BROMINATED DETERGENTS AS TOOLS TO STUDY PROTEIN-DETERGENT INTERACTIONS

In order to study protein-detergent short-range interactions, we analy zed the quenching by brominated detergents of reticulum sarcoplasmic ( SR) Ca2+-ATPase intrinsic fluorescence. For this purpose, 7,8-dibromod odecyl beta-maltoside and 2-O-(10,11-dibromoundecanoyl)sucrose, bromin ated analogs of two non-ionic detergents, the frequently used dodecylm altoside and the newly synthesized 2-O-lauroylsucrose respectively, we re prepared. Rayleigh scattering measurements showed that the brominat ed detergents efficiently and rapidly solubilized SR vesicles like the ir non-brominated analogs although at slightly higher concentrations. Similarly, each analog had a slightly higher critical micellar concent ration than its parent detergent. The partition coefficient K (express ed as the ratio of the molar fraction of detergent in the SR lipid pha se to that in the aqueous phase, at pH 7.5 and 20 degrees C) was simil ar for brominated and non-brominated dodecyl maltoside (3.5-4 X 10(5)) and slightly lower for dibromoundecanoylsucrose (approximate to 10(5) ) than for lauroylsucrose (approximate to 2 X 10(5)). At detergent con centrations too low to solubilize the membrane, the brominated deterge nts rapidly inserted (within seconds) into SR vesicles. In this concen tration range, Ca2+-ATPase fluorescence quenching steadily increased w ith detergent concentration. When the membrane was saturated with dete rgent, the residual fluorescence was about half of its initial value, indicating significant protein-detergent contacts, possibly due to a s lightly higher affinity of Ca2+-ATPase for these detergents than for p hospholipids. For higher detergent concentrations, solubilizing the me mbrane, the fluorescence continued to decrease with detergent concentr ation, with no evidence fur a dramatic change in the average hydrophob ic environment of the protein during the transition from bilayers to a soluble state. For still higher detergent concentrations, above that necessary for membrane solubilization, the fluorescence was further qu enched to a residual relative value of about 20%, corresponding to fur ther delipidation of the protein surface, in agreement with previous r esults [de Foresta, B., le Maire, M., Orlowski, S., Champeil, P., Lund , S., Moller, J. V., Michelangeli, F. & Lee, A. G. (1989) Biochemistry 28, 2558-2567]. Fluorescence quenching for solubilized Ca2+-ATPase wa s quickly reversed upon addition of excess non-brominated detergent. T he effects of the four detergents on the Ca2+-ATPase hydrolysis of p-n itrophenyl phosphate were similar and correlated with the protein-dete rgent contacts evidenced above. In conclusion, both these brominated d etergents appear to be promising tools to study protein-detergent inte ractions at the hydrophobic surface of a membrane protein, either in a membrane or in solubilized complexes.