Table I.
Comparison of Melting Concentration Values (Cm) for Urea-Induced Unfolding and of Melting Temperature Values (Tm) as Determined by Different Approaches on Holo and Apoprotein Forms of hDAAO
| Holo | Holo-apo | Apoprotein | |
|---|---|---|---|
| Urea-induced unfolding |
Cm (M) |
||
| Protein fluorescence: intensity | 2.0 ± 0.2 | — | First: 2.4 ± 0.1 |
| (2.8 ± 0.1)a | Second: 5.6 ± 0.3 | ||
| Wavelength maximum | 3.7 ± 0.2 | — | 3.8 ± 0.2 |
| Far-UV CD (220 nm) | 4.2 ± 0.2 | — | 3.9 ± 0.3 |
| Temperature-induced unfolding |
Tm (°C) |
||
| Trp fluorescence^ | 57.7 ± 0.3 | 52.4 ± 1.2 | 48.6 ± 1.0 |
| FAD fluorescence | — | 53.5 ± 1.1 | — |
| Far-UV CD (220 nm) | 57.0 ± 1.0 | 57.0 ± 1.0 | 51.0 ± 1.0 |
Protein samples were in 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5% glycerol and 5 mM 2-mercaptoethanol or, only for CD measurements, in 50 mM sodium pyrophosphate, pH 8.0, 5% glycerol, at the following concentrations: fluorescence measurements, 0.02 mg protein/mL; far-UV CD and temperature ramp experiments, 0.1 mg protein/mL. Temperature ramp experiments were performed at an identical heating rate (0.5°C/min). Tm values were obtained by calculating the first derivative of the spectroscopic signals and are uncorrected for delay effects. The values determined in the presence of 40 μM FAD are indicated as holo form.
Value determined from the loss of catalytic activity.