“Crude” collagenase refers to the material purified from the fermentation of Clostridium histolyticum bacteria. It is actually a mixture of several different enzymes including collagenase, which act together to break down tissue. This preparation contains collagenase, non-specific proteases, clostripain, neutral protease, and aminopeptidase activities. Crude collagenase is equivalent to the first 40% ammonium sulfate fraction prepared by Mandl.
Molecular mass (SDS-PAGE): 68–130 kDa
As many as seven collagenase proteins can be present, some of these are C-truncated forms of the Type I and Type II collagenases (sometimes called collagenases A and B) that are expressed from two genes, colG and colH.
Molecular mass (sequence): The colG and colH genes have been isolated from C. histolyticum JCM 1403 (ATCC 19401). The colG gene encodes a mature 113,897 Da Type I collagenase and several C-truncated forms. The colH gene encodes the mature 111,963 Da Type II collagenase and one C-truncated form.
Optimal pH: 6.3–8.8. The enzyme is assayed at pH 7.4
Isoelectric Point (pI): Six collagenases purified from crude collagenase had isoelectric points in the range of 5.35–6.20.
Specificity: collagenase recognizes the following peptide sequence where X is most often a neutral amino acid.
Substrates: In addition to the various natural collagen substrates, many synthetic substrates have been prepared:
Z-Gly-Pro-Gly-Gly-Pro-Ala (KM=0.71 mM)
Z-Gly-Pro-Leu-Gly-Pro
N-2,4-Dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg
N-(3-(2-furyl)acryloyl)-Leu-Gly-Pro-Ala (FALGPA)
4-Phenylazobenzoxycarbonyl-Pro-Leu-Gly-Pro-D-Arg
N-succinyl-Gly-Pro-Leu-Gly-Pro 7-amido-4-methylcoumarin (substrate for “collagenase-like peptidase”)
N-(2,4-Dinitrophenyl)-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg amide (substrate for “vertebrate collagenase”)
Activators/Cofactors: Collagenase activity is stabilized by 0.1 mole calcium ions (Ca²⁺) per mole of enzyme. Calcium ions also facilitate binding to the collagen molecule. Zinc ions (Zn²⁺) are required for activity, but are tightly bound to the collagenase during purification. Additional Zn²⁺ should not be necessary as long as no chelator is added during digestion.
Inhibitors:
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA)
Ethylenediaminetetraacetic acid (EDTA)
β-Thujaplicin (Hinokitiol)
Cysteine
2-mercaptoethanol
Glutathione (reduced)
Thioglycolic acid, sodium
8-Hydroxyquinoline-5-sulfonate
This product is supplied as a lyophilized powder.
Traces of calcium chloride and sodium bicarbonate may be present.
Collagenase activity: measured with two substrates; the FALGPA assay measures digestion of a short artificial peptide and the Collagen Digestion Unit (CDU) assay measures digestion of native collagen. Proteolytic activities are assayed with a substrate prepared from casein. Clostripain activity requires a reduced environment so this enzyme has little activity in this preparation.
Specific activity: ≥125 CDU/mg solid
Unit Definition: One Collagen Digestion Unit (CDU) liberates peptides from collagen equivalent in ninhydrin color to 1.0 µmole of L-leucine in 5 hours at pH 7.4 at 37 °C in the presence of calcium ions.
Note: Mandl units have the same description as Collagenase Digestion Units. The conversion factor for Mandl units/Wuensch units to Aladdin units is 1000-2000 to 1.
Specific activity: 0.5–5.0 FALGPA units/mg solid
Unit Definition: One FALGPA Hydrolysis Unit hydrolyzes 1.0 µmole of furylacryloyl-Leu-Gly-Pro-Ala per minute at 25 °C at pH 7.5 in the presence of calcium ions.
Neutral Protease (caseinase) activity: ≤350 units/mg solid
Unit Definition: One Neutral Protease Unit hydrolyzes casein to produce color equivalent to 1.0 µmole tyrosine per 5 hours at pH 7.5 at 37 °C.
Clostripain activity: ≤4 units/mg solid
Unit Definition: One Clostripain Unit hydrolyzes 1.0 µmole of BAEE per minute at pH 7.6 at 25 °C in the presence of DTT.
Precautions and Disclaimer
This product is for R&D use only, not for drug, household, or other uses.
Preparation Instructions
Reconstitution Buffers:
For enzyme assays: 50 mM TES with 0.36 mM CaCl₂, pH 7.4, at 37 °C.
For tissue dissociations: Krebs-Ringer Bicarbonate Buffer with added calcium (0.3–0.5 mM) and BSA (1%).
For tissue culture applications: calcium-free solutions such as Hank’s Balanced Salts or Earle’s Balanced Salt Solution.
To sterile filter solutions of crude collagenase, first centrifuge the solution or filter through a 0.8 µm filter to remove insolubles. This pretreatment will reduce the clogging of the 0.2 µm filter during sterile filtration.
Storage/Stability
This product retains activity for at least one year when stored at –20 °C.
Solutions (10 mg of protein/ml) at neutral pH and with adequate calcium ion concentration (0.3–0.5 mM) will retain activity for over 6 hours on ice. Solutions at –20 °C are stable for several months if frozen quickly in aliquots (10 mg/ml). Freeze-thaw cycles will reduce enzyme activities.
Procedure
This product is suitable for the disaggregation of mouse kidney, human adult and fetal brain, lung, tumor, and many other tissues, particularly epithelium. It is also effective in liver and kidney perfusion studies, digestion of pancreas, isolation of non-parenchymal rat liver cells, and hepatocyte preparation.
The release of cells from tissue depends on the action of both collagenase enzymes and the neutral protease. An important feature for tissue dissociation is the ratio of collagenase to protease activity. Protein concentrations typically vary from 0.1–5 mg/ml and digestion time should be monitored using very gentle agitation to check for tissue dissociation. Collagenase treatment can cause some cells to die. A satisfactory degree of cell dissociation without too much cell death typically can be achieved from 15 minutes to several hours, but can fall outside this range if the protein concentration is not in the typical range.
ZJ26F0938982
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