[Role of arginine and histidine residues in the biological activity of botulinic neurotoxin A]. / Rol' ostatkov arginina i gistidina v biologicheskoi aktivnosti botulinicheskogo neirotoksina tipa A.

Treatment of botulinic neurotoxin A with cyclohexanedione demonstrated that modification of 5 to 10 arginine residues does not change the neurotoxin toxicity, while after modification of 15-20 arginine residues the toxicity is decreased by 40-50% of the original value. Butanedione exerts a stronger detoxicating effect on neurotoxin than cyclohexanedione. The molecular conformation of the modified toxin derivatives and their precipitability upon interaction with antisera against toxin and toxin fragments does not change thereby. The non-toxic derivatives of toxin containing 40 modified arginine residues possess a partial serological affinity for the original toxin in a reaction with antiserum against toxin but do not interact with the antifragment sera. The molecular conformation of these preparations is changed considerably. It is assumed that one or two arginine residues are located near the toxic site of the neurotoxin molecule and are also components of its antigenic determinants. Modification of histidine residues in the neurotoxin molecule by diethylpyrocarbonate is accompanied by a decrease of its toxicity. An additional 10% toxicity is revealed upon modification of 11-13 histidine residues. The molecular conformation of the modified derivatives of neurotoxin and their precipitability do not change thereby. It is probable that 1 or 2 histidine residues are located at or near the toxic site. The data obtained suggest that histidine residues are not localized in antigenic determinants of the neurotoxin molecule.

[Preparation of neurotoxin and hemagglutinin from Clostridium botulinum A and characterization of its neurotoxin]. / Poluchenie neirotoksina i gemaggliutinina Clostridium botulinum tipa A i kharakteristika neirotoksina.

A procedure for preparation of electrophoretically and serologically homogeneous neurotoxin and a highly purified hemagglutinin from the culture fluid of Cl. botulinum A, strain 501 is described. The yield of neurotoxin with specific activity of 80-100 X 10(6) DLM/mg of protein is 5-20%. Neurotoxin has a molecular weight of 150,000, sedimentation coefficient of 7.1S, pI of 6.2-6.3; the maximum of its fluorescence corresponds to 332 nm. The toxin molecule contains 4 SH-groups. Neurotoxin consists of two subunits with molecular weights of 98,000 and 56,000. The storage of neurotoxin at -20 degrees C causes inactivation and electrophoretical heterogeneity of the protein. The inactivation leads to an alteration of the toxin molecule charge, a shift of the lambda max of fluorescence and a loss of the SH-group reactivity without affecting the molecular weight or serological properties of the protein. The data obtained suggest that the conformational state of toxin molecules is essential for its biological activity.

[Effect of urea on botulinum neurotoxin type A]. / Deistvie mocheviny na botulinicheskii neirotoksin tipa A.

Kinetic and concentration-mediated dependences of urea effects on toxicity and upon values of negative extremes (279, 287, 292 nm), shifts of lambda max fluorescence from 332 nm to the red region, decrease of fluorescence intensity, changes in accessibility of SH-groups were found. These data point to direct correlation between conformational changes in neurotoxin in urea solutions and decrease of toxicity. An investigation was carried out on renaturation of conformational changed neurotoxin in 8 M urea solution. In no cases was the toxicity restored, though there was a shift of lambda max fluorescence in protein from 340 to 333 nm, that was the evidence for reverse transition of the neurotoxin molecule from a disordered structure to the globule shape. The data lead to the conclusion that the toxicity region is probably a conformationally depending determinant.

[Effect of photooxidation on biological properties of botulinum neurotoxin A]. / Vliianie fotookisleniia na biologicheskie svoistva botulinicheskogo neirotoksina tipa A.

