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form
essentially salt-free, lyophilized powder
solubility
TESCA buffer (50 mM TES, 0.36 mM Calcium chloride, pH 7.4): soluble
Featured Industry
Diagnostic Assay Manufacturing
shipped in
wet ice
storage temp.
−20°C
产品描述
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 both 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 ranges from 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 reaction varies from 15 minutes to several hours for 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. The enzyme recognizes the sequence -R-Pro-8-X-Gly-Pro-R-, where X is most often a neutral amino acid. Ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA)4; β-mercaptoethanol; glutathione (reduced); thioglycolic acid, sodium; 2,2′-dipyridyl; 8-hydroxyquinoline, cysteine are known to inhibit the enzyme activity.
Caution
也具有梭菌蛋白酶、非特异性中性蛋白酶和胰蛋白酶活性。
Preparation Note
This product is equivalent to first 40% ammonium sulfate fraction of Mandl, I., et al., J. Clin. Invest., 32, 1323 (1953). Solutions are typically prepared at 1-2 mg/mL in TESCA buffer (containing 50 mM TES, 0.36 mM Calcium chloride, pH 7.4 at 37°C.
Unit Definition
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.
Application
The enzyme has also been used along with other proteases for the disaggregation of human tumor, mouse kidney, human brain, ａnd lung epithelial tissues. It is also useful in liver ａnd kidney perfusion studies, digestion of pancreas, isolation of nonparenchymal rat liver cells ａnd hepatocytes. The enzyme from Sigma has been used for the digestion of type I collagen from bovine trabecular ａnd cortical bones.[1] It has also been used as a positive control in FALGPA assay on Group B Streptococci cells.[2]
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