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A procedure for isolation of highly purified hemagglutinin from a toxic complex of culture filtrates of Cl. botulinum type A is described. This procedure includes precipitation with (NH4)2SO4, chromatography on Sephadex G-100, G-200 and DEAE-cellulose, specific adsorption on human erythrocytes and affinity chromatography. Using polyacrylamide gel electrophoresis, it was shown that hemagglutinin is a heteropolymeric protein consisting of a monomer (Mr 53 000) and a trimer (Mr 160 000). The monomer is made up of two subunits with Mr 13 000 and one subunit with Mr 27 000 covalently linked by SS-crosslinks. The number and nature of the SS-crosslinks and SH-groups in the protein molecule were determined and a hypothetical structural model of hemagglutinin was proposed. Using immunochemical analysis, it was shown that some (but not all) serological properties of the highly purified protein from Cl. botulinum type A and of its partially purified counterpart are similar to those of hemagglutinin from Cl. botulinum type B.

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The data on the study of the protective activity of theta hemolysin and Cl. perfringens lecithinase preparations and the corresponding antitoxic sera obtained by indirect immune affinity chromatography are presented. Experiments in mice and guinea pigs indicate that the injection of antihemolytic serum and immunization with anatheta hemolysin ensures the protection of the animals from theta toxin. The enrichment of analecithinase preparation with anatheta hemolysin has been found to increase its protective properties against Cl. perfringens culture and toxin.

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Guinea-pigs were immunized with anatheta-hemolysin preparations adsorbed on aluminum hydroxide, as well as with the mixture of anatheta-hemolysin and type A Cl. perfringens toxoid, purified and concentrated. Anatheta hemolysin preparations were obtained with the use of homogeneous theta hemolysin, as well theta hemolysin of various purification degrees. As a result, antatheta hemolytic guinea-pig sera capable of neutralizing 2,000-8,000 HU of theta-hemolysin were obtained. Tests made to establish the degree of protection in the immunized guinea-pigs did not show that the animals immunized with the mixture of anatheta-hemolysin and type A Cl. perfringens toxoid, purified and concentrated, had any advantages in the degree of protection over the animals immunized with the toxoid alone. But there is no doubt that this component plays a positive role under the conditions of natural gas gangrene when the hemolytic action of Cl. perfringens toxin becomes considerably pronounced.

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Teta-hemolysine was purified from Cl. perfringens strain BP6K 28 as follows: reprecipitation in the isoelectric point of the enzyme with 2 N H2SO4 containing 15% NaCl, DEAE cellulose chromatography, gel filtration on Sephadex G-100-G75 or Bio Gel P-60--P-100, rechromatography on DEAE-Sephadex A-50 and affinity chromatography. The biological activity of homogenous teta-hemolysine, estimated by complete hemolysis of human erythrocytes, exceeded 100, 000 theta E over mg protein but the enzyme was highly labile. Molecular weight of the enzyme was 53,000 daltons.

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The method for obtaining the neurotoxin, or alpha-fraction of the toxin, of Cl. botulinum, type B, is described. In accordance with this method, the toxin was precipitated three times with hydrochloric acid in the isoelectric zone with subsequent extraction with phosphate (pH 6.8) and citrate-phosphate (pH 5.6) buffers, then fractionated in columns with DEAE cellulose (pH 5.6), DEAE Sephadex A-50 (pH 7.2) and Sephadex G-200 (pH 7.2). The homogeneous neurotoxin preparations with molecular weights ranging from 145,000 to 160,000 and having the isoelectric point at pH 5.5 and toxicity 5.0--10.0 x 10(7) Dlm per 1 mg protein were obtained.

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The toxic comples of Cl. botulinum, type F, was separated into the toxic and nontoxic protein fractions by the methods of ion exchange chromatography and gel filtration in accordance with a specially devised purification scheme. Highly purified, electrophoretically and serologically homogeneous toxin with a molecular weight of 150,000 and potency equal to 10 X 10(6) DLM per 1 mg of protein was isolated from the toxic fraction. The nontoxic protein component had faintly pronounced hemagglutinating properties and was essentially different from type A and B hemagglutinins. The toxic complex of Cl. botulinum, type F, was shown to contain a proteolytically active fraction.

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The authors demonstrated an incomplete indentity of Cl. botulinum hemagglutinins of types A and B in the double diffusion reaction in agar gel, and their difference by electrophoretic mobility. Some differences in the interaction of hemagglutinins A and B with human erythrocytes were found by the hemagglutination inhibition method; apparently, of the principal significance in the relization of the reaction of human erythrocyte hemagglutination with hemagglutinins of Cl. botulinum, types A and B, was the OH-group position in the C4 galactose of the mucopolysaccharides of the erythrocyte cell wall. Apart from C4, apparently, for hemagglutinin of types A of significance was the reactive capacity of C1 and C2 galactose atoms, whereas for hemagglutinin of type B--free OH-group in C2 galactose atom.

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A study was made of a possibility of inhibition of biosynthesis of penicillinase in Staph. aureus by acridine derivatives. Acetone preparations of penicillinase were obtained from the cultures of staphylococcus strains 16/160 and 8325 (p11(147) pen 1220) grown in the presence of various subbacterial concentrations of acridine derivatives. The activity of the enzyme was studied in experiment and control by the microiodometric method. Acriflavine and proflavine inhibited the penicillinase biosynthesis from the 4th hour of growth, and rivanol, acrichine, acridines No. 27 and 37--from the 12th hour of the culture growth.

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The authors present the results of a comparative study of desaminase activity in the suspensions of resting cells and in ultrasonic desintegrates of cells of Cl. botulinum types A, B, E and F against a number of amino acids and their amides. It was shown that types A, B, E and F possessed active desamination enzymes; this process, however coursed with a different degree of intensity depending on the substrate. Common for all the 4 types was the presence of desamidase L-asparaginase and L-glutaminase, and also of the desamination enzymes of the aspartic and glutamic acids. Strains of type B had the greatest set of enzymes, and of type A--the least; bacteria of types E and F occupied an intermediate position. None of the types studied contained tryptophandesaminase. Some of desaminases are bound to subcellular structures.

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Five strains of A type Cl. perfringens of different titres of toxicity (BP6K, SR-12, 2836, 2910, 1) possessed a distinct activity of ornithine transcarbamylase. In cells the maximal activity of the enzyme was observed within 3-5 hrs of growth of the culture. The enzyme was partially purified and some of its physico-chemical properties were studied. Ornithine transcarbamylase was termostable. Monoiodine acetate, p-chloromercurybenzoate, semicarbazide, Hg-2+, Cu-2+ and Fe-3+ inhibited the enzymatic activity, but Mg-2+ and Ca-2+ activated the enzyme. Ornithine transcarbamylase was shown to be active in wide region of pH: from pH 6.0 to pH 9.0 and higher. Two peaks of the enzyme activity were observed: the first (a more distinct one) at pH 8.6-8.9 and the second, smaller,--at pH 6.5-6.7.

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