A Collagen
Film Microassay
A quantitative
microassay
ib presented. contact with
Collagen a dried
actively
The
method assay
labeled
the
detection
of bacterial
Collagenase
and
tissue
collagenase
in acetic acid solution forms a thin. tenacious tilm agar surface. The digestion of the film by collagena\e
detected by subsequent dark blue while areas be employed in two screening tation.
fol-
for Tissue
staining with Coomassie of collagenax digestion ways: with collagen-coated
blue. remain
or with collagen-lined capillary has a sensitivity comparable collagen
~uhstrate4
and
requii-e\
to only
Undigested clear. The glas\ coverslips tube\ thou
i\
film is stained technique can as :I rapid
for precise assays using
5 to 60 min
upon
quantiradio-
of incubation.
Previous assays for collagenase have employed either labeled or unlabeled collagen substrate t I .2,3). The “C-radiofibril assay. although the most sensitive of the standard techniques, involves the difficulty and expense of preparing the radioactive substrate. Assays using unlabeled collagen generally suffer from lack of sensitivity. elaborate methodology, or lengthy incubation times (3). We describe a rapid assay employing an unlabeled native collagen substrate deposited as a film on agar-coated glass. The film is incubated in the presence of collagenase and then stained with Coomassie blue. The undigested film stains dark blue, while areas of collagenase activity remain clear. Collagen-coated coverslips are used for rapid screening and are semiquantitative. Collagenase concentration can be measured more precisely using collagen-lined capillary tubes. The areas of collagenase activity are stained with Coomassie blue. and the bound dye is eluted off and its absorbance measured spectrophotometrically. MATERIALS
AND METHODS
The agar used throughout was Special Agar-Noble (Difco Laboratories, Detroit, Mich.). The absorbent tissues were Kimwipes (KimberlyClark. Neenah, Wis.). Acid-extracted guinea pig skin collagen (41, generously provided by Dr. Steven M. Krane, Massachusetts General Hospital. was dissolved at a concentration of 4 mgiml in 0.5V acetic acid. Plain microhematocrit tubes (7.5 mm, 1.1 mm i.d.) were obtained from Curtin Matheson Scientific, Stoneham, Mass.
580
RICHARD
A
LEVENSON
C
E
FIG. 1. Photograph of collagen film assay. (A) Collagen-coated coverslip incubated with three enzyme samples in triplicate at 37°C for I h and then stained with Coomassie blue. Top row: bacterial collagenase. 2.5 pgiml: middle row: mouse bone collagenase. 50 pgiml; bottom row: trypsin, 2 mgiml. Lysis occurs directly under the sample droplet. (B. C, and D) Collagen-lined capillary tubes incubated with bacterial collagenase. 10 pgiml. for 2. 4. and 6 min. (E) Collagen-lined capillary tubes incubated with trypsin. 2 mgiml for I h.
Clostridiurn /zisto/yticurn collagenase (Type III, 425 U/mg, EC 3.4.4.19) was obtained from the Sigma Chemical Company. Highly purified mouse bone collagenase, 7500 U/mg (21, essentially free of caseinolytic activity, was generously provided by Dr. Seizaburo Sakamoto at the Harvard School of Dental Medicine. Trypsin (2x crystallized. salt free, 12.500 Uimg, EC 3.4.4.4) was obtained from Sigma Chemical Company. All 0.005 M CaCl,. pH 7.5. enzymes were buffered in 0.05 M Tris-HCI, Coomassie brilliant blue R-250 (Bio-Rad Laboratories, Richmond, Calif.) was made up as a 2% (w/v) solution in 10% acetic acid and was filtered through Whatman No. 1 Qualitative filter paper. A Gilford spectrophotometer was used. 1. Collugen-Coated
Corlerslips
Agar (1%. w/v) is boiled and then cooled to approximately 50°C. A glass coverslip of any convenient size is dipped into the agar. Excess agar is shaken off and the coverslip is placed on absorbent tissue. After 2-3 min. the agar forms a gel, and the coverslip is trans-
MICROASSAY
FIG. 2. Device for lining gently drawn up and expelled by a short
(l-7
set)
burst
FOR
TISSUE
COLLAGENASE
the capillary tubes with agar and collagen. using the rubber bulb. The excess is purged of compressed
581
The solutions are and the film is dried
air.
