Assaying Collagenase Activity by Specific Labeling of Freshly Generated N-Termini with Fluorescamine at Mildly Acidic pH

Abstract

Collagenases are important enzymes frequently used for isolation of cells. Accurate estimation of collagenase activity is an important requirement. Available techniques for assaying collagenase activity are (i) ninhydrin-based assays, in which ninhydrin reacts with freshly generated N-termini resulting from collagenase action upon collagen, with a colorimetric read-out, (ii) synthetic substrate-based assays, in which collagenase action releases either a radioactive group or a fluorescent moiety. These methods are associated with some drawbacks. Here, we have standardized a simple, cost-effective method for assaying collagenase activity, using fluorescence readouts of N-terminal-specific fluorescamine adduction with peptides and proteins at pH 6.0. Collagenase-mediated degradation of gelatin and collagen was performed at pH 7.0. Following incubation, fluorescamine was added at pH 6.0. A standard plot based on the use of BSA (bovine serum albumin) was used for determination of the moles of freshly-generated N-termini detected, based on fluorescence owing to fluorescamine adduction to N-termini. The method was also tested with two other proteases (trypsin and cathepsin-K). Specific reaction of fluorescamine with α amino groups at pH 6.0 can be used for labeling of freshly generated N-termini in collagen subjected to degradation by a collagenase. The kinetic parameters determined by the fluorescamine assay are comparable to those determined previously. Our improved method of using fluorescamine for protease activity addresses the previously associated drawbacks of this assay, thereby lifting this assay up to an unprecedented level of reliability and convenience. We found that the technique can also be extended to use with other proteases.