A spectroscopic collagenase assay using peroxidase-labeled collagen

A quantitative collagenase assay detecting soluble collagen fragments is described in this paper. Using the reagent N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) type I collagen was conjugated with horseradish peroxidase (POD) which was employed as a reporter enzyme. POD was preferentially linked to the TC B fragment in a ratio of 1.4 mol POD/mol collagen. The conjugation product was immobilized on AH-Sepharose via carbodiimide coupling to form the final collagenase substrate used in the assay. POD activity in the supernatants caused by liberated TC B fragments exhibited a linear relationship for collagenase concentrations up to 100 micrograms/ml bacterial collagenase. Over an incubation period of 4 h the lowest detection limits found were 20 ng/100 microliters for bacterial collagenase and 60 ng/100 microliters for human leukocyte collagenase. Incubation of the assay mixture with 5 micrograms trypsin resulted in 3.8% of the activity released by the equivalent amount of leukocyte collagenase. The assay developed here has been shown to be sensitive and specific for collagenase, with the additional advantage that this method is suited for simple and economic handling.     

A rapid assay method of collagenase activity using 14C-labeled soluble collagen as substrate.

A rapid assay method for vertebrate collagenase (EC 3.4.24.3) activity has been developed using 14C-labeled soluble collagen as substrate. The method is based on the incubation of collagen with enzyme in the presence of glucose to prevent collagen fibril formation followed by selective extraction of the enzyme digestion products into dioxane at a final concentration of 50%. The rate of reaction was about 10 times higher than that obtained by the conventional method using reconstituted collagen fibrils as substrate and the relationship between enzyme activity and concentration was linear over a wider range. When the method was applied to the assay of human granulocyte collagenase, the results showed good correlation with those obtained by the conventional gel method.     

A simple vertebrate collagenase assay using soluble radioactive collagen substrate

A highly sensitive assay for vertebrate collagenase has been developed using [14C]proline- or [3H]proline-labeled collagen as soluble substrate. The substrate was easy to prepare, gave high specific activity (1.4 X 10(6) cpm/mg collagen), and was stable at -20 degrees C for a long period. The digestion reaction for the assay was done at 21 degrees C to minimize the cleavage of collagen by proteases other than collagenase and to protect the 3/4 and 1/4 cleavage fragments of collagen from being further attacked by proteases. The cleaved products were denatured and then separated from undigested native collagen by precipitation with 1 M NaCl at pH 3.5. The conditions selected for denaturation and separation gave better discrimination between the cleaved products and uncleaved substrate than did conditions used in some other assays. The digestion products can be examined further by gel electrophoresis at the end of the assay to confirm the activity of vertebrate collagenase. This assay can also be adapted to assess telopeptidase activity independently of collagenase activity.     

A spectrophotometric collagenase assay

A quantitative collagenase assay using Coomassie blue staining and microtiter spectrophotometry is described. Collagen is gelled and dried onto the bottom of microwells as substrate, washed, incubated with samples, washed again, and then stained. Absorbance at 590 nm increases linearly with increasing amounts of collagen in the range 5-40 micrograms. Bacterial and mammalian collagenases can be detected within 2 h, and 10 ng of bacterial collagenase may be detected in 16 h. For simple screening applications, activity may be detected by eye. The assay is safe, simple, fast, economical, and sensitive.     

A rapid sensitive collagenase assay.

A rapid, sensitive collagenase assay has been developed using¹⁴C-acetylated collagen as a substrate. Acid-soluble calfskin collagen was labeled with [1-¹⁴C]acetic anhydride at pH 8. The acetylated collagen had a specific activity of 6.25 × 10⁵ dpm/mg protein. Collagen was not denatured as evidenced by its resistance to nonspecific proteolysis and sensitivity to bacterial collagenase. Polyacrylamide gel electrophoresis of the acetylated protein showed that the radioactivity was present in the three bands corresponding to the α, β, and γ components of collagen. The rate of release of ¹⁴C from labeled collagen by Clostridium histolyticum collagenase was proportional to enzyme and substrate concentration.     

Spectrophotometric assay for vertebrate collagenase

Collagenase from normal human skin fibroblasts was found to catalyze the hydrolysis of esters and thio esters. This observation led to the development of a rapid, sensitive, continuous spectrophotometric assay for vertebrate collagenase using the thio peptolide Ac-ProLeuGly-S-LeuLeuGly-OC2H5 as substrate in the presence of 4,4'-dithiodipyridine or Ellman's Reagent. A Km of 0.004 M and a kcat of 370,000 h-1 were determined for the thio peptolide-enzyme reaction. The method is able to detect collagenase at concentrations as low as 2 ng/ml.     

