STAPHYLOCOCCUS-AUREUS ALPHA-TOXIN - PRODUCTION OF FUNCTIONALLY INTACT, SITE-SPECIFICALLY MODIFIABLE PROTEIN BY INTRODUCTION OF CYSTEINE AT POSITION-69, POSITION-130, AND POSITION-186

Staphylococcal alpha-toxin, the prototype of an oligomerizing, pore-fo rming cytotoxin, is sensitive to biochemical modifications and cannot be labeled with biotin or fluorescein under preservation of its biolog ical activity. In this study, we have used site-directed mutagenesis t o introduce cysteine residues at positions 69, 130, and 186. Each muta nt was fully and rapidly reactive with several sulfhydryl-specific rea gents, indicating superficial location. Coupling of SH-groups with flu orescein-maleimide or biotin-maleimide was tolerated without loss of h emolytic activity at position 130, and the formed hexamers were visibl e on target cells by fluorescence microscopy and could be detected on electroblots by reaction with streptavidin-peroxidase. At the two othe r positions, modification caused significant loss of activity. However , the labeled proteins still bound to red cells, as shown by fluoresce nce microscopy and electroblotting. Intrinsically labeled alpha-toxin represents a novel tool to study the interaction of this pore-former w ith target membranes.