ferred to another absorbent tissue to prevent sticking. The agar is dried for 15 min at 37°C in a low-humidity warm room to form a transparent, even film. One drop (Z-50 ~1) of collagen in acetic acid solution is sandwiched between two agar-coated coverslips. The coverslips are slid against one another and then carefully separated. The collagen should spread evenly across both coverslips which are then immediately washed with a gentle stream of distilled water. The thin film of collagen adhering to the agar is then dried at room temperature. A stream of compressed air completes the drying in a few seconds. The coverslips so prepared are usable for up to 4 weeks when stored at 4°C. Small drops (2-3 ~1) of the sample solutions are placed on the coverslip and then are incubated at 37°C in 100% humidity. The coverslip is then dipped in distilled water. stained for approximately 30 set in Coomassie blue, and gently washed in distilled water. The collagen film stains dark blue. Complete digestion of the film by collagenase results in clear areas of lysis where the sample was applied: incomplete digestion results in a notable lightening of the film (Fig. I). Quantitation is achieved by ascertaining either the length of time required for complete clearing. or the greatest dilution of enzyme which gives complete clearing after a fixed incubation period. The incubation time required for complete digestion varies from 5 min to 3 h. depending on collagenase concentration.
582
RICHARD
LEVENSON
Agar (1% w/v) is boiled and then cooled to approximately 60°C. It is drawn three-fourths of the way up a microhematocrit tube and then quickly forced out with a blast of compressed air, yielding a uniform dry agar surface. Collagen solution is similarly drawn up and immediately forced out, leaving a thin film on the interior of the tube. A capillary tube holder connected by a Y-valve to a compressed air supply can simplify this procedure (Fig. 2). making it easy to alternate gentle suction with strong blasts of air. The sample solution is then drawn up approximately a quarter of the length of the tubes, and the tubes are incubated at 37°C in 100% humidity. The volume of the sample is not critical, as the volume to surface area ratio of the capillary tube is independent of length. The tubes should be inclined slightly during incubation to keep the sample from moving up the tube. One or more tubes are removed periodically, dipped in distilled water, and filled with Coomassie blue. After 1 min, the dye is forced out with compressed air. Collagenolytic activity is demonstrated by progressive clearing of the originally dark blue staining collagen. Equal lengths are cut from each capillary tube with a glass cutting knife, and the segments are placed in individual test tubes. One milliliter of absolute ethanol is added to the test tubes which are then shaken vigorously for 5 sec. The alcohol elutes the Coomasssie blue which has bound to the remaining collagen, and the absorbance of the dye at 552 nm is recorded. RESULTS In both the coverslip and the capillary tube assay, the collagen film was digested rapidly and consistently by bacterial and mouse bone collagenase. The film of collagen deposited on the coverslips and inside the capillary tubes was approximately 5 pm thick by light and electron microscopy. Despite its thinness. it stained darkly and uniformly.
The collagen-coated coverslips were sensitive to low levels of collagenase. and the time required for complete digestion of the film was dependent on collagenase concentration (Fig. 3). Mouse bone collagenase, 50 pg/ml, caused complete clearing within 30 min. Bacterial collagenase, 10 &ml, caused complete clearing within IS min; 2.5 pg/ml, near the lower limit of detection for this assay, caused complete clearing in approximately 1 h (Fig. 1). Digestion time was independent of small variations in sample volume and digestion was restricted to the area beneath the droplet of enzyme. The specificity of the assay was determined through incubations with
MICROASSAY
FOR
TISSUE
5x3
COLLAGENASE
--Y .I6 .c I E .I4
is .I2 ii-2 a y .I0 0 p .08 IA .06 r b .04 L .02 .Ol 5
7.5 IO 12 15 20
ENZYME Flc;.
3.
Invert
of
clearing
lagenase at concentrations The cover\lips were stained timt: necessary examination for
for each
complete enzyme
time
30
50
CONCENTRATION Verse\
from 5- I00 pLg/ml with Coomassir clearing. concentration.
of
log wa\ blue the
of
100 @g/ml)
enryme
concentration.
Bacterial
co-
incubated on collagen-coated coverslips. after various periods of incubation. The collagen
film
has
determined
by
visual
various controls. Neither distilled water nor buffer had any marked effect on the film even after incubations at 37°C for 34 h. Trypsin (2 mg/ml: Fig. 1) and Pronase ( 1 mgiml: not shown) had little effect after 1 h. As in all collagenase assays. however, the specificity was dependent on the state of the collagen substrate. If the collagen was significantly denatured. nonspecific proteases could rapidly digest areas on the film.
