Process monitoring the inactivation of ricin and model proteins by disinfectants using fluorescence and biological activity.
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.
Cole
Kenneth D
KD
Biochemical Science Division, Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, USA. kenneth.cole@nist.gov
Gaigalas
Adolfas
A
Almeida
Jamie L
JL
D001681:Biological Assay / Q000379:methods*; D002850:Chromatography, Gel / Q000379:methods*; D003666:Decontamination / Q000379:methods; D004202:Disinfectants / Q000737:chemistry*; D004203:Disinfection / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D012276:Ricin / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods*
D004202:Disinfectants; D011506:Proteins; D012276:Ricin