Thermodynamic Measurements for Inactivation of Bioterrorism Agents Ricin and Abrin

1. Measure forward-rate constants for thermal denaturation of ricin and abrin at seven temperatures (60, 65, 70, 75, 80, 85, 90, and 95 C) and three buffer combinations (0.10 M NaCl buffered with 20 mM lactate, pH 3.0; 20 mM acetate, pH 5.0; and 20 mM phosphate, pH 7.0) by monitoring the quenching of intrinsic protein (tryptophan) fluorescence (EX295, EM340) in a thermostatted spectrofluorimeter.
<P>
2. Exploit results (Tm, Delta H) gathered using differential-scanning calorimetry at NCFST to select Tm for toxin proteins and measure reverse-rate constants (protein renaturation) at one temperature and one buffer combination. Calculate Keq and Delta G from ratio of rates. Determine T Delta S from Delta G and Delta H.
<P>
3. Determine the influence of solvent pH on isothermal toxin folding/unfolding equilibria.
<P>
4. Identify time-, pH-, and temperature-dependent reversible and irreversible transitions in ricin conformation and correlate these with changes in toxi-dependent enzyme activity and cytotoxicity.