Although the coverslip method was rapid and simple and required only 3 ~1 of sample, precise quantitation was difficult to achieve. We thus developed collagen-lined capillary tubes, which maintain a virtually constant sample volume to film area ratio regardless of the actual volume of sample present. The amount of Coomassie blue binding to unincubated collagen-lined tubes varied with a standard deviation of less than 3G. The kinetics of the capillary tube system were apparently first order. The amount of Coomassie blue adhering to the collagen remaining inside the tube decreased exponentially in samples from successive incubation periods. Plotted on a semilogarithmic scale. these data yielded a straight line (Fig. 4). It was found that the slopes of these plots were directly proportional to enzyme concentration for bacterial collagenase and for mouse bone collagenase over a defined concentration range (Fig. 5). Slopes from separate runs of the same concentration of mouse bone
584
RICHARD
0
5
1 10
15
LEVENSON
20
TIME
25
30
35
40
45
50
55
60
(min.)
I
I
--
‘0’
FIG. 4. (A) Substrate remaining after incubation with enzyme. Collagen-lined capillary tubes were incubated with 2.5 pg/ml of bacterial collagenase at 37°C. Three tubes were removed at each time point and stained with Coomassie blue for 1 min. Equal lengths from tubes from each time point were cut off and placed together in I ml of ethanol to elute the dye bound to the remaining collagen. Absorbance at 552 nm was measured. (B) Bacterial collagenase. IO fig/ml; single tubes removed at each time point. (C) Mouse bone collagenase. 50 Kg/ml; means 2 S.D. of single tubes. 11 = 5. (D) Trypsin. 100 CLgiml: means + S.D. of single tubes. II = 3. The solid lines were drawn so that the slopes from the linear portions of the curves could be determined.
collagenase (50 pug/ml) had a standard deviation of less than 6%. The slope for this concentration was well defined after an incubation of 30 min; 10 @g/ml of bacterial collagenase could be measured within 10 min (Fig. 4). The specificity of the capillary tube assay for collagenase was deter-
MICROASSAY
,500 I pd’ $. $j ’ In Iii 0 .200 z 150i E 400
FOR
“\,
300
250
TISSUE
\*
10
\ 0 \
0 a2
585
COLLAGENASE
100
"10 1 ir 0
c
10 T 5
IO
Ii5
20
TIME
60
TIME
25
20
3i
4 I.r
45
5 L'
(min.)
90
I20
150
(mtn.)
mined by incubations with various controls. Neither distilled water nor buffer had any discernible effect on the collagen film after incubations of at least 18 h. Less than 10%~of the collagen film was degraded by trypsin (100 pgiml) after an incubation of 60 min. with a further degradation of approximately another 205%in the succeeding 130 min (Fig. 5). By contrast. 0.3 pg/ml of bacterial collagenase completely digested this batch of collagen within 60 min (not shown). Incubation of a partially denatured collagen film with trypsin yielded a biphasic curve: a fairly rapid initial decrease in absorbance. reflecting digestion of the denatured components. was followed by a leveling off of the curve. suggesting resistance of the remaining film to further tryptic digestion (data not shown).
586
RICHARD
0
LEVENSON
IO
5
2 ENZYME
CONCENTRATION
(pg/ml)
25-I
04 0
IO
20
ENZYME
FIG. A’ is an concentration.
5.
(Al
Standard
arbitrary (B)
constant), Standard
K is the same as above). enzyme concentration.
curve:
40
50
60
10
CONCENTRATION
bacterial
determined curve: mouse determined
30
collagenase. from hand-drawn bone collagenase. from
hand-drawn
80
90
100
(pg/ml)
Relative
velocity
(KAIogAsSJIA1.;
semi-log plots Relative velocity semi-log
plots.
