Screening hydrolysis over two-phases.
A new assay is described that monitors hydrolysis with the concurrent transfer of a solvatochromic dye across an oil-water barrier. Through the appropriate design, this transfer is accompanied by a 10(6) gain in fluorescence. This response can be used to effectively screen hydrolytic activity at high-throughput. Using this method, microunits of alkaline phosphatase, glucosidases, as well as several common proteases can be visually detected within an hour through concentration over a 200:1 volumetric ratio of aqueous to organic phases. Development of a water-solublizing protecting group extends this methodology to screen a wide range of processes that undergo cleavage of a covalent bond.
Alkaline Phosphatase↗