Tissue collagenase: a simplified, semiquantitative enzyme assay

Tissue collagenase activity from the ulcerating rabbit cornea has been quantitated in a sensitive capillary tube assay system with an unlabeled, native collagen substrate. In this assay system, initial rates of gel lysis are proportional to enzyme concentration over a defined range of enzyme concentrations. Increased sensitivity to enzyme with an unlabeled substrate has been achieved by restricting diffusion of enzyme to one dimension, in a capillary gel. Corneal collagenase activity has been measured at concentrations down to 0.1 μg/μl. In addition to its high sensitivity to enzyme, the precision and simplicity of the assay and minimal equipment requirements all recommend its use for routine screening of biological fluids for collagenase activity and in the investigation of the effects of inhibitors and stimulators of collagenase activity.     

A handy assay for collagenase using reconstituted fluorescein-labeled collagen fibrils

This report describes an assay for measuring the activity of collagenases using reconstituted fibrils of fluorescein-labeled soluble collagen as substrate. The labeling of commercially available material is easy and not expensive. The assay is very sensitive, reproducible, and is linear with collagenase concentration up to 100% consumption of the reconstituted fibrils. Readings of collagenolytic activity can be determined directly by measuring the fluorescence of the released labeled peptides without further processing of the data.     

Radial diffusion assay of tissue collagenase and its application in evaluation of collagenase inhibitors.

A radial diffusion assay for tissue collagenase (EC 3.4.24.3) has been devised which is simple, sensitive and capable of application to large numbers of samples. The assay employs an agarose matrix containing solubilized lathyritic rat skin collagen as substrate. Fibril formation is induced for 2 h at 37 degrees C subsequent to 41 h digestion at 28 degrees C. The procedure results in sharply defined zones of lysis which may be measured directly or after photography. The characteristics of the procedure are otherwise similar to those reported for other radial diffusion assays. The new method was used to examine the action of 10 compounds which were known or potential inhibitors of tadpole collagenase. The concentration of inhibitor required to produce 50% inhibition is reported for the following compounds: alpha2-macroglobulin, 142 microng/ml; N-acetylcysteine, greater than or equal to 100 mM; cysteine, 8.7 mM; EDTA, 0.46 mM; histidine, greater than or equal to 100 mM; 2,3-dimercaptopropanol, 0.5 mM and mercaptoacetic acid, 70 mM. The procedure also has potential for clinical determinations (e.g. tears, synovial fluid) since assay dishes may be prepared in advance and only 15 micronl of sample is required.     

A collagenase assay using [3H-methyl]collagen

A new assay procedure for collagenase is presented. Highly radioactive substrate is prepared by methylation of native collagen. The ³H-labelled protein is readily attacked by bacterial as well as by mammalian collagenase and resistant to other proteinases. The sensitivity of this assay is higher than that of the enzymic methods hitherto available.     

A sensitive, specific assay for tissue collagenase using telopeptide-free [3H]acetylated collagen

Collagenase is assayed by incubation with soluble, telopeptide-free collagen extracted from rat skin and labeled with [2-3H]acetic anhydride. Collagen is cleaved by collagenase and the resulting fragments are digested with trypsin and chymotrypsin. Undigested collagen is recovered by precipitation with trichloroacetic acid, collected on glass-fiber filters, and quantitated by liquid scintillation spectrometry. This procedure combines features of the Cawston and Barrett (T.E. Cawston and A.J. Barrett, 1979, Anal. Biochem. 99, 340-345) and the Ryh?nen et al. (L. Ryh?nen et al., 1982, Collagen Rel. Res. 2, 117-130) methods. The first method provides a simple way to prepare large quantities of uniform substrate, while the second increases the specificity of the assay by removal of the labeled telopeptides. The assay is reproducible and linear with time and enzyme concentration. It is approximately 10X more sensitive than the Cawston and Barrett method and can readily detect 1-8 mU collagenase (1 unit equals 1 microgram collagen cleaved/min at 30 degrees C). The substrate is resistant to elastase, trypsin, and chymotrypsin and is completely degraded by bacterial collagenase. Collagenase is the only tissue metalloprotease found, to date, that cleaves the substrate.     

A modified collagenase assay method based on the use of p-dioxane.

Radioactive collagen synthesized by human skin fibroblasts in monolayer culture was used as a substrate for collagenase. The high specific activity of this substrate (75,000 cpm/μg) and the use of p-dioxane as a precipitant of the undigested collagen permit this enzyme to be assayed with collagen in solution at 35°C and pH 7.5. The dilutions used are sufficient to prevent the collagen molecules from aggregating, thus precluding the use of inhibitors of gel formation which tend to decrease the activity of the enzyme. Using a 1-h incubation, the procedure is reproducible (SD ± 2.3%) and linear over the range from 10 to 100 ng of bacterial collagenase. Vertebrate collagenase activity is also easily measured with this method.     