in
vs
enzyme
(KLdogA,j2,xr. duplicate,
vs
DISCUSSION This collagen film assay is rapid and sensitive to low levels of bacterial and mammalian collagenase. A total of less than 7.5 ng of bacterial collagenase or SOng of mouse bone collagenase can be detected within I hr ustng the coverslip technique. The assay does not require labeled substrate, recovery of breakdown products, lengthy incubations, or complex processing. The digestion of the collagen film by both bacterial and mouse bone collagenase appears to obey first-order kinetics (Figs. 4, 5). The nonlinearity observed near reaction completion may be due to product inhibition. Reaction rate is proportional to enzyme concentration for bacterial collagenase (Fig. 5A). This is true for mammalian collagenase in the concentration range O-50 pug/ml. At higher concentrations, the reaction may be substrate limited, since so little collagen is present in the film. The precision of the assay is good. Reaction rate or time-to-clearing
MICROASSAY
FOR
TlSSliE
COLLAGENASE
587
determinations vary with a standard deviation of less than 10% when the same collagen preparation is used. Although both the coverslips and the capillary tubes can be prepared at least a month in advance. altering their sensitivity or ability to bind the films slowly “age.” stain. Individual runs should be calibrated against a standard enzyme concentration. Although the quantitation provided by the capillary tubes is relatively good, perhaps the most useful application of the collagen film assay would be the employment of the coverslips for rapid detection of collagenase activity in column eluates. With IO coverslips, one can assay more than 90 column fractions. The exact nature of the substrate in the collagen film has not been determined. The formation of the collagen film is apparently due to the electrostatic interactions between the collagen and constituents of the agar. Certain sulfated polysaccharides, similar to those present in agar. have been shown to accelerate the formation of fibrils from a solution of collagen monomers (5.6). Agarose. which is free of polyanions. binds the collagen film poorly in comparison to the agar. The thickness of the collagen film is not affected by minor variations in the amount of agar applied, the amount of collagen solution applied, or the manner in which excess amounts of these compounds are removed. A trypsin control should be included in every assay. Most incubations can be run in 20 min or less. depending on the activity of the collagenase sample: therefore, it may be possible to ignore the slower manifestations of nonspecific proteolytic attack. If longer incubations are necessary. it may be advisable to subtract from the slope generated by the collagenolytic sample the slope generated by the proteolytic control. Sometimes it is difficult to identify a unique slope in the trypsin incubation. The time points approach linearity but display considerable scatter. It has been shown that collagen can trap soluble proteins within the spaces found periodically along the fibrils (7). The scatter observed may be due to adsorption of the trypsin to the collagen film and its subsequent staining by the Coomassie blue. The assay has certain limitations. During long incubations, the dried agar on the coverslips rehydrates and swells slightly. This can weaken the collagen film, which must then be treated gently during the staining procedure. The collagen film may also be removed by nonenzymatic means. Dissociative agents such as urea and guanidine hydrochloride actively separate the collagen from the agar. This effect can be recognized by ragged edges around the cleared areas and outside the area of sample application. due to diffusion of the small molecular weight compounds. Crude tissue extracts may consequently require preliminary purification before this assay can be employed. Since the products of digestion are not recovered, there is no
588
RICHARD
LEVENSON
convenient way to express enzyme activity in terms of the standard units now widely used for animal collagenases, namely, micrograms of collagen digested per minute per milligram of enzyme (or other variable). This difficulty can be surmounted if the assay is calibrated with an enzyme preparation of known activity. A new collagen film assay for tissue collagenase is presented. The simplicity. speed, and sensitivity of this assay recommend its use in the isolation and purification of animal collagenases. ACKNOWLEDGMENTS I wish to acknowledge the advice. support. and generous assistance of Drs. Judah Folkman. Richard Kornbluth. Seizaburo Sakamoto. and Stephen M. Krane. 1 am also indebted to Dr. Michael Klein for his kind perseverance.
REFERENCES I. Nagai. Y.. Lapiere. C. M., and Gross, J. (1966) Biockr~mi.stvy 5, 3173-3130. 2. Sakamoto. S.. Sakamoto, M.. Goldhaber, P.. and Glimcher, M. (1975) Biocherrr. Biophys. Rcs. Cornmrrn. 63, 172- 17X. 3. Berman, M. B.. Manabe. R.. and Davison. P. F. (1973) Anal. Biwhc~nz. 54, Z-534. 4. Glimcher. M. J.. Francois. C. J.. Richards. L.. and Krane, S. M. (1964) Bioclrim. Bioplrys. Ac,ta 93, 585-602. 5. Gelman. R. A.. and Blackwell. J. t 1974) Bioc~hirn. Bioph.vs. Ac,ftr 342, 245-261. 6. Obrink, B. (1973) Brv. J. Bioc/reru. 34, 129-137. 7. Vieth. W. R. (1971) in Recent Developments in Separation Science. Li. N.. ed.). Vol. I. pp. 181-201, CRC Press, Cleveland.
