THERMOSTABILIZATION OF PROTECTIVE ANTIGEN - THE BINDING-COMPONENT OF ANTHRAX LETHAL TOXIN

Protective antigen (PA) is the binding component of anthrax lethal tox in produced by Bacillus anthracis, and constitutes a major ingredient of the vaccine against anthrax. PA and lethal factor when added togeth er are cytolytic to mouse macrophages and J774G8 macrophage cell lice. This in vitro lethal toxicity assay is very useful in understanding t he molecular mechanism of action of lethal toxin. Effective utilizatio n of PA is, however, hampered due to its thermolability. On prolonged storage at 37 degrees C, PA was found to lose its activity almost comp letely. The effect of solvent additives like trehalose, sorbitol, xyli tol, sodium citrate and magnesium sulphate on the thermal stabilizatio n of PA was examined. The results indicated an increase in the stabili ty of PA when the incubation at 37 degrees C was carried out in the pr esence of solvent additives used in the 1-3 M range. Magnesium sulphat e helped retain the activity up to 82.7% against the control in which no additive was used, as judged by cytolytic assay using J774G8 macrop hage cell line. Trehalose or sodium citrate also showed an appreciable protection of PA activity, while sorbitol or xylitol were not very ef fective. Competitive binding assay using radiolabeled PA showed that P A had lost capacity of binding to macrophage cells on prolonged incuba tion at 37 degrees C. Circular dichroism results at 4, 18 and 37 degre es C indicated an increase in secondary structure at 37 degrees C rela tive to that al 4 or 18 degrees C, supporting the activity data.