Table 5.

Summary of collagenase activity assays

Method	Substrate	Enzyme activity	Benefit	Drawbacks	References
Rosen assay	Collagen	After a reaction at pH 7.4 and 37 °C (presence of calcium ions) for 5 h, the amount of peptides released from collagen was equal to the amount of color development of 1 μmol of leucine and ninhydrin, which was a CDU (one collagen digestion unit).	Qualitative and quantitative accuracy and reliability	The reaction time is long, and ninhydrin easily fades after color development	[40,89]
Synthetic peptide	FALGPA/Pz peptide	One FALGPA unit refers to the hydrolysis of 1 μmol of FALGPA per minute at 25 °C pH 7.5 (calcium ions present).	Rapid reaction, the substrate is stable and invariable, suitable for large-scale inspection	Does not directly reflect the hydrolytic activity of collagenase toward natural collagen	[86,90]
Electrophoresis assay	Gelatin	/	Rapid qualitative analysis	Unquantifiable	[86]
Azocoll assay	Azocoll	The light absorption value at A520 is increased by 1.0 per minute, which is defined as one unit of enzyme activity.	The operation is simple, the specificity is high, the colored hydrolysate is stable	Does not directly reflect the hydrolytic activity of collagenase toward natural collagen	[57]
Isotope labeling	14C-labeled collagen	Scintillation counting is performed to characterize enzyme activity, and collagenase activity is represented by the dpm value.	The sensitivity is high, suitable for in vivo testing	The equipment is expensive and produces radioactive contamination	[91]
Fluorescence labeling	FITC-collagen	/	Qualitative and quantitative accuracy and reliability	The substrate is expensive, the solvent affects the fluorescence intensity	[92,93]