Description
Application
Clostridiopeptidase A has been used in a study to describe a quantitative method to assess small amounts of collagen based on MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry (MS) subsequent to digestion of collagen.
This product has been used in the isolation of pancreatic ductal adenocarcinoma (PDAC) cell lines and the tissue culture of pancreatic fibroblasts.
Biochem/physiol Actions
Collagenase is activated by four gram atom calcium per mole enzyme. It is inhibited by ethylene glycol-bis(beta-aminoethyl ether) - N, N, N′,N′-tetraacetic acid, beta-mercaptoethanol, glutathione, thioglycolic acid and 8-hydroxyquinoline.
Effective release of cells from tissue requires the action of collagenase enzymes and the neutral protease. Collagenase is activated by four gram atom calcium (Ca²⁺) per mole enzyme. The culture filtrate is thought to contain at least 7 different proteases ranging in molecular weight from 68-130 kDa. The pH optimum is 6.3-8.8. The enzyme is typically used to digest the connective components in tissue samples to liberate individual cells. Collagenase treatment can cause some cells to die. Typically, concentrations varying from 0.1 to 5 mg/mL are used for digestion. The duration of the reaction varies from 15 minutes to several hours to yield a satisfactory efficiency of cell dissociation without causing too much cell death. Krebs Ringer buffer with calcium and BSA is preffered and Zn²⁺ is required for activity.
Preparation Note
A stock solution may be prepared by dissolving 0.05-0.1 mg/mL of collagenase in 50 mM TES buffer containing 0.36 mM calcium chloride (TESCA), pH 7.4, at 37 °C.
Analysis Note
Also contains clostripain, nonspecific neutral protease, and tryptic activities.
Other Notes
One collagen digestion unit (CDU) liberates peptides from collagen from bovine achilles tendon equivalent in ninhydrin color to 1.0 μmole of leucine in 5 hours at pH 7.4 at 37 °C in the presence of calcium ions. One FALGPA hydrolysis unit hydrolyzes 1.0 μmole of furylacryloyl-Leu-Gly-Pro-Ala per min at 25°C. One Neutral Protease unit hydrolyzes casein to produce color equivalent to 1.0 μmole of tyrosine per 5 hr at pH 7.5 at 37°C. One Clostripain Unit hydrolyzes 1.0 μmole of BAEE per min at pH 7.6 at 25°C in the presence of DTT.
