Collagenase - Assay

Source:
Clostridium histolyticum
CAS:
9001-12-1
EC:
3.4.24.3

Collagenase Assay

Method
Assay methods utilizing labelled collagen have been reported by Gisslow and McBride (1975), Robertson et al. (1972) and Sakamoto et al. (1972). Since true collagenase attacks the helical region of the molecule, change in optical rotary dispersion reflects collagen degradation (Keil et al. 1975). Worthington collagenase products CLS and CLSPA are assayed as described below:

Method: A modification of the procedure of Mandl et al. (1953). Collagenase is incubated for 5 hours with native collagen. The extent of collagen breakdown is determined using the Moore and Stein (1948) colorimetric ninhydrin method. Amino acids liberated are expressed as micromoles leucine per milligram collagenase. One unit equals one micromole of L-leucine equivalents from collagen in 5 hours at 37°C and pH 7.5 under the specified conditions.

Reagents
  • 0.05 M TES [tris(hydroxymethyl)-methyl-2-aminoethane sulfonate] buffer with 0.36 mM calcium chloride, pH 7.5
  • 4% Ninhydrin in methyl cellosolve with 7.1 mM stannous chloride
  • 0.2 M Sodium citrate, pH 5.0
  • Ninhydrin-citric acid mixture: Prepare by mixing 50 ml of the 4% ninhydrin in methyl cellosolve containing 7.1 mM stannous chloride with 50 ml of 0.2 M citrate pH 5.0. Allow mixture to stir for 5 minutes.
  • 50% n-Propanol
  • Substrate: Worthington bovine achilles tendon collagen (Code: CL) and vitamin free casein
  • 50% (w/v) Trichloroacetic acid
Enzyme

Dissolve enzyme at a concentration of 1 mg/ml in 0.05 M TES with 0.36 mM calcium chloride, pH 7.5. Dilutions run are 1/10 and 1/20 in the above buffer.

Procedure

Weigh 25 mg of Worthington bovine collagen into each of four test tubes. Include at least two tubes to serve as blanks which will contain no enzyme. Add 5.0 ml of 0.05 M TES buffer to the tubes and incubate at 37°C for 15 minutes. Start the reaction by adding 0.1 ml of enzyme dilution to appropriate tubes.

After 5 hours, stop the collagenase reaction by transferring 0.2 ml of solution (leaving behind the collagen) to test tubes containing 1.0 ml of ninhydrin-citric acid mixture. Include an enzyme blank (collagen incubated with 0.1 ml TES buffer in place of enzyme). Heat for 20 minutes in a boiling water bath. After cooling, dilute with 5 ml of 50% n-propanol. Let stand for 15 minutes and read absorbance at 600 nm. From an L-leucine standard curve determine micromoles amino acid equivalent to leucine liberated.

Non-specific protease activity (i.e. caseinase activity) is determined using the above assay and substituting 25 milligrams vitamin free casein for collagen. The reaction is stopped after 5 hours by the addition of 0.5 ml of 50% trichloroacetic acid. After centrifugation, 0.2 ml of the supernatant is transferred to 1.0 ml of ninhydrin and treated as above. Caseinase activity is calculated as collagenase activity.

Calculation

Collagenase Products

Description
Activity
Code
Cat. #
Price
Size
Description
Collagenase, Purified
Source:
Clostridium histolyticum
Chromatographically purified. ≤ 50 caseinase units per milligram. Supplied as a lyophilized powder.
Store at 2-8°C.
Activity
≥500 units per mg dry weight
Code
CLSPA
Cat. #
LS005275
Description
Collagenase Vial, NCIS
Source:
Clostridium histolyticum
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.
Activity
≥500 units per mg dry weight
Code
CLSPANK
Cat. #
LK003240
Description
Collagenase/Elastase Vial (CLSH)
Source:
Clostridium histolyticum
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.
Activity
≥20,000 units collagense/30 units elastase per vial
Code
CLSH
Cat. #
LK002067
Description
Collagenase, Type 1
Source:
Clostridium histolyticum
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.
Activity
≥125 units per mg dry weight
Code
CLS-1
Cat. #
LS004194
Description
Collagenase, Type 2
Source:
Clostridium histolyticum
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.
Activity
≥125 units per mg dry weight
Code
CLS-2
Cat. #
LS004174
Description
Collagenase, Type 3
Source:
Clostridium histolyticum
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.
Activity
≥100 units per mg dry weight
Code
CLS-3
Cat. #
LS004180
Description
Collagenase, Type 4
Source:
Clostridium histolyticum
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.
Activity
≥160 units per mg dry weight
Code
CLS-4
Cat. #
LS004186
Description
Collagenase, Type 5
Source:
Clostridium histolyticum
Partially purified to contain higher collagenase and caseinase activities. A dialyzed, lyophilized powder.
Store at 2-8°C.
Activity
≥450 units per mg dry weight
Code
CLS-5
Cat. #
LS005280
Description
Collagenase, Type 6
Source:
Clostridium histolyticum
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.
Activity
≥400 units per milligram dry weight
Code
CLS-6
Cat. #
LS005318
Description
Collagenase, Type 7
Source:
Clostridium histolyticum
Prepared to contain 4-5X more collagenase, FALGPA and Caseinase activities than Collagenase 1-4 and with moderate clostripain and tryptic activities.
 
