Table 2.
The distance of closet approach between the donor fluorescein dyes in the active-sites of FIX/FIXa derivatives and the acceptor rhodamine dyes incorporated into PCPS vesicles and anisotropy (r) values for the labeled proteins.
| Donor Enzyme | Ro | L | r | r(+PCPS) |
|---|---|---|---|---|
| Fl-FPR-FIXa | 49 ±1 | 75 ±2 | 0.10 | 0.11 |
| Fl-FPR-FIXa-desEGF1 | 46 ±1 | 65 ±2 | 0.10 | 0.12 |
| Fl-FIXa-Cys195 | 49 ±1 | 81 ±3 | 0.14 | 0.15 |
| Fl-FIX-Cys195 | 49 ±1 | 84 ±3 | 0.14 | 0.15 |
The Ro and L values (in Å) were determined based on the efficiency of energy transfer due to decreases in the emission intensity of the donor fluorescein dyes in the active-sites of proteins (20–50 nM) upon their titration with PCPS vesicles containing the acceptor OR dyes with densities of 0.9–2.5 × 10−4 OR/Å2 in 0.1 M NaCl, 0.05 M Hepes, pH 7.4 containing 5 mM Ca2+, assuming a random orientation of transition dipoles for donor and acceptor dyes (κ2 = 2/3) as described in “Materials and methods”. The anisotropy values (r) for fluorescein in the active-sites of the proteins were determined at excitation and emission wavelengths of 493 nm and 521 nm, respectively.