BLUE COLLAGENASE ASSAY

Abstract

A method of measuring soluble or insoluble cell or tissue-associated collagenase activity. The substrate includes native (fibrillar) collagen fragments that were stained with Coomassie Brilliant Blue R-250. Incubation with collagenase can be observed in real-time by the generation of digested smaller fragments. The degraded blue fragments are obtained by filtration through class fibers, onto which intact collagen fibrils are retained. The filtrate containing the blue collagen fragments is incubated with a detergent in order to extract the blue dye, and the mixture is centrifuged in order to separate the dye in the supernatant from the pellet, which contains de-stained collagen fragments and other insoluble materials contained in the test samples (such as bacterial cells or tissues). The amount of dye extracted is quantified by measuring the amount of dye extracted from these fragments, i.e., the absorbance at 600 nm using a spectrophotometer or an ELISA reader.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This nonprovisional application is a continuation of and claims priority to U.S. Provisional Patent Application No. 62/020,179, entitled "Blue Collagenase Assay", filed Jul. 2, 2014, by the same inventor, the entirety of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates, generally, to collagenases. More specifically, it relates to blue collagenase assays.

[0004] 2. Brief Description of the Prior Art

[0005] Collagenases are defined as proteases that can digest native collagen in the triple helix region. There is no spectrophotometric assay that uses native collagen fibrils ["Collagenase", Worthington Biochemical Company, I.U.B, 3.4.24.3, C.A.S. 9001-12-1]. Commonly used substrates in collagenase assay kits include synthetic peptides, or denatured collagen such as acid soluble collagen and heat-gelling collagen or gelatin. Since denatured collagen can also be degraded by gelatinase and/or protease such as trypsin, assays based on the digestion of these substrates are not specific for the determination of collagenase activity, and hence are referred to by those of ordinary skill in the art as collagenolytic activity, which can be broadly defined as just the ability to lyse collagen, gelatin, and other proteins containing proline.

[0006] Collagen can be stained in blue tissue section by blue dyes such as aniline blue dyes (e.g., MSDS: CI#: 42755; Synonym: Acid blue 22 China blue; Chemical Name: Not available; Chemical Formula: C32H27N309S3.2Na) and Coomassie Brilliant Blue R-250 (MSDS: C14: 42660; Synonym: Acid Blue 83; Acid Brilliant Blue; CI Acid Blue 83; Chemical Name: Benzenemethanaminium, N-[4-[[4-[(4-ethoxyphenyl)amino]phenyl][4-[ethyl[(3-sulfophenyl)methyl]am- ino]phenyl]methylene]-2,5-cyclohexadien-1-ylidene]-ethyl-3-sulfo-, inner salt, monosodium salt; Chemical Formula: C46-17144-N3-O7-S2-Na). This property has been applied to the preparation of blue collagen-coated plastic plates (gelled collagen and dried onto plastic plates) for the observation of collagen degradation assay by oral squamous carcinoma cells in cancer research (Aznavorian et al., Cancer Research, American Association for Cancer Research, 2001:61:6264-6275), and adapted to spectrophotometric measurement of the plates (Nethery et al., "A spectrophotometric collagenase assay", 1986, Anal Biochem 159(2):390-5). Since the collagen is denatured in the heat-gelling process, the use of inhibitors of other enzymes is applied in order to deduce that the activity observed is that of collagenase (Aznavorian et al., Cancer Research, American Association for Cancer Research, 2001:61:6264-6275).

[0007] The interest in the detection and measurement of collagenase activity and/or use of Coomassie dye has generated the publication of numerous assays. Examples include EP 2016421 B1; U.S. Pat. No. 6,057,160; U.S. Pat. No. 4,219,337; U.S. Pat. No. 4,023,933; EP 0319334 A2; U.S. Pat. No. 4,176,009; EP 1039299 B1; Sedmak et al. A rapid, sensitive, and versatile assay for protein using Coomassie brilliant blue 0250, Analytical Biochemistry. Volume 79, Issues 1-2, May 1, 1977, pp. 544-552; Burokerkilgore et al. A Coomassie Brilliant Blue 0-250-Based Colorimetric Assay for Measuring Activity of Calpain and Other Proteases. Analytical Biochemistry. Volume 208, Issue 2, Feb. 1, 1993, pp. 387-392; and Teratoa et al. A rapid assay method of collagenase activity using 14C-labeled soluble collagen as substrate. Biochimica et Biophysica Acta (BBA)--Enzymology. Volume 445, Issue 3, Oct. 11, 1976, pp. 753-762. However, each of these references has their own respective drawbacks and would be incapable, individually or in combination, of measuring soluble or insoluble cell or tissue-associated collagenase activity using Coomassie dye.

