Comparison of C12E8 and 5,6-Br2C12E8 properties: Critical micellar concentration, steady-state interaction with SR vesicles as deduced from light scattering, and effects on protein intrinsic fluorescence.
(A) Detergent cmc (arrow) for C12E8 and 5,6-Br2C12E8, as determined from the spectral changes of 40 μM methyl orange (expressed as ΔA415 nm− ΔA500 nm, in absorbance units) in the presence of increasing concentrations of these detergents. (B) Perturbation by C12E8 and 5,6-Br2C12E8 of the 90° light scattering (at 290 nm) by SR vesicles (at 4 μg protein/ml). In Panels A and B, closed symbols correspond to 5,6-Br2C12E8, open symbols correspond to non-brominated C12E8. (C and D) Perturbation by C12E8 (C) and 5,6-Br2C12E8 (D) of light scattering by SR vesicles, as in Panel B, but here recorded upon continuous delivery of concentrated detergent from a small syringe into the spectrophotometer cuvette, and in the presence of different concentrations of membranes (20, 50 or 100 μg/ml of protein, short dash, long dash, and continuous lines, respectively). Recorded signals were not corrected for the resulting small dilution of membranes (10% at 0.5 mM detergent). The few data points for “negative” detergent concentrations correspond to data recorded before actuation of the syringe. (E and F) Perturbation by C12E8 (E) and 5,6-Br2C12E8 (F) of the intrinsic fluorescence signal for SR vesicles under the above conditions (continuous delivery of detergent and in the presence of different concentrations of membranes, again at 20, 50 or 100 μg/ml of protein). In Panels D and F, after addition of Br2C12E8 up to 0.44 mM to the 100 μg/ml SR suspension, increasing amounts of non-brominated C12E8 were finally added to the ~2 ml suspension, up to about 6 mM (2, 2, 4, 8, 16, and finally 32 μl of a very concentrated solution of C12E8−100 mg/ml, i.e. 186 mM). Blank values (buffer only, in the absence of membranes) were subtracted from the “fluorescence” signal, but detergent-induced dilution and photolysis were not corrected for.