Leonard Spero

The role of sulfhydryl groups in the activity of type A botulinum toxin

Abstract Both crystalline botulinum toxin, Type A, (mol. wt. 900 000) and a lower molecular weight form (about 130 000) derived from it, have 4-SH groups and approx. 1 disulfide bridge per 10 5 g protein. Both forms were increasingly inactivated over a 24-h period to one-tenth of the original toxicity by p- mercuribenzoate (PMB) but iodoacetamide caused a smaller reduction and N- ethylmaleimide had no effect at all. Reversal of the PMB inactivation by reduced glutathione could not be achieved. It is concluded that reaction of -SH groups may affect conformational stability of botulinum toxin, but these groups are not an integral part of its active center.

Photooxidation of crystalline Clostridium botulinum type A toxin in the presence of methylene blue.

Abstract Photooxidation of crystalline botulinum type A toxin in the presence of traces of methylene blue results in a very rapid detoxification of the toxin. The combining power of the photochemically produced toxoid with the toxin antibody in vitro was not reduced as compared to the original toxin. Only a more extensive photooxidation of the toxin resulted in a moderate reduction of this property. Preliminary tests indicate that the protein detoxified by means of photooxidation is antigenic.

Microheterogeneity of staphylococcal enterotoxin B

Abstract Four components have been demonstrated in staphylococcal enterotoxin B by isoelectric focusing in polyacrylamide gels. In a typical preparation their relative concentrations from the most to least cathodic were 6, 56, 31 and 7%. The components were not stable conformers nor were the differences in isoionic points due to bound ligand. A comparison of the measured isoionic points with values calculated for varying numbers of excess basic groups indicated that each component differed from the succeeding one by a single charge. Sequential conversion of the components from the more to the less alkaline forms was obtained by exposure to pH 9.0 at 37 °C. The data gave an excellent fit for two consecutive first-order reacions in whicc the specific reaction rate constants were nearly identical. The reaction was much slower at pH 7.3 and 37 °C and no change at all was observed over a period of 32 days at pH 9.0 and 1 °C. Amide hydrolysis is the mechanism for these conversions, but the rate is too slow to account for the appearance of four components during the comparatively short period of bacterial fermentation. It is suggested, therfore, that only the most cathodic component is synthesized by the organism and that this is converted to the other forms enzymatically.

The Secondary Structure of Staphylococcal Enterotoxins A, B and C,

The circular dichroism (CD) of staphylococcal enterotoxins A, B and C was measured. The CD of enterotoxins B and C were almost identical from 250 to 320 nm, but differed from the CD of enterotoxin A. The spectrum of enterotoxin A in this wavelength region contained the same bands with respect to both location and sign, but with significant differences in intensity. The CD spectra of enterotoxins B and C were also much more alike from 190 to 250 nm. Although all three enterotoxins had a major negative extremum at 215--218 nm, its magnitude was equal in enterotoxins B and C, but was substantially decreased in enterotoxin A. The secondary structure of the enterotoxins contained little alpha-helix as analyzed with CD models. A secondary structure of entertoxin B compured from a scheme based on a joint prediction histogram of five separate methods, placed 29 residues in alpha-helices, 71 in beta-pleated sheets, 88 in beta-turns and 55 in aperiodic conformation.

The alkaline inactivation of botulinum toxin

Abstract In contrast to early observations on crude solutions of toxin, the purified toxin from Cl. botulinum, type A, was found to be quite stable at moderate alkalinity. The rate of inactivation increased by a factor of 4 × 104 over one pH unit above pH 10. Over a limited range, the inactivation rate was inversely proportional to the ninth power of the hydrogen-ion concentration. It is proposed that this rate is dependent upon the degree of a specific ionization and that the group involved is the ϵ-amino group of lysine.

Stabilization of native structure by the closed disulfide loop of staphylococcal enterotoxin B

Abstract The extent and rate of reduction of aqueous staphylococcal enterotoxin B by dithiothreitol in the absence or presence of the denaturant guanidine hydrochloride were directly proportional to the extent and rate of enterotoxin B unfolding. Thus, the single disulfide bond of native enterotoxin B must be positioned in a region of tertiary structure. Reduction followed by alkylation with iodoacetamide or iodoacetic acid of enterotoxin B unfolded in 8 M urea resulted, after refolding in dilute alkaline buffer, in aggregation of the alkylated monomer with a significant increase in viscosity. Also, the stability of alkylated enterotoxin B toward denaturation by guanidine hydrochloride was greatly diminished as compared to unmodified enterotoxin B. However, the refolded, alkylated enterotoxin derivatives retained the ability to completely precipitate rabbit antibody specific for native toxin. Tryptic hydrolysis of a single peptide bond in the 92–112 disulfide loop also significantly lowered the stability of the protein to guanidine denaturation. It is proposed, therefore, that the closed disulfide loop of enterotoxin B stabilizes a minor domain of structure whose disruption increases the conformational flexibility of the toxin molecule without extensive denaturation and loss of biologic specificity.

