ANALYTICAL

BIOCHEMISTRY

193.20-23

(1991)

A Method to Detect Proteinase Unprocessed X-ray Films

Laboratory of Bacteriology and Immunology, The Rockefeller

Activity

Using

Ambrose L. Cheung, Patrick Ying, and Vincent A. Fischetti

University,
1230

York Avenue, New York, New York 10021

Received

September

11, 1990

Routine assays to detect proteinases in biological samples are generally tedious and time-consuming. To expedite the recognition of proteinases, we have developed an assay utilizing the gelatin on the surface of an unprocessed Kodak X-Omat AR film as the proteolytic substrate. A positive reaction is indicated by a clear zone on the film after it has been rinsed with running water. This proteinase assay has been found to be inexpensive, rapid, and simple. Besides its ease of use, this assay has been found to be quantitatively reproducible with a well-defined endpoint. More importantly, this assay method is applicable to a variety of proteolytic enzymes under diverse pH (5-8.5) and salt conditions (up to 5 M NaCI) and has a sensitivity similar to that of azocoll. Since the assay does not require sophisticated equipment, it is useful as a general laboratory procedure. 0 1991 Academic Press, Inc.

and quantitative. In addition, this method has been found to have a sensitivity comparable to that of azocoll when dilutions of trypsin and chymotrypsin were used as standards.

MATERIALS

AND

METHODS

Trypsin, chymotrypsin, collagenase, and pepsin were purchased from Worthington Biochemical Corporation (Freehold, NJ). Zwittergent 3-14 was obtained from Calbiochem (San Diego, CA). Azocoll, soybean trypsin inhibitor, papain, and subtilisin BPN (Type XXVII) were from Sigma Chemical Co. (St. Louis, MO). All other chemicals including PMSF, SDS, Tris-base, Triton X-100, potassium thiocyanate, and guanidine were of reagent grade from Sigma.

Azocoll, an insoluble powdered cowhide conjugated to a bright red dye, is often used as a substrate in the determination of proteolytic activities (l-3). The extent of hydrolysis of the denatured collagen in the cowhide by a variety of proteinases is generally measured by a colorimetric assay which analyzes the release of colored peptides from the substrate into solution. Under carefully controlled experimental conditions (l), this assay is linear, reliable and reproducible. However, the method is fairly tedious and time-consuming and requires a spectrophotometer for quantitative analysis. To expedite the detection of proteinases among laboratory samples, we have utilized gelatin, another form of denatured collagen, on the surface of unprocessed XOmat XAR-5 films (Kodak Corp., Rochester, NY) as a proteolytic substrate. With the appearance of the plastic support below the gelatin coating (i.e., a clear zone) at the site of sample application on the film as a positive endpoint, this assay has been found to be quick, simple,
20

Assaying X-ray

Proteinase Film

with

Gelatin

Coating

on X-Omat

The gelatin coating on unprocessed X-Omat XAR-5 film (20 X 25 cm) of Kodak Chemical Corp. was used as a substrate for a variety of proteolytic enzymes. For the standard assay, 50 ~1of a sample containing proteinases was applied onto the film which was then incubated in an incubator for 1 h at 42C. The appearance of the plastic below the gelatin film as indicated by a clear zone at the site of application after the film was washed with running water was considered positive. Washing with tap water at ambient temperature from a laboratory faucet for 1 min was found to be adequate in the detection of clear zones in our studies.

1 Abbreviations sodium dodecyl say.

used: sulfate;

PMSF, ELISA,

phenylmethylsulfonyl enzyme-linked

fluoride; immunosorbent

SDS, as-

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1991 of reproduction

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reserved.

UNPROCESSED

X-RAY

FILM

METHOD

TO

DETECT

PROTEINASES

21

10

2.5

1.25

0.6

0.3

Soybean Trypsin Inhibitor

FIG. 1. Digestion of the gelatin coating on an X-ray film with trypsin. Trypsin at various concentrations in azocoll buffer (1 mg/ml to 0.3 wg/ml) was applied (50 ~1) onto X-Omat XAR-5 film and incubated at 42C for 1 h. After incubation, the film was washed with running water to produce clear zones at the site of application. Soybean trypsin inhibitors (2 mg/ml) were added to some samples (trypsin at 1 mg/ml) to demonstrate inhibition. Because of the sensitivity of the assay, only dilutions of trypsin within a range of 10 to 0.3 pg/ml are shown.

