Tiie Joixful of Riologic~l Ciiemistry

The in vitro exposure of staphylococcal enterotoxin B to trypsin resulted in the formation within 30 min of a product (enterotoxin-T) unchanged in molecular weight, but with threonine as a second NH2 terminus. Upon reduction of the -SSbridge in enterotoxin-T, two fragments were separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The peptide bond between Lys-97 and Thr-98 was unequivocally identified by automated sequence determination as the site of cleavage by trypsin. This LysThr bond is positioned in the 21.residue disulfide loop of enterotoxin B, and enterotoxin-T is thus a “nicked” molecule (COLLIER, R. J., AND KANDEL, J. (1971) J. Biol. Chem. 246, 1496-1503). Native enterotoxin B obtained directly from bacterial cell culture was also nicked to a small extent, but not at the trypsin-susceptible bond. In the absence of denaturant the two reduced fragments did not separate and were thus held together by strong noncovalent forces. Enterotoxin B and -T demonstrated very similar amino acid composition, sedimentation in the ultracentrifuge, gel filtration on Sephadex G-50, and reducibility of the disulfide bond by dithiothreitol. They were also indistinguishable in the quantitative precipitin reaction, gave a reaction of complete identity in Ouchterlony immunodiffusion, and showed equivalent emetic activity in rhesus monkeys. Hence, no significant physicochemical or biological difference could be found between enterotoxin B and -T. Although there is an analogy between the structures and reactivity to trypsin of diphtheria toxin and enterotoxin B, it is unlikely that the staphylococcal enterotoxins undergo a comparable physiological activation since the antigenic variant staphylococcal enterotoxin A was found to be completely resistant to trypsin.