A functional role for cysteine disulfides in the transmembrane transport of diphtheria toxin

Diphtheria toxin was modified at one or both of its cysteine disulfide bridges by iodoacetamide, methylmethanethiosulfonate, and atomic mercury. The products of these reactions were characterized and tested for toxicity in vitro and in vivo. All were toxic in vitro, but had lost almost all cytotoxic activity toward HeLa cells. It was possible to show from in vivo protection experiments that modification of the cysteine disulfide in the B-chain interfered with cell surface binding, while modification of the cysteine disulfide linking the A and B domains inhibits a step subsequent to binding in the intoxication process. The latter finding supports a functional role for this interdomain cysteine disulfide in the membrane transport process.