Isothermal denaturation of aqueous staphylococcal enterotoxin B by guanidine hydrochloride, urea, and acid pH

Abstract

The denaturation of staphylococcal enterotoxin B at 23 I : :1" by aqueous guanidine hydrochloride, urea, or dilute HCI was studied by measuring change in the intrinsic viscosity and near-ultraviolet difference spectrum of the protein. Prolonged exposure (hours to days, depending on denaturant concentration) of enterotoxin B to guanidine up to 6 M or urea up to 9 M was required for unfolding to reach equilibrium. Refolding of denatured toxin to native protein after dilution of denaturant was complete within minutes to a few hours. Thus, the native conformation of enterotoxin B C ertain strains of Sfup/zj,lococcirs uurrus elaborate watersoluble exoproteins which are capable of inducing an acute gastroenteritis in nian (Casman e f d., 1963). One of these exoproteins, staphylococcal enterotoxin B, can be obtained in high purity from the culture supernatants of appropriate staphylococcal strains (Schantz ef a / . . 1965). SEB' is also ii nonspecific mitogen. stimulating lymphocytes in riri'o to undergo blastoid transformation with increased mitotic activity (Peavy er a/., 1970) and to release macrophage migration inhibition factor (Kaplan: 1972). SEB. a globular protein with a molecular weight of 28,500, i s composed of one peptide chain containing 239 amino acid residues (Huang and Bergdoll. 1970; Schantz er ([/.. 1Y65). The single intramolecular disulfide bond of SEB spans a stretch of 21 amino acids b2-tween residues 92 and 112; 32 of the 34 aromatic residues in thy toxin niolecule are located outside of the closed disulfide loop. The basic and acidic residues are distributed along the entire length of the SEB chain. Our laboratory is presently investigating structural features of the native or modified SEB molecule requisite for in cico enterotoxic or immunogenic (Warrcn i'r d., 1973) and in i'ifro mitogenic activity, An iniportant part of this effort has been a study of the stability of SEB under various solvent conditions. Detailed information on the denaturation of other protein enterotoxins or mitogens is presently not available in the literature. Characteristics of the isothermal denaturation of a protein give important insights into structural features of thc protein in its native state . The native conformation of SEB in 0.15 hi KCI was found to be stable toward disruption by Gdn'HCI and urea in that unfolding of the protein in aqueous solutions of these denaturants showed a large time dependency. However, the protein was relatively susceptible to acid denaturation. drastic alteration in SEB structure having occurred helow pH 3.5.is favored by a large activation energy of denaturation and a comparatively small activation energy of renaturation. These isothermal denaturation experiments indicate a very stable core of structure in native enterotoxin B. However, toxin in 0.15 M KCI was rapidly denatured following addition of HCI to a p H of below 3.5. Below p H 2.3 slow association of acid-denatured toxin into a 7.6s aggregate was detected. The lability of enterotoxin to acid pH suggests stabilization of stru...

β Structure of aqueous staphylococcal enterotoxin B by spectropolarimetry and sequence-based conformational predictions

“…The excellent agreement for the amount Staphylococcal enterotoxin B is a bacterial exoprotein which can be purified to molecular homogeneity (Schantz et al, 1965) and is composed of a single polypeptide chain of 239 amino acid residues (Huang and Bergdoll, 1970). The native SEB* 1 molecule in aqueous solution shows an unusual kinetic resistance toward isothermal denaturation by Gdn-HCl or urea, yet appears to be rapidly denatured upon exposure to an acid environment (pH <3.5) in the absence of Gdn-HCl or urea (Warren et al, 1974a). To further elucidate structures of SEB in native and denatured states, a systematic CD study of SEB under a variety of solvent conditions has been performed.…”

“…Application of the Chou-Fasman sequence analysis (1974a,b) to SEB indicated that most of the native ß structure is composed of numerous ß segments held in a repeating, antiparallel configuration by intervening ß bends. This highly cooperative (3-sheet configuration probably contributes to the kinetic stability of native SEB toward perturbation by Gdn-HCl or urea (Warren et al, 1974a). Circular dichroism also revealed that disruption of native SEB structure by acid pH resulted in a several-fold increase of a helix and not an increase in the amount of random structure.…”

Rapid purification of staphylococcal enterotoxin B by high-pressure liquid chromatography

“…Culture filtrates of S. aureus 10-275 served as a source of crude SEB. Strain 10-275 is a high-SEB-producing mutant derivative of strain S6 (4,27,28).…”

Immunological studies on DNA‐lysine photoadduct

“…Melting curves were recorded at a fixed wavelength of 260 nm. Perecent denaturation was evaluated and the apparent first order rate constant for the thermal denaturation process was calculated as described earlier (13,18).…”