Guanidination and nitroguanidination of staphylococcal enterotoxin B

Guanidination of the free amino groups of staphylococcal enterotoxin B with 3,5-dimethyl-1-guanylpyrazole converted 31-32 of 33 epsilon-amino groups and 30% of the N-terminal residue. This product, although markedly reduced in solubility, suffered no gross change in conformation and retained full biological activity. A derivative prepared by reaction with O-methylisourea with only one lysyl residue unaltered lost most of its emetic activity. Nitroguanidination with 3,5-dimethyl-1-nitroguanylpyrazole converted up to 28 of the epsilon-amino groups and essentially all of the N-terminus. This material was greatly reduced in ability to produce emesis and like the O-methylisourea prepared guanidinated enterotoxin, gave only a line of partial identity in double diffusion. The loss of activity is attributed to unfolding and it is concluded that the free amino groups of enterotoxin B do not critically participate in either its antigenic determinants or its active center for emesis.

Effect of guanidinium salts on the toxicity of botulinum toxin

Abstract : The effect of guanidinium salts on the stability of botulinum toxin and the mechanisms through which denaturation by these salts occurs is described. Some salts are effective in reducing toxicity at low concentrations; in others, toxicity is retainedeven in saturated solution. The nature of the interaction is complex, involving more than a change in folding or conformation. The hypothesis that salt solutions with high thermodynamic water activity labilize the hydrogen-bonded structure of the protein is shown to be invalid. Also shown to be inapplicable is the hypothesis that a direct effect of the anion on the quanidinium cation leads to a reduction of its thermodynamic activity and ability to break hydrogen bonds. The protective mechanism appears to operate via binding of anions across clusters of cationic sites on the charged protein to preserve spatial configurations and charge distributions.

The pH dependence of enterotoxin polymerization by formaldehyde.

Abstract Toxoiding of staphylococcal enterotoxin B is accomplished by polymerizing the exotoxin into large, water-soluble antigen complexes in neutral or acid formaldehyde solution (Warren, J. R., Spero, L. and Metzger, J. F. (1973) J. Immunol. 111, 885). Experimental data for the presumptive nature of the amino acid sites in the enterotoxin B molecule at which intermolecular cross-linking by formaldehyde occurs are now reported. The polymerization of enterotoxin B in aqueous formaldehyde buffered at pH 7.5 was almost completely inhibited by added lysine; polymerization of enterotoxin at pH 5.0 was inhibited by added lysine or N-acetyltyrosine. Small model compounds for arginine, asparagine, glutamine, histidine and tryptophan residues were without effect. These results suggest that enterotoxin was polymerized at neutral pH by cross-linking between lysine residues, at acid pH by cross-linking between pairs of lysine and tyrosine residues. Thus, a high degree of chemical specificity and a marked pH dependence of this specificity are the major features of enterotoxin polymerization by formaldehyde.

Analysis of the near-ultraviolet circular dichroic spectra of staphylococcal enterotoxins A, B and C☆

The near-ultraviolet CD spectra from 260 to 300 nm of staphylococcal enterotoxins A, B and C were resolved empirically into Gaussian curves. Each spectrum contained the same six components with maximum ellipticities at virtually identical wavelengths. The rotatory strength of the bands of enterotoxin A, however, differed significantly from that of enterotoxins B and C. Each Gaussian curve was identified as corresponding to a prominent CD transition of an aromatic chromophore: two phenylalanine bands from its 1Lb transition (0-0 and 0 + 930 cm-1); four tyrosine bands from its 1Lb transition (the 800 cm-1 primary vibronic progression) with the weakest of these overlapping a phenylalanine band; and one 1Lb tryptophan band. At alkaline pH, tyrosylate CD bands arose, centered at 247-249 nm and at 295-298 nm. The intensity of the 295-298 nm bands indicated that most of the tyrosine CD in the neutral enterotoxins was contributed by buried residues. The tryptophan contribution to the CD of enterotoxin A was positive, while that of B and C was negative. Also indicative of a different milieu was the ready oxidation of the A toxin by N-bromosuccinimide and the unavailability of the single tryptophan residue in the other two toxins to this reagent. The environment of the disulfide bond was markedly diverse in the three enterotoxins. Enterotoxin C was refractory to reduction by mercaptoethanol under conditions where enterotoxins B and A were readily reduced. The contribution of the disulfide of enterotoxin B to its CD spectrum was positive with an intensity of about 9000 deg . cm2 . dmol-1 and was centered near 273 nm. The difference spectrum between native and reduced enterotoxin A was much smaller and appeared to be conformational in origin.