Film Microassay
A quantitative
microassay
ib presented. contact with
Collagen a dried
actively
The
method assay
labeled
the
detection
of bacterial
Collagenase
and
tissue
collagenase
in acetic acid solution forms a thin. tenacious tilm agar surface. The digestion of the film by collagena\e
detected by subsequent dark blue while areas be employed in two screening tation.
fol-
for Tissue
staining with Coomassie of collagenax digestion ways: with collagen-coated
blue. remain
or with collagen-lined capillary has a sensitivity comparable collagen
~uhstrate4
and
requii-e\
to only
Undigested clear. The glas\ coverslips tube\ thou
i\
film is stained technique can as :I rapid
for precise assays using
5 to 60 min
upon
quantiradio-
of incubation.
Previous assays for collagenase have employed either labeled or unlabeled collagen substrate t I .2,3). The “C-radiofibril assay. although the most sensitive of the standard techniques, involves the difficulty and expense of preparing the radioactive substrate. Assays using unlabeled collagen generally suffer from lack of sensitivity. elaborate methodology, or lengthy incubation times (3). We describe a rapid assay employing an unlabeled native collagen substrate deposited as a film on agar-coated glass. The film is incubated in the presence of collagenase and then stained with Coomassie blue. The undigested film stains dark blue, while areas of collagenase activity remain clear. Collagen-coated coverslips are used for rapid screening and are semiquantitative. Collagenase concentration can be measured more precisely using collagen-lined capillary tubes. The areas of collagenase activity are stained with Coomassie blue. and the bound dye is eluted off and its absorbance measured spectrophotometrically. MATERIALS
AND METHODS
The agar used throughout was Special Agar-Noble (Difco Laboratories, Detroit, Mich.). The absorbent tissues were Kimwipes (KimberlyClark. Neenah, Wis.). Acid-extracted guinea pig skin collagen (41, generously provided by Dr. Steven M. Krane, Massachusetts General Hospital. was dissolved at a concentration of 4 mgiml in 0.5V acetic acid. Plain microhematocrit tubes (7.5 mm, 1.1 mm i.d.) were obtained from Curtin Matheson Scientific, Stoneham, Mass.
580
RICHARD
A
LEVENSON
C
E
FIG. 1. Photograph of collagen film assay. (A) Collagen-coated coverslip incubated with three enzyme samples in triplicate at 37°C for I h and then stained with Coomassie blue. Top row: bacterial collagenase. 2.5 pgiml: middle row: mouse bone collagenase. 50 pgiml; bottom row: trypsin, 2 mgiml. Lysis occurs directly under the sample droplet. (B. C, and D) Collagen-lined capillary tubes incubated with bacterial collagenase. 10 pgiml. for 2. 4. and 6 min. (E) Collagen-lined capillary tubes incubated with trypsin. 2 mgiml for I h.
Clostridiurn /zisto/yticurn collagenase (Type III, 425 U/mg, EC 3.4.4.19) was obtained from the Sigma Chemical Company. Highly purified mouse bone collagenase, 7500 U/mg (21, essentially free of caseinolytic activity, was generously provided by Dr. Seizaburo Sakamoto at the Harvard School of Dental Medicine. Trypsin (2x crystallized. salt free, 12.500 Uimg, EC 3.4.4.4) was obtained from Sigma Chemical Company. All 0.005 M CaCl,. pH 7.5. enzymes were buffered in 0.05 M Tris-HCI, Coomassie brilliant blue R-250 (Bio-Rad Laboratories, Richmond, Calif.) was made up as a 2% (w/v) solution in 10% acetic acid and was filtered through Whatman No. 1 Qualitative filter paper. A Gilford spectrophotometer was used. 1. Collugen-Coated
Corlerslips
Agar (1%. w/v) is boiled and then cooled to approximately 50°C. A glass coverslip of any convenient size is dipped into the agar. Excess agar is shaken off and the coverslip is placed on absorbent tissue. After 2-3 min. the agar forms a gel, and the coverslip is trans-
MICROASSAY
FIG. 2. Device for lining gently drawn up and expelled by a short
(l-7
set)
burst
FOR
TISSUE
COLLAGENASE
the capillary tubes with agar and collagen. using the rubber bulb. The excess is purged of compressed
581
The solutions are and the film is dried
air.
