Crude collagenase preparations contain several isoforms of two different collagenases, a sulfhydryl protease, clostripain, a trypsin-like enzyme, and an aminopeptidase. This combination of collagenolytic and proteolytic activities is effective at breaking down intercellular matrices, the essential part of tissue dissociation. One component of the complex is a hydrolytic enzyme which degrades the helical regions in native collagen preferentially at the Y-Gly bond in the sequence Pro-Y-Gly-Pro, where Y is most frequently a neutral amino acid. This cleavage yields products susceptible to further peptidase digestion. Crude collagenase is inhibited by metal chelating agents such as cysteine, EDTA or o-phenanthroline but not DFP. It is also inhibited by α2-macroglobulin, a large plasma glycoprotein. Ca2+ is required for enzyme activity. Particular enzymatic profiles of each collagenase have been correlated with the tissues from which the cells for study were obtained (or with the uses to which the cells are put) and as a result of the correlations several types of crude collagenases have been established by Worthington: Types 1, 2, 3, and 4.
• Type 1 crude collagenase has the original balance of collagenase, caseinase, clostripain and tryptic activities. • Type 2 contains higher relative levels of protease activity, particularly clostripain. • Type 3 contains lowest levels of secondary proteases. • Type 4 is designed to be especially low in tryptic activity to limit damage to membrane proteins and receptors. • Type 5 contains higher collagenase and caseinase values. • Type 6 contains high collagenase activity with a caseinase to collagenase ratio ~2:1. Designed to be enriched for Type II (col H) collagenase relative to Type I (col G). • Type 7 contains collagenase and caseinase activities four-fold higher than collagenase Types 1 and 2.
• Purified collagenase, Code: CLSPA, contains minimal secondary proteolytic activities along with high collagenase activity.
NEW!Animal Free Types AFA, AFB and AFC, STZ1, and STZ2 collagenases are derived from cultures grown in medium completely devoid of animal based components and designed for bioprocessing applications where introduction of potential animal derived pathogens must be prevented. Levels of secondary proteases are similar to Types 1 and 2 collagenase.
• CLSAFA is the original AFA grade designed to have collagenase and secondary proteases similar to Types 1 and 2 collagenase. • CLSAFB contains higher collagenase and caseinase activities than CLSAFA. • CLSAFC has especially low tryptic activity similar to Type 4 collagenase. • STZ1 & STZ2, 0.22μ filtered STEMxyme® AOF Collagenase/Neutral Protease (Dispase®) blends for primary and stem cell isolation.
Worthington also offers 0.22µm filtered preparations of each type in pre-packaged form for direct reconstitution and use in cell isolation and culture procedures. Correlations between enzyme type and effectiveness with different tissues have been good, but not perfect, due in part to the variable parameters of use. Nevertheless most researchers consider the tissue-typing of crude collagenase lots to be a valuable service. A detailed description of the Worthington collagenase and contaminant assays can be found in the Worthington Enzyme Manual. In addition, tissue specific references and detailed isolation conditions can be found in the Worthington Tissue Dissociation Guide.
The collagenase assay is a modification of Mandl wherein collagenase is incubated for five hours with native collagen and the extent of collagen breakdown is determined using the Moore and Stein, JBC, 176, 367, (1948) colorimetric ninhydrin method. Amino acids released are expressed as micromoles leucine per milligram collagenase.
Uses: Crude collagenases are widely used in enzymatic primary cell isolation and tissue dissociation procedures. Most researchers employ either crude collagenase preparations such as Types 1, 2, 3, and 4 or chromatographically purified collagenase (Code: CLSPA); the latter usually combined with secondary enzymes such as elastase, hyaluronidase, etc. For best results, the precise mixture of proteolytic activities must be tailored to the tissue to be dissociated. Correlations between type and effectiveness with different tissues have been good, but not perfect, and may be dependent partly on parameters of use and objectives as well as lot-to-lot variations. For more information see the Collagenase Sampling Program information. Worthington also publishes a Tissue Dissociation Guide which provides additional information regarding the enzymes used for these applications and specific references for numerous cell and tissue types.
Unit Definition: One Unit releases one micromole of L-leucine equivalents from collagen in 5 hours at 37°C, pH 7.5
Cell Isolation Optimizing System (CIT) Hepatocyte Isolation System (HIS) Neonatal Cardiomyocyte Isolation System (NCIS) Papain Dissociiation System (PDS/PDS2)
Description
Activity
Code
Size
Cat. No.
Price
Collagenase, Purified
Chromatographically purified. ≤ 50 caseinase units per milligram. Supplied as a lyophilized powder.
Store at 2-8°C.
Collagenase Vial, NCIS
A component of the NCIS kit. This material is 0.22 micron membrane filtered and lyophilized in autoclaved vials. A vial reconstituted with 5 ml of HBSS or equivalent yields a solution of 300 units/ml of collagenase, Code: CLSPA. Suitable for cell isolation and culture applications.
Store at 2-8°C.
Collagenase/Elastase Vial (CLSH)
Enzyme vial of HIS kit that contains 20,000 units of collagenase (Code: CLS-1) and 30 units of elastase (Code: ESL), filtered through 0.22 micron membrane and lyophilized.
