Effect of single and double peptide bond scission by trypsin on the structure and activity of staphylococcal...

Two peptide bonds of staphylococcal enterotoxin C, were hydrolyzed concurrently at quite different rates during limited digestion with trypsin. A Lys-Val at about position 92 in the disulfide loop was the first bond cleaved, followed by a Lys-Asx at about position 57 on the NH2-terminal side of the loop. Preparations of singly cleaved material (enterotoxin C1-T1) contained about 93% of the cleaved protein and 7% unreacted enterotoxin. Preparations of the doubly cleaved material (enterotoxin C1-T2) consisted of 98% enterotoxin C1-T2 and 2% enterotoxin C1-T1. In the absence of denaturant, enterotoxin C1-T2 behaved as a single particle. It gave a single peak on Sephadex G-75 with a sedimentation coefficient of 2.85 S and a molecular weight of 29,100 by sedimentation equilibrium. Circular dichroic spectra indicated only minor conformational differences between enterotoxins C1-T2 and C1. However conformational stability was significantly affected with the unfolding of enterotoxin C1-T2 in 4 M guanidine hydrochloride proceeding at about twice the rate of native enterotoxin. Enterotoxin C1-T2 was separated into 6,500 and 22,000 molecular weight polypeptides by gel filtration on Sepharose 6B in 6 M guanidine hydrochloride. Complementation (as measured by CD spectra, serologic activity and mitogenicity) of the two polypeptides was readily achieved from solution in 6 M guanidine hydrochloride by dialysis against phosphate buffer. The 22,000 molecular weight polypeptide was further separated into two peptides (Mr = 4,000 and 19,000 after alkylation of the reduced disulfide bridge. Summation of the amino acid composition of the constituent peptides of enterotoxin C1-T2 agreed well with the composition of enterotoxin C1. A comparison of the 6,500 and 4,000 molecular weight polypeptides from enterotoxin C1-T2 with structurally equivalent segments of enterotoxin B suggested structural homology between the two antigenic variants. Enterotoxins C1, C1-T1, and C1-T2 gave reactions of complete identity in Ouchterlony immunodiffusion and were indistinguishable in the quantitative precipitin reaction. Enterotoxins C1-T1 and C1-T2 were highly mitogenic but were slightly less potent than the native enterotoxin. Enterotoxin C1-T2 had equivalent emetic activity to enterotoxin C1 in rhesus monkeys. It is suggested that the exceptional lability to limited enzymic hydrolysis exemplified by enterotoxin C1 is associated with beta turn structures at protein surfaces.