Rapid,sensitive fluorimetric assay for proteases using fluorescein-labelled albumin coupled to Fractogel activated with 2-fluoro-1-methylpyridinium salt in flow-injection analysis

A rapid and sensitive flow-injection method is described for the fluorimetric determination of the proteolytic activity of a number of enzymes. The substrate, fluorescein-labelled bovine serum albumin coupled to a 2-fluoro 1-methylpyridinium salt-activated Fractogel support, is packed in a small reactor (35 × 2 mm i.d.) and inserted into a flow manifold. Under the reaction conditions the amount of fluorescein released from enzymatic cleavage of the substrate is found to be directly proportional to the activity of the proteolytic enzyme. Sample throughput is 30–35 h^?1. The calibration range for trypsin is linear up to at least 0.35 μg ml^?1. Linear responses were also obtained for otherproteolytic enzymes such as papain, chymotrypsin, ficin and bromelain.