ferred to another absorbent tissue to prevent sticking. The agar is dried for 15 min at 37°C in a low-humidity warm room to form a transparent, even film. One drop (Z-50 ~1) of collagen in acetic acid solution is sandwiched between two agar-coated coverslips. The coverslips are slid against one another and then carefully separated. The collagen should spread evenly across both coverslips which are then immediately washed with a gentle stream of distilled water. The thin film of collagen adhering to the agar is then dried at room temperature. A stream of compressed air completes the drying in a few seconds. The coverslips so prepared are usable for up to 4 weeks when stored at 4°C. Small drops (2-3 ~1) of the sample solutions are placed on the coverslip and then are incubated at 37°C in 100% humidity. The coverslip is then dipped in distilled water. stained for approximately 30 set in Coomassie blue, and gently washed in distilled water. The collagen film stains dark blue. Complete digestion of the film by collagenase results in clear areas of lysis where the sample was applied: incomplete digestion results in a notable lightening of the film (Fig. I). Quantitation is achieved by ascertaining either the length of time required for complete clearing. or the greatest dilution of enzyme which gives complete clearing after a fixed incubation period. The incubation time required for complete digestion varies from 5 min to 3 h. depending on collagenase concentration.
582
RICHARD
LEVENSON
Agar (1% w/v) is boiled and then cooled to approximately 60°C. It is drawn three-fourths of the way up a microhematocrit tube and then quickly forced out with a blast of compressed air, yielding a uniform dry agar surface. Collagen solution is similarly drawn up and immediately forced out, leaving a thin film on the interior of the tube. A capillary tube holder connected by a Y-valve to a compressed air supply can simplify this procedure (Fig. 2). making it easy to alternate gentle suction with strong blasts of air. The sample solution is then drawn up approximately a quarter of the length of the tubes, and the tubes are incubated at 37°C in 100% humidity. The volume of the sample is not critical, as the volume to surface area ratio of the capillary tube is independent of length. The tubes should be inclined slightly during incubation to keep the sample from moving up the tube. One or more tubes are removed periodically, dipped in distilled water, and filled with Coomassie blue. After 1 min, the dye is forced out with compressed air. Collagenolytic activity is demonstrated by progressive clearing of the originally dark blue staining collagen. Equal lengths are cut from each capillary tube with a glass cutting knife, and the segments are placed in individual test tubes. One milliliter of absolute ethanol is added to the test tubes which are then shaken vigorously for 5 sec. The alcohol elutes the Coomasssie blue which has bound to the remaining collagen, and the absorbance of the dye at 552 nm is recorded. RESULTS In both the coverslip and the capillary tube assay, the collagen film was digested rapidly and consistently by bacterial and mouse bone collagenase. The film of collagen deposited on the coverslips and inside the capillary tubes was approximately 5 pm thick by light and electron microscopy. Despite its thinness. it stained darkly and uniformly.
The collagen-coated coverslips were sensitive to low levels of collagenase. and the time required for complete digestion of the film was dependent on collagenase concentration (Fig. 3). Mouse bone collagenase, 50 pg/ml, caused complete clearing within 30 min. Bacterial collagenase, 10 &ml, caused complete clearing within IS min; 2.5 pg/ml, near the lower limit of detection for this assay, caused complete clearing in approximately 1 h (Fig. 1). Digestion time was independent of small variations in sample volume and digestion was restricted to the area beneath the droplet of enzyme. The specificity of the assay was determined through incubations with
MICROASSAY
FOR
TISSUE
5x3
COLLAGENASE
--Y .I6 .c I E .I4
is .I2 ii-2 a y .I0 0 p .08 IA .06 r b .04 L .02 .Ol 5
7.5 IO 12 15 20
ENZYME Flc;.
3.
Invert
of
clearing
lagenase at concentrations The cover\lips were stained timt: necessary examination for
for each
complete enzyme
time
30
50
CONCENTRATION Verse\
from 5- I00 pLg/ml with Coomassir clearing. concentration.
of
log wa\ blue the
of
100 @g/ml)
enryme
concentration.
Bacterial
co-
incubated on collagen-coated coverslips. after various periods of incubation. The collagen
film
has
determined
by
visual
various controls. Neither distilled water nor buffer had any marked effect on the film even after incubations at 37°C for 34 h. Trypsin (2 mg/ml: Fig. 1) and Pronase ( 1 mgiml: not shown) had little effect after 1 h. As in all collagenase assays. however, the specificity was dependent on the state of the collagen substrate. If the collagen was significantly denatured. nonspecific proteases could rapidly digest areas on the film.
