Process Monitoring the Inactivation of Ricin and Model Proteins by Disinfectants Using Fluorescence and Biological Activity

Abstract

It is important to develop rapid and reliable processes to monitor the decontamination of toxins released to the environment. The inactivation of the protein toxin ricin by the disinfectants bleach (sodium hypochlorite) and monochloramine was measured by the effect on mammalian cell cytotoxicity. The effect of the disinfectants on the native fluorescence (due mainly to tryptophan and to a lesser extent tyrosine) of ricin was also measured in parallel. Reactions of the disinfectants resulted in a decrease in the native fluorescence that was measured in real time in a noninvasive manner. We compared the inactivation of two well-characterized model enzymes to the behavior of ricin. The model enzymes studied were lysozyme, a small basic enzyme stabilized with internal disulfide bonds, and heart-muscle-type lactate dehydrogenase (LDH), a large protein composed of four subunits. The biological activities of the model enzymes were measured in parallel with their fluorescence. Gel electrophoresis showed a large number of modifications of the proteins caused by the disinfectants reflected in changes in mobility and the formation of higher-order aggregates. Size-exclusion chromatography showed that the disinfectants did not break down the subunit structure of ricin but instead resulted in an increased size and heterogeneity of the protein. Size-exclusion chromatography of LDH indicated that the subunits were dissociated and that higher-order aggregates were also formed. Bleach caused a rapid inactivation of biological activity correlated with a rapid decrease in the fluorescence. Monochloramine required much higher concentrations for significant effects and the kinetics of the reactions were slow, with half-life values of the decrease on the order of minutes. Each protein showed individual differences in responses to the disinfectants, but there was a consistent correlation between the loss of fluorescence and the decrease in biological activity. These results indicate that the monitoring the fluorescence is a useful process with limitations that can be used to monitor the inactivation of toxins using disinfectants.

Ricinus communis Intoxications in Human and Veterinary Medicine—A Summary of Real Cases

“…Functional assays for ricin have traditionally been done by animal toxicity tests and in vitro cytotoxicity assays [130,284]. Later, cytotoxicity assays have been amended to detect ricin out of complex matrices [282,283,285,286]. Using functionally blocking antibodies, these tests enable the discrimination of ricin from other cytotoxins.…”

N-Chlorotaurine, a Long-Lived Oxidant Produced by Human Leukocytes, Inactivates Shiga Toxin of Enterohemorrhagic Escherichia coli

“…As a consequence of these chemical reactions, gel electrophoresis with NCT-treated Stx2 revealed bands with lower, but also higher molecular weight (Figure 5). The latter phenomenon has recently been observed with the toxin ricin, lactate dehydrogenase, and lysozyme treated with hypochlorous acid or (to a lesser extent) with monochloramine and has been explained by formation of higher-order aggregates [27]. Moreover, ovalbumin-chlorination by hypochlorous acid has been found to promote formation of aggregates stabilized by non-covalent bonds [32].…”

“…Decades later, the active chlorine compounds NaOCl and monochloramine were shown in few studies to inactivate aflatoxins, endotoxins and ricin, mainly in connection with their use as water disinfectants [27], [28], and recently as irrigation solutions for root canals in odontology [29]. The finding that mainly NCT, but also other chloramines are produced by leukocytes as long-lived oxidants with antiinflammatory and antimicrobial properties, implicated their importance for the human defense system [1], [4], [5], [30].…”

Evaluation of the Effectiveness of Surfactants and Denaturants to Elute and Denature Adsorbed Protein on Different Surface Chemistries

“…[5][6] For biodefense applications, the removal of adsorbed proteins is important for surface decontamination. [7][8] Surfactants like sodium dodecyl sulfate, and denaturants like guanidine hydrochloride are commonly used chemical excipients for the elution of proteins from surfaces. [9][10][11] In addition to the above additives, amino acids and their derivates, [12][13] inorganic salts, 14-15 organic solvents, 16 and osmolytes 17 have also been used as eluents.…”