Detection of collagenase activity in oral bacteria

Collagenolytic activity of 12 species of oral bacteria was assessed using two methods of detection. Except for two species, all bacterial strains tested were capable of degrading at least one general protein substrate. Results of collagenolytic activity in a growth assay indicate that Bacteroides gingivalis is the only bacterium capable of degrading collagen when the substrate is sterilized using ethylene oxide. However, if the substrate is sterilized by autoclaving, in the presence or absence of the growth medium, other bacterial species could be shown to be collagenolytic. Collagenolytic activity was also demonstrated when whole or broken cells were used in a [14C]collagen assay. Results from this assay and from inhibition studies indicate that collagenolytic activity can either be the result of the combined activities of both a specific collagenase and nonspecific proteases (B. gingivalis) or nonspecific proteases only (other strains in this study), although in the latter case, the time taken to hydrolyze collagen can be 10 times longer than with a specific collagenase.     

Regulation of collagenase production by steroids in uterine smooth muscle cells: An enzymatic and immunologic study

An enzyme-linked immunosorbent assay (ELISA) has been developed for rat collagenase. The assay is capable of measuring the enzyme from a variety of rat cell sources at concentrations of 10–;50 ng/ml, approximately 500–;1,000-fold more sensitive than radiolabelled collagen fibril assay systems. The assay is specific to collagenase from the rat: enzymes from human, tadpole, mouse, and bacterial sources failed to cross-react significantly with rat enzyme. The assay is reproducible and accurate, and is capable of detecting enzyme in the presence of serum or tissue inhibitors. Using the ELISA, we have examined the effect of a variety of hormones on the production of collagenase by rat myometrial smooth muscle cells in culture. Of all the reproductive hormones examined, only progesterone and its synthetic derivative medroxyprogesterone acetate were capable of inhibiting the production of the enzyme by these cells. The maximally effective concentration of progesterone was 1 x 10^?6M, and that of medroxyprogesterone acetate was 1 x 10^?7M. The effect of the steroid was selective: no effect on cell proliferation or on general protein synthesis was observed. In addition to the progestational steroids, the glucocorticoids were also capable of inhibiting the production of collagenase by the cells at similar nominal concentrations. However, the myometrial cells were found actively to metabolize progesterone but not hydrocortisone in culture. Thus, the effective inhibitory concentration of progesterone was approximately ten-fold lower than that of hydrocortisone. The results of this study support the concept that progesterone plays a major role in preventing the production of collagenase in the rat uterus.     

A collagen film microassay for tissue collagenase.

A quantitative microassay for the detection of bacterial and tissue collagenase is presented. Collagen in acetic acid solution forms a thin, tenacious film upon contact with a dried agar surface. The digestion of the film by collagenase is detected by subsequent staining with Coomassie blue. Undigested film is stained dark blue while areas of collagenase digestion remain clear. The technique can be employed in two ways: with collagen-coated glass coverslips as a rapid screening method or with collagen-lined capillary tubes for precise quantitation. The assay has a sensitivity comparable to those assays using radioactively labeled collagen substrates and requires only 5 to 60 min of incubation.     

The role of the C-terminal domain in collagenase and stromelysin specificity.

Recombinant human interstitial collagenase, an N-terminal truncated form, delta 243-450 collagenase, recombinant human stromelysin-1, and an N-terminal truncated form, delta 248-460 stromelysin, have been stably expressed in myeloma cells and purified. The truncated enzymes were similar in properties to their wild-type counterparts with respect to activation requirements and the ability to degrade casein, gelatin, and a peptide substrate, but truncated collagenase failed to cleave native collagen. Removal of the C-terminal domain from collagenase also modified its interaction with tissue inhibitor of metalloproteinases-1. Hybrid enzymes consisting of N-terminal (1-242) collagenase.C-terminal (248-460) stromelysin and N-terminal (1-233) stromelysin.C-terminal (229-450) collagenase, representing an exchange of the complete catalytic and C-terminal domains of the two enzymes, were expressed in a transient system using Chinese hamster ovary cells and purified. Both proteins showed similar activity to their N-terminal parent and neither was able to degrade collagen. Analysis of the ability of the different forms of recombinant enzyme to bind to collagen by ELISA showed that both pro and active stromelysin and N-terminal collagenase.C-terminal stromelysin bound to collagen equally well. In contrast, only the active forms of collagenase and N-terminal stromelysin.C-terminal collagenase bound well to collagen, as compared with their pro forms.     