[0008] Accordingly, what is needed is an improved assay, kit, and/or vessel for detection of activity of blue collagenase. However, in view of the art considered as a whole at the time the present invention was made, it was not obvious to those of ordinary skill in the field of this invention how the shortcomings of the prior art could be overcome.

[0009] All referenced publications are incorporated herein by reference in their entirety. Furthermore, where a definition or use of a term in a reference, which is incorporated by reference herein, is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply,

[0010] While certain aspects of conventional technologies have been discussed to facilitate disclosure of the invention, Applicants in no way disclaim these technical aspects, and it is contemplated that the claimed invention may encompass one or more of the conventional technical aspects discussed herein.

[0011] The present invention may address one or more of the problems and deficiencies of the prior art discussed above. However, it is contemplated that the invention may prove useful in addressing other problems and deficiencies in a number of technical areas. Therefore, the claimed invention should not necessarily be construed as limited to addressing any of the particular problems or deficiencies discussed herein.

[0012] In this specification, where a document, act or item of knowledge is referred to or discussed, this reference or discussion is not an admission that the document, act or item of knowledge or any combination thereof was at the priority date, publicly available, known to the public, part of common general knowledge, or otherwise constitutes prior art under the applicable statutory provisions; or is known to be relevant to an attempt to solve any problem with which this specification is concerned.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] For a fuller understanding of the invention, reference should be made to the following detailed description, taken in connection with the accompanying drawings, in which:

[0014] FIG. 1A depicts a collagen suspension incubated with trypsin,

[0015] FIG. 1B depicts a collagen suspension incubated with C. histolyticum collagenase.

[0016] FIG. 2 depicts collagenase activity in S. mutans (Sm).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0017] In the following detailed description of the preferred embodiments, reference is made to the accompanying drawings, which form a part thereof, and within which are shown by way of illustration specific embodiments by which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the invention.

[0018] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. As used in this specification and the appended claims, the term "or" is generally employed in its sense including "and/or" unless the context clearly dictates otherwise.

[0019] Blue collagenase assay is a method of measuring soluble or insoluble cell or tissue-associated collagenase activity. The substrate includes native fibrillar collagen fragments that were stained with Coomassie Brilliant Blue R-250. Incubation with collagenase can be Observed in real-time by the generation of digested smaller fragments, and can be obtained by filtration, and can be quantitated by measuring the amount of dye extracted from these fragments.

[0020] The present method is amenable to the preparation of a blue collagenase assay kit, which would contain reaction tubes containing blue collagen substrate, tips plugged. with glass wool for filtration and collection tubes containing the extraction solution, and a bottle of concentrated substrate buffer.

[0021] In an embodiment, the current invention is a novel method to assay for collagenase activity using collagen fibrils (e.g., Type I) from Bovine Achilles tendon (SIGMA-ALDRICH, Product no. C9879). This method allows for both the qualitative observation of collagenase activity and the quantitative activity assay using a spectrophotometer. It is contemplated and shown herein that this method is applicable to both soluble and cell-associated collagenase.

[0022] In certain embodiments, the method presented herein is unique in staining lyophilized collagen with Coomassie Brilliant Blue R-250 (though other dyes are contemplated herein) at saturation level and in its native state without solubilization by acid and/or gelling by heat. Further, collagen fibrils can be cut with sharp scissors, and the dye can be extracted from the digested collagen fragments by Polysorbate 20 (TWEEN 20) for quantitative measurement of the absorbance at the optimal wave length of 600 nm, at which particulates also get absorbed. As such, the assay is applicable either to both soluble and insoluble cell or to tissue-associated collagenase. Application of the Blue Collagenase Assay to soluble Clostridium histolyticum collagenase and to cell-associated Streptococcus mutans collagenase is described in preparation.

[0023] In application, the present method can be applied to the staining other insoluble proteins, such as gelatin granules and other types of collagen for use as a substrate for the analysis of gelatinase and other collagenase types. The present method can also be used to analyze metalloproteinases in humans and animals.

EXAMPLE 1

[0024] As an example, application of the blue collagenase assay to soluble Clostridium histolyticum collagenase (see FIGS. 1A-IB) and cell-associated Streptococcus mutans collagenase (see FIG. 2) is described herein.

[0025] The method generally includes the following steps:

[0026] 1. The collagen fibrils are stained using Coomassie Brilliant Blue R-250 (C46-H44-N3-07-S2-Na).

