Fig. 4.

Transducin binding to monomeric rhodopsin. (A) Superposition of SEC elution profiles: G_t holoprotein (Gαβγ) or rhodopsin (R; inactive ground state) alone (dashed lines) or as mixture (solid lines). (B) SEC elution profiles as in A obtained with R*. Detection occurred at the absorption maxima of protein (280 nm), R* (380 nm), and rhodopsin (500 nm). R*·G_t complex formation is indicated by a shift of the main elution peak. For this peak, a value of 3.6–3.8 was determined for the absorption ratio A₂₈₀/A₃₈₀ (black line), which is consistent with a 1:1 stoichiometry between R* and G_t in the R*·G_t complex (theoretical values are 3.56 for R*:G_t = 1:1 and 2.52 for R*:G_t = 2:1; see Materials and Methods). (C) SEC elution profile of R*/G_t mixture detected at 380 nm (absorption maximum of R*) and 253 nm (absorption maximum of GDP). The ratio between peak areas corresponding to R* and GDP, respectively, ranges from 3.0 to 3.2 for different runs (theoretical values are 3.07 for R*:G_t = 1:1 and 6.13 for R*:G_t = 2:1; see Materials and Methods).