Time-resolved absorbance difference spectra were collected at delays from 1 to 128 μs after photolysis of membrane and detergent suspensions of rhodopsin at 20°C. Fitting both sets of data with two exponential decays plus a constant showed a similar fast process (lifetime 11 μs in membrane, 12 μs in 5% dodecyl maltoside) with a small but similar spectral change. This demonstrates that the Lumi I → Lumi II process, previously characterized in detergent suspensions, has similar properties in membrane without significant effect of detergent. The slower exponential process detected in the data is quite different in membrane compared to detergent solubilized samples, showing that the pronounced effect of detergent on the later rhodopsin photointermediates begins fairly abruptly near 20 μs. Besides affecting the late processes, the data collected here shows that detergent induces a small blue shift in the 1 μs difference spectrum (the Lumi I minus rhodopsin difference spectrum). The blue shift is similar to one induced by chloride ion in the E181Q rhodopsin mutant and may indicate that the ionization state of Glu181 in rhodopsin is affected by detergent.
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Vol. 82 • No. 6