[0027] 2. The Blue collagen fibrils are suspended in collagenase substrate buffer (50 mM Tris, 50 mM NaCl, 10 mM CaCl₂, pH7.5).

[0028] 3. The blue collagen fibrils in suspension are incubated with the test sample at 37° C. on a rotator.

[0029] 4. The collagenase activity results in the production of blue collagen particulates that can be readily observed.

[0030] 5. After the incubation time of three (3) hours or longer, the mixture is filtered through glass wool/fibers to which the blue collagen fibrils are retained, and the small blue fragments are collected in the filtrate,

[0031] 6, To determine the amount of digested collagen, the blue dye is extracted by incubation of the filtrate in the presence of Polysorbate 20 (PATEN 20) added at 2%, at 37° C. on a rotator,

[0032] 7. The mixture, containing the blue dye, is centrifuged at 12,000×g for 5 minutes.

[0033] 8. Absorbance of the blue supernatant is measured at 600 nm using a spectrophotometer or ELISA reader and expressed as equivalent U of Clostridium histolyticum collagenase.

[0034] For analysis, a standard curve was established with C. histolyticum collagenase used at various concentrations: 5 U/mL (1), 10 U/mL (2), 15 U/mL (3) and 20 U/mL (4) S. rautans GS-5 cell suspension was assayed in parallel using 1 mL of a bacterial suspension corresponding to 5 OD at 600 nm. All samples were tested in quadruplicates. Each column represents the mean of 4 measurements with standard error bar.

[0035] The reaction mixture (2 mL) consisted of 250 μl of blue collagen suspension in 1 mL Collagenase substrate buffer (CSB) and each test sample was diluted in CSB up to 1 mL. Each sample was assayed in quadruplicates. Incubation was for 4 h at 37° C. on a rotator.

[0036] The deduced activity of Sm collagenase was 9 C. histolyticum equivalent collagenase units (9 Ch eq.U) in a sample containing S. mutans GS5 cells at an absorbance of 5 OD at 600 nm, corresponding to a relative specific activity of 1.8 Ch eq. U/OD of bacteria,

EXAMPLE 2

1. Staining of Collagen

[0037] Type I fibrillar Collagen from Bovine Achilles Tendon (SIGMA-ALDRICH, product no. C9879) was cut with a pair of sharp scissors into shorter fibrils. The fibrils were passed through a 1 mm sieve and collected as dry collagen.

[0038] The dry collagen was mixed with 0.2% Coomassie Brilliant Blue in a solution of 10% acetic acid, 40% methanol in deionized water (standard method for staining protein bands in polyacrylamide gel), at a ratio of 500 mg collagen in 30 mL acetic acid-methanol-water solution, in a 50 mL conical centrifuge tube on a rotor at room temperature for 30 mins to 1 hour until all the collagen was saturated with the blue dye, after which time acetic acid-methanol-water (10:40:50) was added up to 50 mL.

[0039] The blue collagen suspension was centrifuged at 200×g for 2 minutes, and the supernatant was discarded. The excess dye was removed by rinsing with the solution of 10% Acetic acid, 40% methanol in deionized water.

[0040] With repeated mixing, the blue collagen was extensively washed with PBS (phosphate buffered saline, pH 7.2.) added up to 50 mL, followed by centrifugation and pouring off the supernatant containing floating denatured collagen until the supernatant came out clear and colorless.

[0041] The blue collagen was washed once with a solution of collagenase substrate buffer (50 mM Tris, 50 mM sodium chloride and 10 mM. Calcium chloride, pH 7.5, with sodium azide added at 0.02%) and then re-suspended in the same, but fresh, substrate buffer.

[0042] Aliquots of the blue collagen suspension (250 μl) were distributed into graduated 2 mL microcentrifuge tubes (FISHER SCIENTIFIC Co.) using 1 mL pipettor and a cut-off pipet tip to pipet up the collagen fibrils. The filled microcentrifuge tubes were stored in the refrigerator until used. The blue collagen suspension is stable, even when stored at room temperature (25° C.). Alternatively, the blue collagen can be lyophilized.

[0043] Quality control for specificity can be performed by incubation with Trypsin.

