines. As an alternative to Ampholines, we used simple buffers to generate the isoelectric focusing pH gradient and used this SPE C in a comparative study to evaluate the effects of Ampholine contamination on the biological and biochemical properties of this toxin. We found that Ampholine contamination overestimates protein concentration; consequently, the biological activity of SPE C was actually greater than reported. The most serious effect of Ampholines in SPE C was on amino acid analysis. The presence of Ampholines causes an apparent increase in neutral amino acids and a decrease in basic amino acids.