Assaying Proteinases with Azocoll The method of Chavira et al. (1) was used to assay for azocoll hydrolysis with proteinases. Briefly, azocoll (0.25 g), an insoluble proteinase substrate, was suspended in 50 ml of azocoll buffer (0.05 M Tris, 1 mM CaCl,, pH 7.8) and stirred rapidly in a 50-ml beaker for at least 30 min. Azocoll solutions (1 ml) were rapidly withdrawn from the stirring suspension with a large bore micropipet and dispensed into microfuge tubes to which 50 ~1 of a proteinase sample had been added. The tubes were then allowed to rotate at 28 rpm at 37C for 1 h. Reactions were stopped by icing the samples for 3 min followed by centrifugation in an Eppendorf microfuge (16,000g for 2 min) at 4C. The clear supernatant was removed and read spectrophotometrically at A,,, as previously described (1). Supernatants from tubes containing buffer alone served as negative controls. Determination of Extracellular subtilis Proteinases from Bacillus

or soybean trypsin inhibitor (2 mg/ml) and a buffer control did not exhibit a clear zone under the same conditions (Fig. 1). In general, the sensitivity of the test can be enhanced by gently rubbing (not scratching) the ball of the thumb over the surface of the film while it was being rinsed with water. Since trypsin and chymotrypsin are commonly used enzymes in a variety of laboratory procedures, they were chosen as prototype enzymes to test the effect of temperature and time of incubation of enzymes on the gelatin substrate on unprocessed X-ray films. Results demonstrated that the sensitivity of the assay increased with the rise in incubation temperature (Fig. 2). Because of excessive drying of the film at 6OC, all subsequent experiments were carried out at 42C. When the time of incubation of the gelatin substrate with trypsin (10-l pglml) and chymotrypsin (10-l pg/ml) was increased from 1 to 6 h at hourly intervals, no further increase in sensitivity was observed. The effect of pH on the gelatin substrate with trypsin and chymotrypsin was also examined. Within a pH range of 5 to 8.5, the level of activity of both enzymes on the gelatin substrate remained unchanged. Based on these results, all subsequent assays were performed at 42C for 1 h at a pH where the tested enzyme is active. Using a variety of enzymes, the sensitivity of the gelatin substrate was compared to that of azocoll. The enzymes tested included three serine proteinases (trypsin, chymotrypsin, subtilisin BPN), a metalloproteinase (collagenase), a thiol proteinase (papain), and an acid proteinase (pepsin at pH 5.5). With the exception of

001

B. subtilis RN5789, a strain which secretes extracellular proteinases, was a gift from S. Projan (Public Health Research Institute, New York, NY). The bacteria were grown in 50 ml of brain heart infusion broth for 15 h at 37C with mixing (200 rpm). Cells were centrifuged (2000g for 25 min) and the cell-free supernatant fraction was recentrifuged to remove any residual bacteria, concentrated to 600 ~1 in an Amicon cell (mol wt cutoff, 500) (Danver, MA) and used in assays without further treatment.
RESULTS

E ~\ 9 i z > m z m h rs E? 1

010

100

1000

CHYMO W TRYPSIN 23 37 42 60

Dilutions of trypsin (from 1 mg/ml to 0.3 pg/ml) were used to study its effect on the gelatin substrate on XOmat XAR-5 film. As shown in Fig. 1, a clear zone was observed at the site of application after the film was washed with running water. In contrast, trypsin (1 mg/ ml) mixed with either PMSF (1 mM final concentration)

Incubation

temperature

FIG. 2. The effect of incubation temperatures on the enzymatic digestion of gelation coating on X-ray films. Dilutions of trypsin and chymotrypsin (lo-O.3 pg/ml) were applied onto a X-ray film and incubated at different temperatures (given in C) for 1 h. Bars represent the concentrations of enzymes which produced clear zones on the X-ray film.

22

CHEUNG,

YING,

AND

FISCHETTI TABLE Compatibility 2 to Gelatin Substrate

trypsin and chymotrypsin (in azocoll buffer), reactions of these enzymes with the gelatin substrate were allowed to occur in buffer as described in the Worthington Enzyme Manual (4). As shown in Table 1, the level of sensitivity was comparable between two systems. With the exception of pepsin, the sensitivity of the gelatin substrate to these enzymes is comparable to that of azo~011. In contrast, the acid proteinase pepsin, like azocoll, was not very sensitive to detection by the gelatin substrate. To ascertain whether salts, detergents, or chaotropic agents interfere with digestion of the substrate by trypsin or chymotrypsin, enzymes (final concentration at 5-l pug/ml in azocoll buffer) were added to azocoll buffer containing the respective reagents (final volume at 50 ~1). The activity of the enzyme in these reagents was compared with that of enzyme in buffer alone. Results demonstrate that with the exception of SDS at O.l%, which inactivated the enzyme, the activities of these enzymes in these reagents were identical with that of the buffer control. This finding with 0.1% SDS was also confirmed by concomitant assays for proteinases with azocoll. As displayed in Table 2, most of the commonly used laboratory reagents are compatible with this assay. In contrast, buffers which contained either 3 M guanidine or 1 M potassium thiocyanate produced clear zones on the film in the absence of enzymes and are therefore incompatible with the assay system. To evaluate the usefulness of this assay in an experimental system, we took advantage of the fact that B. subtilis secretes a multiplicity of extracellular proteinases. Using strain RN5789, a derivative of B. subtilis 168 (S. J. Projan, personal communication), we examined the effect of extracellular proteinases on the gelatin substrate. Our data revealed that the assay produced well-defined endpoints (i.e., clear zones) up to a 1:16