Photooxidation of botulinic neurotoxin A in the presence of methylene blue is associated with a decrease in toxicity down to complete detoxication. During neurotoxin photooxidation, when the toxicity makes up to 1 to 3% of the original one, the conformation of the neurotoxin molecule and its antigenic properties remain unchanged. Under these conditions, using diethylpyrocarbonate, a specific reagent for histidine, the photooxidized neurotoxin was found to contain 5-6 oxidized histidine residues per molecule of neurotoxin; this was accompanied by changes in the UV absorbance spectrum around 280 nm. It was assumed that the main decrease in neurotoxin toxicity during photooxidation is probably due to oxidation of tryptophane, since the differential UV spectra suggest that the higher the extremum around 280 nm, the greater the decrease of toxicity; chemical modification of histidine residues alone causes no noticeable detoxication.

[Changes in biological properties of botulinum neurotoxin a induced by chemical modification of its molecule by tryptophan and tyrosine]. / Izmenenie biologicheskikh svoistv botulinicheskogo neirotoksina tipa A pri khimicheskoi modifikatsii ostatkov triptofana i tirozina v ego molekule.

Using spectrophotometric titration of botulinic neurotoxin A by N-bromosuccinimide, the oxidation of one tryptophane residue was shown to induce an almost complete detoxication of the toxic protein. The conformation of the toxin molecule remained thereby unchanged, as well as the precipitation capacity of the modified toxin after oxidation of two tryptophane residues. The toxin with three or more modified tryptophane residues did not produce precipitation bands with antiserum against original toxin. Nitration of the tyrosine residues in the neurotoxin molecule with tetranitromethane gradually decreased its toxicity. All nitrous derivatives of toxin (both toxic and non-toxic ones) containing 2-18 modified tyrosine residues revealed a precipitating capacity in a reaction with antiserum against original toxin and anfragment sera. The non-toxic toxin nitrous derivatives with 15-18 modified tyrosine residues possessed partial serological affinity for original toxin in a reaction with antiserum against toxin and did not interact with antisera against toxin fragments. The conformation of molecules of toxin nitrous derivatives with 4-5 modified tyrosine residues was not changed irrespective of a 80% loss of the enzyme toxicity.

[Isolation and characterization of the polypeptide fragments obtained by limited proteolysis of botulinic toxin type A]. / Vydelenie i kharakteristika polipeptidnykh fragmentov, poluchennykh pri orgranichennom proteolize botulinicheskogo toksina tipa A.

A limited proteolysis of the botulinic toxin of A type by subtylopeptidase A resulted in two high molecular weight non-toxic fragments. The peptide with mol. weight of 100,000 is made up of two subunits with mol. weights of 52,000 and 48,000. The second peptide whose mol. weight is 40,000 is a single-chained one. The high molecular weight peptide has one S--S bond and two SH-groups, whereas the one with a lower molecular weight--no S--S bond and 1.3--1.5 SH-groups. Dansylation of the first fragment revealed two N-terminal amino acids (histidine, arginine) in toxin, which suggests the localization of the first fragment at the N-end of the toxin molecule. Using immunochemical analysis with monospecific antiserum against original toxin and antifragment sera, the antigenic determinants from the fragments were shown to be serologically different. A structural model of botulinic toxin of A type is proposed.

[Obtaining purified preparations of individual Shigella newcastle K- and O-antigens by using an ultracentrifugation method]. / Poluchenie ochishchennykh preparatov individual'nykh K- i O-antigneov shigell N'iukasl s ispol'zovaniem metoda ul'tratsentrifugirovaniia

A modified method of obtaining the purified K- and O-antigens of Sh. newcastle with the use of ultracentrifugation is described. Serologically individual K- and O-antigens were obtained and their chemical composition was studied. Polysaccharide of the K-antigen consisted of the galacturonic acid, galactose, glucose, rhamnose xylose and hexosamine; it contained no heptoses. The O-antigen contained galactose, glucose, mannose, rhamnose, heptose and hexosamine; in difference to K-antigen was equal to 150000--160000. Thus, the K- and the O-antigen differed not only in the electrophoretic and diffuse mobility and immunological specificity, but also in the chemical composition.