Chemical studies on staphylococcal alpha-toxin and its fragments

Abstract

THE CRYSTALLIZED staphylococcal alpha-toxin, a single protein with a mol . wt of 36,000, exhibits hemolytic, dermonecrotic and lethal activity (WATANABE and KATO, 1974, 1976) . However, the relationship between its chemical structure and its mechanism of action at the molecular level is not fully understood. Since the purified alpha-toxin is devoid of free SH-groups and disulfide bridges, the toxin was subjected to proteolytic treatment with the aim of obtaining split products which might be more amenable to structural studies . WATANABE and KATO (1978) reported that the purified alpha-toxin treated with trypsin yielded two components by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis . A heavy chain fragment (mol . wt 17,000) had lethal activity in mice but lacked hemolytic and dermonecrotic activities, whereas a light fragment (mol . wt 14,000) was nontoxic and unstable . The present effort was undertaken to identify the Nand C-terminal amino acids of alpha-toxin and to correlate the results with the fragment chain structure of the toxin molecule. The crystallized alpha-toxin and heavy and light chain fragments were prepared as described previously (WATANABE and KATO, 1976, 1978). The time course of the digestion of the purified alpha-toxin by trypsin was followed by a slab-polyacrylamide gel electrophoresis (Fig . 1), The position of the heavy and light chain fragments of alpha-toxin was established by staining a few gels . The relevant regions ofthe unstained gels were removed, pooled, homogenized with 0-03% SDS and extracted overnight with the detergent . The extracts were lyophilized and used for the analyses of amino acid compositions and Nterminal amino acid residues . The amino acid compositions of alpha-toxin and its heavy and light chain fragments are summarized in Table 1. Amino acid analyses indicate different contents of lysine, histidine, threonine, methionine and tyrosine in the two fragments. The lethal heavy chain fragment is distinctive in containing only one residue of histidine. In experiments where we wanted to examine the effects of photo-oxidation of the histidine residue (WEIL et al., 1951), exposure of the heavy chain fragment to visible light in the presence of methylene blue at pH 8-0 resulted in a 60% loss of lethal activity in mice . The total of the mol . wts and amino acid residues of the two fragments is 31,000 and 268, respectively (Table 1) . When the alpha-toxin molecule is cleaved simultaneously at more than one position by trypsin, one or several small peptide(s) of no more than ten amino acids each (e .g . 26 amino acid residues in Table 1) are lost during fragment isolation procedures . Amino-terminal analysis, performed by the dansyl chloride method (GRAY, 1967), of alpha-toxin and its heavy chain and light chain fragments indicated alanine, isoleucine and alanine residues, respectively (Table 2). The sequences of the carboxymethylated alpha-