The dichroic activity at 222 nm for SsADH in the absence (●) and presence of NADH (○), and for N249Y mutant (△), was measured at various temperatures in 17 mM sodium pyrophosphate, pH 9.8. Inset, the dichroic activity of the three ADH forms as measured at 25 °C in 50 mM sodium phosphate, pH 6.8 (open bars), and 17 mM sodium pyrophosphate, pH 9.8, in the absence (dotted bars), and presence (hatched bars) of 0.1 M NaCl. Experimental tracings were accumulated and corrected for blanks by computer calculations, as described in the Experimental section. Enzymes and NADH concentrations were 0.5 and 20 μM respectively. The results are expressed as the means±S.D. for three independent experiments.