A fluorescence-based detergent binding assay for protein hydrophobicity.
Protein hydrophobicity is often detected by binding of protein to micelles of a mild detergent. A new fluorescence method for detection of this binding is presented. The method is based on a long-range quenching of tryptophan fluorescence by energy transfer to a pyrene-labeled phospholipid probe incorporated into micelles of Brij 96. The method is rapid, simple, and requires only a few micrograms of protein. Strongest quenching is obtained when both pyrene probe and brominated Brij 96, a short-range quencher, are combined. To define the best assay conditions the physical properties and quenching behavior of micelles with or without these probes have been compared. It is shown that both quenchers accurately measure binding of model compounds and protein toxins to micelles. Comparison of quenching by the different probes can be used to derive information on tryptophan location relative to the micelle core.
London
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D001966:Bromine; D066298:In Vitro Techniques; D008823:Micelles; D010714:Phosphatidylethanolamines; D010938:Plant Oils; D011092:Polyethylene Glycols; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D008823:Micelles; D010714:Phosphatidylethanolamines; D010938:Plant Oils; D011092:Polyethylene Glycols; C028809:N-(1-pyrenesulfonyl)dipalmitoyl-L-alpha-phosphatidylethanolamine; D014364:Tryptophan; C033084:polyethylene glycol oleyl ether; D001966:Bromine