2. Establishment of a Standard Curve Using Soluble C. Histolytieum Collagenase of Known Enzyme Activity

[0044] C. hislolyticum collagenase with known enzyme activity (SIGNMA ALDRICH) and the samples to be tested were analyzed in parallel. C. histolyticum collagenase was selected as a test sample for the establishment of a standard curve. C. histolyticum collagenase Type I of known enzyme activity (SIGMA-ALDRICH) was solubilized in substrate buffer at 1U per μL. A standard curve is established with the C. histoyticum collagenase, which is used as a reference. Depending on the amount of enzyme in the test samples a standard curve can be established with C. histolyticum enzyme concentrations from 20-300 Units/mL. A quick test can be performed with C. histolyticum and the test samples in order to determine the appropriate range of the concentrations needed for the standard curve. This is facilitated by the observation of collagen degradation in real time.

[0045] Up to 1.75 mL of sample can be used and added to each tube containing the blue collagen (total volume of the reaction mix is 2 mL).

[0046] The tubes were incubated at 37° C. on a rotator that are placed in an incubator for 3 hours or as long as needed depending on enzyme concentration. Digestion of the blue collagen by collagenase was periodically observed, resulting in a dose-dependent generation of small blue collagen particles. At the end of the incubation period, the reaction mixture from each tube was filtered through loosely packed glass wool.

[0047] After the incubation period, the reaction mixture was poured into 1 mL pipet tip containing loosely packed glass fibers, and the filtrate containing small blue collagen fragments were collected into a fresh 2 mL-microcentrifuge tube containing 200 μl of 20% Tween 20 in substrate buffer was added.

[0048] The tubes were then incubated at 37° C. on a rotator for about 2 hours until dye was extracted from the collagen particles.

[0049] After the extraction period, the tubes were centrifuged at 10,000×g for 5 minutes to sediment the de-stained collagen fragments and other insoluble components in the test samples. The supernatant containing the dye from each tube is measured for the absorbance at 600 nm using a spectrophotometer or an ELISA reader. Tubes incubated with buffer alone (1 mL substrate buffer containing 2% Polysorbate 20 (TWEEN 20)) were used as a blank.

[0050] A standard curve is established with the C. histolyticum collagenase (U/mL versus OD at 600 nm), and the corresponding enzyme activity of the test sample is derived from the curve and expressed in Ch-equivalent U, while the relative specific activity is calculated as follows: Ch-equivalent U/mg of protein (if known), or in the case of bacterial suspension (OD 600 mn of bacteria).

[0051] As a side note, due to the heterogeneity of the collagen fibrils, a standard curve typically is established for each lot of blue collagen.

3. Activity Assay of Bacterial Cell-Associated Collagenase

[0052] Streptococcus mutans whole cells were used as a model of insoluble collagenase.

[0053] Bacterial cells were collected by centrifugation and the cell was suspended in substrate buffer at a concentration of 5 OD₆₀₀ nm per mL.

[0054] One (1) mL of bacterial cell suspension was added to each blue collagen substrate tube, and substrate buffer was added up to 2 mL. The assay was performed in quadruplicate.

[0055] Subsequent steps were performed, as described previously for C. histolyticum collagenase.

[0056] Enzyme activity unit was derived from the C. histolyticum standard curve and expressed as C. histolyticum equivalent units.

[0057] The advantages set forth above, and those made apparent from the foregoing description, are efficiently attained. Since certain changes may be made in the above construction without departing from the scope of the invention, it is intended that all matters contained in the foregoing description or shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

[0058] It is also to be understood that the following claims are intended to cover all of the generic and specific features of the invention herein described, and all statements of the scope of the invention that, as a matter of language, might be said to fall therebetween.

Claims

1. An assay for measuring soluble or insoluble cell or tissue-associated collagenase activity, comprising: obtaining dry collagen fibrils suitable for dying; staining said collagen fibrils with blue dye at saturation level; suspending said blue collagen fibrils in a collagenase substrate buffer; incubating said blue collagen fibril suspension with a test sample at 37.degree. C. on a rotator, wherein collagenase activity results in production of blue collagen particulates that can be readily observed in a resulting mixture; filtering said mixture to retain said blue collagen fibrils, wherein said hlu collagen particulates are digested and collected in a resulting filtrate; extracting said blue dye from said filtrate to determine an amount of said digested blue collagen particulates; and measuring absorbance of said digested blue collagen particulates.

2. An assay as in claim 1, wherein the step of measuring absorbance is performed using a spectrophotometer at an optimal wave length of 600 nm.

3. An assay as in claim 1, wherein the step of staining said collagen fibrils is performed using 0.2% Coomassie Brilliant Blue in a solution of acetic acid-methanol-water (10:40:50, v:v:v), at a ratio of 500 mg collagen in 30 mL acetic acid-methanol-water solution.

Images