Store at 2-8°C.
≥20,000 units collagense/30 units elastase per vial
Collagenase, Type 1
The original balance of enzymatic activities. Each lot assayed for collagenase, caseinase, clostripain and tryptic activities. Suggested for epithelial, liver, lung and adrenal primary cell isolations. A dialyzed, lyophilized powder.
Store at 2-8°C.
Collagenase, Type 2
Prepared to contain higher clostripain activity. Suggested for bone, heart, liver, thyroid and salivary primary cell isolation. Supplied as a dialyzed, lyophilized powder.
Store at 2-8°C.
Collagenase, Type 3
Lower in secondary proteolytic contaminant activities but with typical collagenase activity. Suggested for mammary primary cell isolation. A dialyzed, lyophilized powder.
Store at 2-8°C.
Collagenase, Type 4
Prepared to contain lower tryptic activity levels to limit damage to membrane proteins and receptors but with normal to above normal collagenase activity. Suggested for pancreatic islet primary isolation. A dialyzed, lyophilized powder.
Store at 2-8°C.
Collagenase, Type 6
Prepared to contain above normal collagenase, FALGPA and Caseinase activities and designed to be enriched with Type II (col H) collagenase.
Store at 2-8°C.
Collagenase, Type 7
Prepared to contain 4-5X more collagenase, FALGPA and Caseinase activities than Collagenase 1-4 and with moderate clostripain and tryptic activities.
Collagenase, Type 1, Filtered
Collagenase, Type 1 (Code: CLS-1), which is filtered through a 0.22 micron membrane and lyophilized in vials.
Store at 2-8°C.
Collagenase, Type 2, Filtered
Collagenase, Type 2 (Code: CLS-2), which is filtered through a 0.22 micron membrane and lyophilized in vials.
Store at 2-8°C.
Collagenase, Type 3, Filtered
Collagenase, Type 3 (Code: CLS-3), which is filtered through a 0.22 micron membrane and lyophilized in vials to contain 50 milligrams per vial.
Store at 2-8°C.
Collagenase, Type 4, Filtered
Collagenase, Type 4 (Code: CLS-4), which is filtered through a 0.22 micron membrane and lyophilized in vials.
Store at 2-8°C.
Collagenase, Type 5, Filtered
Collagenase Type 5 (Code: CLS-5), which is filtered through a 0.22 micron membrane and lyophilized in vials.
Store at 2-8°C.
Collagenase, Purified, Animal Free
Prepared from cultures grown in medium completely devoid of animal based components and designed for bioprocessing applications where introduction of animal derived pathogens must be prevented. Chromatographically purified. ≤ 50 caseinase units per milligram. Supplied as a lyophilized powder.
Store at 2-8°C.
Collagenase CLSAFA Animal Free/AF
Prepared cultures grown in medium completely devoid of animal based components and designed for bioprocessing applications where introduction of animal derived pathogens must be prevented.
Store at 2-8°C.
Collagenase, CLSAFA, Filtered
Collagenase, Animal Free/AF (Code: CLSAFA) which is filtered through a 0.22 micron membrane and lyophilized in vials.
Store at 2-8°C.
Collagenase CLSAFB Animal Free/AF
Prepared from cultures grown in medium completely devoid of animal based components and designed for bioprocessing applications where introduction of animal derived pathogens must be prevented.
Store at 2-8°C.
Collagenase, CLSAFB, Filtered
Collagenase, Animal Free/AF (Code: CLSAFB) which is filtered through a 0.22 micron membrane and lyophilized in vials.
Store at 2-8°C.
Collagenase CLSAFC Animal Free/AF
Prepared from cultures grown in medium completely devoid of animal based components and designed for bioprocessing applications where introduction of animal derived pathogens must be prevented.
Store at 2-8°C.
Collagenase, CLSAFC, Filtered
Collagenase Animal Free/AF (Code: CLSAFC) which is filtered through a 0.22 micron membrane and lyophilized in vials.
Store at 2-8°C.
Collagenase CLSAFD Animal Free/AF
Prepared from cultures grown in medium completely devoid of animal based components and designed for bioprocessing applications where introduction of animal derived pathogens must be prevented.
Store at 2-8°C.
STEMxyme® 1, Collagenase/Neutral Protease (Dispase)
A specialized combination of Animal Free Clostridium histolyticum collagenase (Code: CLSAFB) and animal free Bacillus polymyxa neutral protease with a minimum of 250 CLS units and 1,000 caseinase units per mg dry weight. Designed for stem cell and other primary cell isolations and bioprocessing applications where introduction of potential animal derived pathogens must be prevented.
Store at 2-8°C.
STEMxyme® 2 Collagenase/Neutral Protease (Dispase)
A specialized combination of Animal Free Clostridium histolyticum collagenase (Code: CLSAFB) and animal free Bacillus polymyxa neutral protease with a minimum of 250 CLS units and 2,000 caseinase units per mg dry weight. Designed for stem cell and other primary cell isolations and bioprocessing applications where introduction of potential animal derived pathogens must be prevented.
Store at 2-8°C.
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