 
Store at 2-8°C.
Activity
≥1,000 units per milligram dry weight
Code
CLS-7
Cat. #
LS005332
Description
Collagenase, Type 1, Filtered
Source:
Clostridium histolyticum
Collagenase, Type 1 (Code: CLS-1), which is filtered through a 0.22 micron membrane and lyophilized in vials.
Store at 2-8°C.
Activity
≥125 units per mg dry weight
Code
CLSS-1
Cat. #
LS004214
Description
Collagenase, Type 2, Filtered
Source:
Clostridium histolyticum
Collagenase, Type 2 (Code: CLS-2), which is filtered through a 0.22 micron membrane and lyophilized in vials.
Store at 2-8°C.
Activity
≥125 units per mg dry weight
Code
CLSS-2
Cat. #
LS004202
Description
Collagenase, Type 3, Filtered
Source:
Clostridium histolyticum
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.
Activity
≥100 units per mg dry weight
Code
CLSS-3
Cat. #
LS004206
Description
Collagenase, Type 4, Filtered
Source:
Clostridium histolyticum
Collagenase, Type 4 (Code: CLS-4), which is filtered through a 0.22 micron membrane and lyophilized in vials.
Store at 2-8°C.
Activity
≥160 units per mg dry weight
Code
CLSS-4
Cat. #
LS004210
Description
Collagenase, Type 5, Filtered
Source:
Clostridium histolyticum
Collagenase Type 5 (Code: CLS-5), which is filtered through a 0.22 micron membrane and lyophilized in vials.
Store at 2-8°C.
Activity
≥450 units per mg dry weight
Code
CLSS-5
Cat. #
LS005286
Description
Collagenase CLSAFA Animal Free/AF
Source:
Clostridium histolyticum
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.
Activity
≥150 units per mg dry weight
Code
CLSAFA
Cat. #
LS004152
Description
Collagenase, CLSAFA, Filtered
Source:
Clostridium histolyticum
Collagenase, Animal Free/AF (Code: CLSAFA) which is filtered through a 0.22 micron membrane and lyophilized in vials.
Store at 2-8°C.
Activity
≥150 units per mg dry weight
Code
CLSAFAS
Cat. #
LS004118
Description
Collagenase CLSAFB Animal Free/AF
Source:
Clostridium histolyticum
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.
Activity
≥300 units per mg dry weight
Code
CLSAFB
Cat. #
LS004145
Description
Collagenase, CLSAFB, Filtered
Source:
Clostridium histolyticum
Collagenase, Animal Free/AF (Code: CLSAFB) which is filtered through a 0.22 micron membrane and lyophilized in vials.
Store at 2-8°C.
Activity
≥300 units per mg dry weight
Code
CLSAFBS
Cat. #
LS004124
Description
Collagenase CLSAFC Animal Free/AF
Source:
Clostridium histolyticum
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.
Activity
≥200 units per mg dry weight
Code
CLSAFC
Cat. #
LS004138
Description
Collagenase, CLSAFC, Filtered
Source:
Clostridium histolyticum
Collagenase Animal Free/AF (Code: CLSAFC) which is filtered through a 0.22 micron membrane and lyophilized in vials.
Store at 2-8°C.
Activity
≥200 units per mg dry weight
Code
CLSAFCS
Cat. #
LS004130
Description
STEMxyme® 1, Collagenase/Neutral Protease (Dispase)
Source:
Clostridium histolyticum/Bacillus polymyxa
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.
Activity
≥250 CLS/1,000 CAS units per milligram dry weight
Code
STZ1
Cat. #
LS004106
Description
STEMxyme® 2 Collagenase/Neutral Protease (Dispase)
Source:
Clostridium histolyticum/Bacillus polymyxa
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.
Activity
≥250 CLS/2,000 CAS units per milligram dry weight
Code
STZ2
Cat. #
LS004112