Although the coverslip method was rapid and simple and required only 3 ~1 of sample, precise quantitation was difficult to achieve. We thus developed collagen-lined capillary tubes, which maintain a virtually constant sample volume to film area ratio regardless of the actual volume of sample present. The amount of Coomassie blue binding to unincubated collagen-lined tubes varied with a standard deviation of less than 3G. The kinetics of the capillary tube system were apparently first order. The amount of Coomassie blue adhering to the collagen remaining inside the tube decreased exponentially in samples from successive incubation periods. Plotted on a semilogarithmic scale. these data yielded a straight line (Fig. 4). It was found that the slopes of these plots were directly proportional to enzyme concentration for bacterial collagenase and for mouse bone collagenase over a defined concentration range (Fig. 5). Slopes from separate runs of the same concentration of mouse bone
584
RICHARD
0
5
1 10
15
LEVENSON
20
TIME
25
30
35
40
45
50
55
60
(min.)
I
I
--
‘0’
FIG. 4. (A) Substrate remaining after incubation with enzyme. Collagen-lined capillary tubes were incubated with 2.5 pg/ml of bacterial collagenase at 37°C. Three tubes were removed at each time point and stained with Coomassie blue for 1 min. Equal lengths from tubes from each time point were cut off and placed together in I ml of ethanol to elute the dye bound to the remaining collagen. Absorbance at 552 nm was measured. (B) Bacterial collagenase. IO fig/ml; single tubes removed at each time point. (C) Mouse bone collagenase. 50 Kg/ml; means 2 S.D. of single tubes. 11 = 5. (D) Trypsin. 100 CLgiml: means + S.D. of single tubes. II = 3. The solid lines were drawn so that the slopes from the linear portions of the curves could be determined.
collagenase (50 pug/ml) had a standard deviation of less than 6%. The slope for this concentration was well defined after an incubation of 30 min; 10 @g/ml of bacterial collagenase could be measured within 10 min (Fig. 4). The specificity of the capillary tube assay for collagenase was deter-
MICROASSAY
,500 I pd’ $. $j ’ In Iii 0 .200 z 150i E 400
FOR
“\,
300
250
TISSUE
\*
10
\ 0 \
0 a2
585
COLLAGENASE
100
"10 1 ir 0
c
10 T 5
IO
Ii5
20
TIME
60
TIME
25
20
3i
4 I.r
45
5 L'
(min.)
90
I20
150
(mtn.)
mined by incubations with various controls. Neither distilled water nor buffer had any discernible effect on the collagen film after incubations of at least 18 h. Less than 10%~of the collagen film was degraded by trypsin (100 pgiml) after an incubation of 60 min. with a further degradation of approximately another 205%in the succeeding 130 min (Fig. 5). By contrast. 0.3 pg/ml of bacterial collagenase completely digested this batch of collagen within 60 min (not shown). Incubation of a partially denatured collagen film with trypsin yielded a biphasic curve: a fairly rapid initial decrease in absorbance. reflecting digestion of the denatured components. was followed by a leveling off of the curve. suggesting resistance of the remaining film to further tryptic digestion (data not shown).
586
RICHARD
0
LEVENSON
IO
5
2 ENZYME
CONCENTRATION
(pg/ml)
25-I
04 0
IO
20
ENZYME
FIG. A’ is an concentration.
5.
(Al
Standard
arbitrary (B)
constant), Standard
K is the same as above). enzyme concentration.
curve:
40
50
60
10
CONCENTRATION
bacterial
determined curve: mouse determined
30
collagenase. from hand-drawn bone collagenase. from
hand-drawn
80
90
100
(pg/ml)
Relative
velocity
(KAIogAsSJIA1.;
semi-log plots Relative velocity semi-log
plots.
