Chemistry and Biology of Proteins - F. Haurowitz 1953

Toxins (protein)
Bacterial toxins

Exotoxins. Exotoxins are toxic substances produced within Bacterial Cells and subsequently released into the surrounding environment or the host Organism. The exotoxins of Corynebacterium diphtheriae, Clostridium botulinum, and Clostridium tetani have been successfully purified and isolated in crystalline form. All highly potent toxins are globulin-type Proteins.

Diphtheria toxin is purified by fractional precipitation with ammonium sulfate. The purified toxin has a Molecular Weight of 74,000, and its molecular axial ratio is 4.7 : 1 [2]. Treatment with formaldehyde leads to a loss of toxicity, indicating that the free amino groups of the toxin play a crucial role in its biological activity [3]. Diphtheria toxin is a globulin-like protein that is inactivated by heat and Proteolytic Enzymes. It readily combines with the iron of the porphyrin ring and may serve as the protein component of the respiratory enzyme in Corynebacterium diphtheriae [1].

Crystalline tetanus toxin is obtained by precipitation with methyl alcohol at a slightly acidic reaction, low Ionic strength, and temperatures ranging from —8° to —5° [4]. This toxin is a highly labile globulin with an isoelectric point at pH 5.1 and a sedimentation constant (s) of 4.5. Upon prolonged storage, it is inactivated even at 0° [5], converting into a non-toxic, dimeric, easily flocculating compound that nevertheless retains The ability to be precipitated by its specific antibody [5].

The molecular weight of crystalline Clostridium botulinum exotoxin is approximately 1,000,000 [6, 7], its isoelectric point lies at pH 5.6, and its dissymmetry constant is f/f₀ =1.45; the axial ratio a/b reaches a very high value of the order of 10 [6, 7]. In the precipitate formed by the reaction of the toxin with its corresponding antibody, the antigen-to-antibody ratio is not constant, even though the toxin appears to be a homogeneous substance; the maximum number of antibody molecules bound by a single antigen molecule reaches 60 [6].

Partial purification of Clostridium welchii toxin [8] has been achieved using fractionation with ammonium sulfate and ethyl alcohol. This toxin appears to be an a-lecithinase that cleaves phosphorylcholine from lecithin or sphingomyelin [9]. Scarlet fever toxin was isolated by adsorption on Lloyd's reagent, followed by extraction with an alkaline buffer and precipitation from the extract with ammonium sulfate. The toxin is stable to brief heating at 100° and is not degraded by Trypsin or Pepsin; its molecular weight is approximately 14,000, and its isoelectric point is at pH 5.55. Similar to diphtheria toxin, the presence of free amino groups is essential for the biological activity of this toxin [10, 11].

The MECHANISM OF ACTION of exotoxins remains unclear. Some of these toxins exhibit a high affinity for The Nervous system and induce paralysis. The lethal dose of botulinum toxin required to kill a single mouse is 0.00005 у, or approximately 20 million toxin molecules [12]. Such an infinitesimally small quantity suggests that the toxin acts as an enzyme [13], catalyzing the Formation of other toxic products or triggering a chain reaction [12].

The toxins of Corynebacterium diphtheriae, Clostridium botulinum, and Clostridium tetani are typical Antigens. Upon injection of these substances into rabbits, large quantities of antitoxins are produced, which are capable of binding to the toxins and neutralizing their toxic properties. Toxins and antitoxins neutralize each other in definite proportions, making it possible to determine their concentrations by titrating one against the other.

Gramicidin and tyrocidine. Toxins of this type differ from those described in the previous section. They were discovered in the soil bacterium Bacillus brevis [14, 15]. They can be extracted from these Bacteria using ethyl alcohol, but not Water. The addition of ether to the alcoholic extract precipitates a toxin designated as tyrocidine, while the second toxin, termed gramicidin, remains in solution. Gramicidin and tyrocidine are Peptides with an unusual Structure; they lack both free amino groups and free carboxyl groups and are most likely cyclopeptides [16]. Unlike other naturally occurring Peptides and Proteins, gramicidin and tyrocidine contain d-Amino Acids [18], as well as Ornithine and ethanolamine [18].

The simplest of these cyclopeptides is gramicidin S [19]. It is an l-valyl-l-ornithyl-l-leucyl-d-phenylalanyl-l-prolyl cyclopeptide [18, 20]¹.

The gramicidin molecule isolated from B. brevis has the following composition: d-leu₆, l-try₆, d,l-val₄, l-ala₄, gly₂; in addition, the peptide contains 2 molecules of ethanolamine [18]. Countercurrent distribution analysis established that, contrary to initial assumptions, gramicidin is not a homogeneous substance but rather a mixture of several peptides [21, 22]. Tyrocidine has the following composition: phe₃, glu₂, asp₂, orn₂, try₂, pro₂, leu₂, val₂, ammonia₃ [15]. Gramicidin exhibits exceptionally potent toxicity against Gram-positive bacteria, whereas tyrocidine acts on both Gram-positive and Gram-negative bacteria. Although both preparations display bacteriostatic activity, they cannot be used for therapeutic purposes due to their toxicity to humans². Similar antibiotic Polypeptides have been isolated from other bacteria [22]. One of these, aerosporin (polymyxin A), is of particular interest because its composition includes a,y-diaminobutyric acid [23].

Endotoxins. Bacterial endotoxins have been found in Eberthella typhosa, Vibrio cholerae, and various types of Salmonella dysenteriae. Endotoxins are extracted from bacteria using trichloroacetic acid or diethylene glycol. Unlike many proteins, they are resistant to the action of trypsin [25] and are not inactivated by heat.

Endotoxins are complexes composed of a carbohydrate, a phospholipid, and a protein or peptide; they are present in the smooth S-forms of bacteria and absent in the rough R-forms [25]. In addition to the O-antigen, a so-called H-antigen has been found in bacterial flagella [26]. The endotoxin of dysentery bacteria of the Shiga type can be extracted with diethylene glycol. This endotoxin is dissociated into its constituent components upon treatment with 90% phenol [27]. The serological Specificity of this endotoxin is determined by its carbohydrate component, which Functions as a hapten (see Chapter XIV) and acquires antigenic properties upon conjugation with a protein [27]. A similar endotoxin has been isolated from the Flexner-type dysentery bacterium [28].

¹ Regarding The structure of gramicidin S, see the footnote on page 43. — Ed. note.

² The author's assertion that gramicidin cannot be used for therapeutic purposes due to its toxicity is incorrect. In surgical practice, gramicidin has found widespread application in wound treatment, where its toxic effects are hardly manifested owing to its slow absorption. The toxicity of gramicidin restricts only its intravenous or subcutaneous administration. — Ed. note. Endotoxins have also been isolated from Brucella melitensis [29] and Eberthella typhosa [30]. The latter endotoxin, purified by acetone fractionation, contains 50–60% CARBOHYDRATES, 3–4% Lipids, and approximately 30% peptide-like material [30].

Tuberculin, obtained from tubercle bacilli, is also a protein derivative. Fractionation of tuberculin with ethyl alcohol and by Electrophoresis yielded two Polysaccharides, three proteins (A, B, and C), and a cyclopeptide (D). The typical cutaneous reaction to the intradermal administration of tuberculin is elicited by protein A [31, 32]. It is noteworthy that proteins from tubercle bacilli are toxic only to infected organisms and are harmless to normal ones [33].