A continuously recording spectrophotometric assay has been developed for Clostridium histolyticum collagenase based on the hydrolysis of 2-furanacryloyl-L-leucylglycyl-L-prolyl-L-alanine (FALGPA). The hydrolysis of this peptide by collagenase obeys Michaelis-Menten kinetics with V = 1.8 X IO5 pkatal/kg and K,,, = 0.5 mM. FALGPA is hydrolyzed more rapidly by collagenase than any other commonly used synthetic substrate, but is not cleaved by any of the well-known proteinases such as trypsin, thermolysin, or elastase. The assay itself is rapid, convenient, and sensitive, and should greatly facilitate detailed kinetic studies of collagenase.
Clostridium histolyticum
' To whom correspondence should be sent. ' To simplify presentation, Clostridium histolyticum collagenase is simply referred to as collagenase.
- or fluorescein fluorescence (35) from soluble, insoluble, or gelled collagen labeled with these substances.
A second class of assays follows the hydrolysis of synthetic peptides. The most frequently used substrates are Cbz3-Gly-Pro-Gly-Gly-Pro-Ala (8) Cbz-Gly-Pro-Leu-Gly-Pro (9), and Pz-Pro-Leu-Gly-Pro-D-Arg (11). The rate of hydrolysis of the first two peptides is monitored calorimetrically by measuring the release of free amino groups (Gly-ProAla and Gly-Pro, respectively) after reaction with ninhydrin. For the latter peptide, the chromophoric Pz-Pro-Leu fragment produced on hydrolysis is extracted into ethyl acetate and its concentration determined calorimetrically.