Preparation of fluorescent derivatives of lipases and their use in fluorescence energy transfer studies in hydrocarbon/water interfaces.
Fluorescein isothiocyanate reacted with a chromobacter and pseudomonad lipase to yield mono-substituted, fully active, enzymes. With the carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) in the non-aqueous phase, fluorescence energy transfer was used to follow the lipase and similarly labelled model proteins in and out of the interface in heptane, and heptane/di-O-palmitoyl-rac-glycerol (a substrate analogue), emulsions. Competitive binding, and displacement by other proteins could also be followed.
D001419:Bacteria / Q000201:enzymology; D004735:Energy Transfer; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D006838:Hydrocarbons; D006863:Hydrogen-Ion Concentration; D008049:Lipase; D011549:Pseudomonas / Q000201:enzymology; D013050:Spectrometry, Fluorescence; D013499:Surface Properties; D013861:Thiocyanates; D014867:Water
D005452:Fluoresceins; D006838:Hydrocarbons; D013861:Thiocyanates; D014867:Water; D008049:Lipase; D016650:Fluorescein-5-isothiocyanate