A continuous spectrophotometric assay for Clostridium histolyticum collagenase

A continuously recording spectrophotometric assay has been developed for Clostridium histolyticum collagenase based on the hydrolysis of 2-furanacryloyl-l-leucylglycyl-l-prolyl-l-alanine (FALGPA). The hydrolysis of this peptide by collagenase obeys Michaelis-Menten kinetics with V = 1.8 × 10⁵μkatal/kg and K_m = 0.5 mm. FALGPA is hydrolyzed more rapidly by collagenase than any other commonly used synthetic substrate, but is not cleaved by any of the well-known proteinases such as trypsin, thermolysin, or elastase. The assay itself is rapid, convenient, and sensitive, and should greatly facilitate detailed kinetic studies of collagenase.     

Method to analyze collagenase and gelatinase activity by fibroblasts in culture

Summary The net amount of collagen produced and deposited by fibroblasts in cell culture is determined by the rate of collagen synthesis as well as the rate of collagen degradation. Although collagen synthesis can be analyzed by several techniques, it is more difficult to measure collagen degradation. Breakdown of collagen depends upon the activity of a family of structurally and catalytically related mammalian enzymes termed matrix metalloproteinases (MMPs). Interstitial collagenase (MMP1) initiates the cleavage of fibrillar collagen, whereas gelatinases (MMP2 and MMP9) digest the denatured collagen fragments. A method has been developed to quantitate the activity of collagenase (MMP1) and gelatinase (MMP9) in conditioned medium from fibroblast cell cultures. The assay, which uses the fluorogenic substrate Dnp-Pro-Cha-Gly-Cys(Me)-His-Ala-Lys(Nma)NH₂, is technically simple and amenable to high throughput analysis. Addition of specific inhibitors of the metalloproteinases allows for simultaneous measurement of both collagenase and gelatinase activity.     

The gelatinolytic activity of rat uterus collagenase

The collagenase produced by rat uterine cells in culture has been examined for its ability to degrade denatured collagen. Acting as a gelatinase, rat uterus collagenase was able to successfully degrade the denatured chains of collagen types I through V. In addition, the enzyme produced multiple cleavages in these chains and displayed values for Km of 4-5 microM, compared to values of 1-2 microM when native collagen was used as substrate. Furthermore, rat uterus collagenase degraded the alpha 2 chain of denatured type I collagen at a significantly faster rate than the alpha 1 chain, as previously observed for human skin fibroblast collagenase. In contrast to the action of human skin collagenase, however, the rat uterus enzyme was found to be a markedly better gelatinase than a collagenase, degrading the alpha chains of denatured type I guinea pig skin collagen at rates some 7-15-fold greater than native collagen. Human skin collagenase degrades the same denatured chains at rates ranging from 13-44% of its rate on native collagen. Rat uterus collagenase, then, is approximately 50 times better a gelatinase than is human skin collagenase. In addition to its ability to cleave denatured collagen chains at greater rates than native collagen, the rat uterus collagenase also attacked a wider spectrum of peptide bonds in gelatin than does human skin collagenase. In addition to cleaving the Gly-Leu and Gly-Ile bonds characteristic of its action on native collagen, rat uterus collagenase readily catalyzed the cleavage of Gly-Phe bonds in gelatin. The rat enzyme was also capable of cleaving Gly-Ala and Gly-Val bonds, although these bonds were somewhat less preferred by the enzyme. The cleavage of peptide bonds other than Gly-Leu and Gly-Ile appears to be a property of the collagenase itself and not a contaminating protease. Thus, it appears that the collagenase responsible for the degradation of collagen during the massive involution of the uterus might also act as a gelatinase to further degrade the initial products of collagenolysis to small peptides suitable for further metabolism.     

A fluorescent screening assay for collagenase using collagen labeled with 2-methoxy-2,4-diphenyl-3(2H)-furanone

This report describes the use of the compound 2-methoxy-2,4-diphenyl-3(2H)-furanone to label collagen as a substrate for the detection of mammalian collagenase in a fluorescent assay which is suitable for screening large numbers of samples. The compound 2-methoxy-2,4-diphenyl-3(2H)-furanone presents distinct advantages over other fluorophores, since both the unbound reagent and its hydrolysis products are nonfluorescent. The labeling procedure uses commercially available collagen, is fast and simple, and gives a 90% yield of labeled substrate. The fluorescent collagen substrate is stable and retains fluorescence over a wide range of pH. The assay detects, reproducibly, metal-dependent collagenase activity in microliter volumes of conditioned media from cultured neoplastic cells or in chromatographic fractions from such media.