Enzymological studies at high hydrostatic pressure generally involve temperature, pH and pressure as variables, owing to the effect of adiabatic compression and the ionization volume of the buffer system. In the case of NAD dependent oxidoreductases this implies that the extinction coefficient of the coenzyme may be affected by p, T and pH, apart from the spectral change accompanying the redox reaction.
Measurements of the pressure dependence of the absorbance of NADH show a slight red shift and a 1% decrease (3% increase) of the absorbance at 339 nm (360 nm) at 2 kbar. The pH dependence at the given wavelengths amounts to -(2.4 ± 0.1)% per pH unit (25 °C), while the intrinsic temperature effect (after correction for thermal expansion) is of the order of -0.2% per degree (20-30 °C).
Applying buffers with negligible ionization volume, 366 nm is the optimum wavelength for high pressure studies up to 2 kbar because here the pressure dependent spectral changes of the NADH absorption vanish.
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