Collagenase from Cl. histolyticum
Cl. histolyticum is known to produce a mixture of collagenases which are able to digest the triple helical collagen molecule in situ. The individual collagenases can be divided into two groups.

Cleavage specificity
Group I enzymes obviously cleave type I collagen near the C-terminus with subsequent proteolysis in the area of the N-terminus. Group II enzymes have a cleavage specificity similar to that of mammalian collagenases. They are able to cleave type I collagen across all three chains within the macromolecule.
    The collagen molecule is a triple helix which contains the repetitive sequence -Pro(Hyp)-XGly-Pro(Hyp)-, where X is a neutral amino acid. Collagenase is specific for the X-Gly bond in this sequence, and is thus also able to cleave denatured collagen and other polypeptides with a related primary structure.
    SERVA collagenases also contain a number of other enzymatic activities, including clostripain, which cleaves peptides preferentially at the carboxyl side of arginine residues, a tryptic activity, which acts preferentially at the carboxyl side of arginine and Iysine residues, and neutral non-specific proteases. The particular suitability of SERVA collagenase preparations for degrading intercellular material is partly due to the presence of balanced amounts of these enzymes.

Properties

Cofactors
Collagenase requires calcium ions both for full catalytic activity and binding to the collagen molecule.

Inhibitors
Inhibitors of collagenase include cysteine, EDTA, o-phenanthroline, 8-hydroxyquinoline-5-sulfonate, bipyridyl and 2,3-dimercaptopropanol. It is also inhibited by tris buffer above pH 7.5 ⁽⁵⁾. Collagenase is not inhibited by diisopropylphosphorofluoridate.

Stability and Storage
Collagenase is soluble in water and dilute salt solutions. It is reversibly inactivated at high pH values, and irreversibly inactivated at low pH values. This product should be stored at 4° C to maintain its high activity. Under these conditions, collagenase remains stable without loss of activity for at least two years.

Enzymatic Activities
The collagenolytic activity of SERVA preparations is assayed in our quality control department by two different methods:

As the extraneous activities of these preparations are known to contribute to their performance, three further activities are assayed.
Clostripain ⁽⁷⁾ and trypsin like ⁽⁸⁾ activities are both measured by their ability to hydrolyse N-benzolyl-L-arginine ethyl ester (BAEE), but clostripain activity is determined in the presence of an activator, DTT (dithiothreitol).
Neutral proteinases are determined by their ability to hydrolyse dimethylcasein, liberated amino acids being determined with 2,4,6-trinitrobenzene sulfonic acid (TNBS) ⁽⁹⁾.
Full details of all of the above assay procedures are available on request.

Other units:
FALGPA units: 1 nkatal is the amount of enzymic activity which catalyzes the hydrolysis of 1 nmol of 2-furanacryloyl-L-leucyl-glycylL-prolyl-L-alanine (FALGPA) per second at 25° C, pH 7.5.
Mandl units: 1 U liberates 1 µmol amino acid from collagen expressed as leucine per 5 hours at 37° C, pH 7.5.

Extraneous activities:
Trypsin-like proteases: 1 U catalyzes the hydrolysis of 1 µmol BAEE per minute at 25 ° C, pH 7.6.
Clostripain: 1 U catalyzes the hydrolysis of 1 µmol BAEE per minute at 25° C, pH 7.6, after activation with 1 mM calcium acetate and 2.5 mM dithiothreitol.
Other proteinases: 1 DMC-U catalyzes the cleavage of 1 µmol peptide bond from dimethyl casein per minute at 25° C, pH 7.0, expressed in terms of newly formed terminal amino groups (determined with TNBS).

The isolation of islets from pancreatic tissue
In recent years, collagenase has been used successfully for the isolation of viable pancreatic islets from several different species ^(10,11,12).
     Until now, a number of factors have limited the usefulness of this method in the transplantation of human islets. These include marked variations in the activity of collagenase preparations, the presence of b -cell toxic factors, the absence of any correlation between enzyme activity and islet yield and the influence of collagenase on the immunogenicity and chemotactic behaviour of the islets.