in
vs
enzyme
(KLdogA,j2,xr. duplicate,
vs
DISCUSSION This collagen film assay is rapid and sensitive to low levels of bacterial and mammalian collagenase. A total of less than 7.5 ng of bacterial collagenase or SOng of mouse bone collagenase can be detected within I hr ustng the coverslip technique. The assay does not require labeled substrate, recovery of breakdown products, lengthy incubations, or complex processing. The digestion of the collagen film by both bacterial and mouse bone collagenase appears to obey first-order kinetics (Figs. 4, 5). The nonlinearity observed near reaction completion may be due to product inhibition. Reaction rate is proportional to enzyme concentration for bacterial collagenase (Fig. 5A). This is true for mammalian collagenase in the concentration range O-50 pug/ml. At higher concentrations, the reaction may be substrate limited, since so little collagen is present in the film. The precision of the assay is good. Reaction rate or time-to-clearing
MICROASSAY
FOR
TlSSliE
COLLAGENASE
587
determinations vary with a standard deviation of less than 10% when the same collagen preparation is used. Although both the coverslips and the capillary tubes can be prepared at least a month in advance. altering their sensitivity or ability to bind the films slowly “age.” stain. Individual runs should be calibrated against a standard enzyme concentration. Although the quantitation provided by the capillary tubes is relatively good, perhaps the most useful application of the collagen film assay would be the employment of the coverslips for rapid detection of collagenase activity in column eluates. With IO coverslips, one can assay more than 90 column fractions. The exact nature of the substrate in the collagen film has not been determined. The formation of the collagen film is apparently due to the electrostatic interactions between the collagen and constituents of the agar. Certain sulfated polysaccharides, similar to those present in agar. have been shown to accelerate the formation of fibrils from a solution of collagen monomers (5.6). Agarose. which is free of polyanions. binds the collagen film poorly in comparison to the agar. The thickness of the collagen film is not affected by minor variations in the amount of agar applied, the amount of collagen solution applied, or the manner in which excess amounts of these compounds are removed. A trypsin control should be included in every assay. Most incubations can be run in 20 min or less. depending on the activity of the collagenase sample: therefore, it may be possible to ignore the slower manifestations of nonspecific proteolytic attack. If longer incubations are necessary. it may be advisable to subtract from the slope generated by the collagenolytic sample the slope generated by the proteolytic control. Sometimes it is difficult to identify a unique slope in the trypsin incubation. The time points approach linearity but display considerable scatter. It has been shown that collagen can trap soluble proteins within the spaces found periodically along the fibrils (7). The scatter observed may be due to adsorption of the trypsin to the collagen film and its subsequent staining by the Coomassie blue. The assay has certain limitations. During long incubations, the dried agar on the coverslips rehydrates and swells slightly. This can weaken the collagen film, which must then be treated gently during the staining procedure. The collagen film may also be removed by nonenzymatic means. Dissociative agents such as urea and guanidine hydrochloride actively separate the collagen from the agar. This effect can be recognized by ragged edges around the cleared areas and outside the area of sample application. due to diffusion of the small molecular weight compounds. Crude tissue extracts may consequently require preliminary purification before this assay can be employed. Since the products of digestion are not recovered, there is no
588
RICHARD
LEVENSON
convenient way to express enzyme activity in terms of the standard units now widely used for animal collagenases, namely, micrograms of collagen digested per minute per milligram of enzyme (or other variable). This difficulty can be surmounted if the assay is calibrated with an enzyme preparation of known activity. A new collagen film assay for tissue collagenase is presented. The simplicity. speed, and sensitivity of this assay recommend its use in the isolation and purification of animal collagenases. ACKNOWLEDGMENTS I wish to acknowledge the advice. support. and generous assistance of Drs. Judah Folkman. Richard Kornbluth. Seizaburo Sakamoto. and Stephen M. Krane. 1 am also indebted to Dr. Michael Klein for his kind perseverance.
REFERENCES I. Nagai. Y.. Lapiere. C. M., and Gross, J. (1966) Biockr~mi.stvy 5, 3173-3130. 2. Sakamoto. S.. Sakamoto, M.. Goldhaber, P.. and Glimcher, M. (1975) Biocherrr. Biophys. Rcs. Cornmrrn. 63, 172- 17X. 3. Berman, M. B.. Manabe. R.. and Davison. P. F. (1973) Anal. Biwhc~nz. 54, Z-534. 4. Glimcher. M. J.. Francois. C. J.. Richards. L.. and Krane, S. M. (1964) Bioclrim. Bioplrys. Ac,ta 93, 585-602. 5. Gelman. R. A.. and Blackwell. J. t 1974) Bioc~hirn. Bioph.vs. Ac,ftr 342, 245-261. 6. Obrink, B. (1973) Brv. J. Bioc/reru. 34, 129-137. 7. Vieth. W. R. (1971) in Recent Developments in Separation Science. Li. N.. ed.). Vol. I. pp. 181-201, CRC Press, Cleveland.