Isolation of cardiomyocytes
Collagenase has also been used in the isolation of cardiomyocytes suitable for the preparation of cultures. If tissue disaggregation is accomplished by perfusion of the intact organ with enzyme solution, then collagenase is always the enzyme of choice either alone, or in conjunction with other enzymes such as hyaluronidase ⁽¹³⁾, or trypsin. Collagenase can also be used when disaggregation is achieved by incubating small pieces of tissue in enzyme solution ⁽¹⁴⁾ .

Isolation of hepatocytes
Isolated hepatocytes are used in tumour promotion and initiation studies, for studying cellular control mechanisms and in drug and carcinogen assay systems.
     SERVA "Collagenase Hep Plus" has been independently tested for use in hepatocyte isolation. Hepatocytes were isolated from rat liver by perfusion with a solution of SERVA "Collagenase Hep Plus" (Cat. no. 17447) for 12 - 13 minutes. The viability of the isolated cells was determined using both trypan blue and fluorescein diacetate, and was in all cases found to be in excess of 90 %. Plating efficiency was shown to be greater than 85 % after 2 hours.

Other tissues
Collagenase has also been successfully used for the isolation of cells from bone ⁽¹⁵⁾, thyroid glands ⁽¹⁶⁾, and ovarian and uterine tissues ⁽¹⁷⁾

References

(2) Bond, M.D. & Van Wart, H.E. (1984) Biochemistry 23, 3092-99. Relationship between the individual collagenases of Clostridium histolyticum: evidence for evolution by gene duplication.

(3) French, M.K. et al. (1987) Biochemistry 26,681 -7. Limited proteolysis of type I collagen at hyperreactive sites by class I and 11 Clostridium histolyticum collagenases: Complementary digestion patterns

(4) Bond, M.D. & Van Wart, H.E. ( t 984) Biochemistry 23, 3085-91. Characterization of the individual collagenases from clostridium histolyticum.

(5) Van Wart, H.E. & Steinbrink, D.R. (1981) Anal. Biochem. 113, 356-65. A continuous spectrophotometric assay for clostridium histolyticum collagenase.

(6) Wünsch, E. & Heinrich, H.G. (1963) Hoppe-Seyler's Z. Physic Chem.333,149-51. Determination of collagenase.

(7) Kézdy, F.J. et al. (1965) Biochemistry 4,2302-8. Titration of a/ centres in thrombin solutions. Standardization of the enzyme.

(8) Bergmeyer, H.U. (1983) Methods of enzymatic analysis Vol ll 3rd ed.,319-20. Trypsin.

(9) Lin, Y.-C. et al. (1969) J. Biol. Chem.244,789-93. Action of proteolic teolytic enzymes on N,N-dimethyl proteins. Basis for a microassay for proteolytic enzymes.

(11) Noel, J. et al. (1982) Metabolism 31 (2),184-87. A method for large-scale, high-yield isolation of canine pancreatic islets of Langerhans.

(12) Izumi, R. et al. (1985) Transplantation Proceedings 17 (1), 383-84. Isolation of human pancreatic islets from cryopreserved pancreas.

(13) Nag, A.C. & Cheng, M. (1981) Tissue & Cell 13 (3),515-23. Adult mammalian cardiac muscle cells in culture.

(14) Lindl, T. & Bauer, J. (t 987) Zell- und Gewebekultur: Einführung in die Grundlagen sowie ausgewählte Methoden und Anwendungen
86-7 Fischer, Stuttgart .

(15) Brand, J.S. & Hefley, T.J. (1984) Collagenase and the isolation of cells from bone, in: Cell separation methods and selected a applications, Vol.3, (t 2) 265-83, Acad. Press.

(16) Kerkhof, P.R. (1982) Journal of tissue culture methods 7 (1), 23-6. Preparation of primary cultures of ovine thyroid gland cells.

(17) Marcus, G.J. et al. (1984) Endocrine Research 10 (2),151 -62. Enzymatic dissociation of ovarian and uterine tissues.

Reference:
1. Van Wart, H.E. & Steinbrink, D.R. (1981) Anal. Biochem. 113,356-65

17449.04 2.5 g