of Various Reagents on X-ray Films Reagents

Compatibility Yes Yes Yes Yes Yes No No

3M 5 M 2% 2% 5M 3 M 1M

Tris NaCl Triton X-100 Zwittergent urea guanidine potassium thiocyanate

Note. Dilutions of trypsin and chymotrypsin in azocoll buffer in the presence of various reagents were applied onto the film and incubated as described. In addition, various reagents in buffer without enzymes were also included. To ascertain if enzyme inhibition occurs, concomitant assays for proteinase detection with azocoll were also conducted if necessary. As expected, SDS at a concentration of 0.1% inhibits trypsin and chymotrypsin in both gelatin and azocoll assays.

dilution of the culture supernatant. This level of proteinase activity in the supernatant is equivalent to that of trypsin at a concentration of 10.08 pg/ml as determined by the production of clear zones from known enzyme dilutions. This result correlated closely with that of the extracellular proteinases of strain RN5789 on azocoll when trypsin was used as a positive standard.
DISCUSSION

TABLE

1 of Gelatin to Azocoll measurable hvdml) Substrate

Comparison

of the on X-ray

Sensitivity
Films Lowest

concentration

Gelatin substrate on X-ray films Trypsin Chymotrypsin Papain Collagenase Pepsin (pH 5.5) Subtilisin BPN 0.63 10.0 5.0 5.0 500 1.0

Azocoll 0.63 15.0 10.0 5.0 ND 1.0

Note. Dilutions of various enzymes in appropriate buffers (see text) were applied onto the film and incubated for 1 h at 42C. The lowest measurable concentration at which a clear zone was produced was reported for each enzyme. ND, not detected.

The use of gelatin, a denatured form of collagen, as a substrate to detect proteolytic activities is not new. The application of gelatin on chromatographic paper (5), microscopic slides (6), polyacrylamide gels (7), and ELISA wells (8) as a proteolytic substrate has been reported. In distinction to previous studies, our assay took advantage of a prefabricated system whereby the preexisting gelatin substrate on X-Omat XAR-5 film was used as a substrate to detect the presence of a variety of proteinases. The presence of a well-defined endpoint (i.e., a clear zone) together with the simplicity of the assay allows this procedure to be performed by laboratory personnel with minimal training. Based on our experience with this assay, there appeared to be little variation in results between different lots of film. In addition to the benefit of a rapid and reproducible assay (results available in 1 h), this method also has a very low cost overhead. In particular, a box of 50 films (size 20 X 25 cm) may accommodate up to 250 assays (40 samples each). In comparison to azocoll, the cost is 306 vs $8 per assay based on the current acquisition costs at our institution. Another useful feature of this assav svstem is the flexibility of the method in regard to proteolytic activities from a variety of enzymes including serine proteinases, metalloproteinase, thiol proteinase and acid proteinase. Notably, the sensitivity of this assay is comparable to
-

UNPROCESSED

X-RAY

FILM

METHOD

TO

DETECT

PROTEINASES

23
is an investigator of the American Affiliate. We thank Steve J. Projan

that of azocoll, a widely used substrate for the assay of proteinase (1). The similarity in sensitivity between these two substrates may have stemmed from the fact that they are both denatured forms of collagen. With the exception of guanidine and potassium thiocyanate, this laboratory procedure is compatible with most laboratory reagents under a variety of pH and molarity conditions, Although it is rather surprising to find that 3 M guanidine but not 5 M urea produced clear zones on the film in the absence of enzymes, the differential effect on the detachment of gelatin from the plastic support of the X-ray film may have been due to the more chaotropit characteristics of guanidine in comparison to urea. Finally, this technique does not require expensive equipment (e.g., spectrophotometer) as with casein (9) and azocoll (1).
ACKNOWLEDGMENTS This work York Heart was supported in part by Grants-In-Aid Association and the American Heart from the New Association and

NIH Grant AI11822. A. L. Cheung Heart Association, New York City for providing B. subtilis RN5789.

REFERENCES
1. Chavira, Biochem. 2. Studabaker, R. Jr., Burnett, T. J., and Hageman, J. Chromatogr. Szulmajster, (1972) Chem. 185, J. H. (1984) 497503. Eur. J. BioAd.

136,446-450.
J. F. (1979) E., and Manual Biol. J. (1979) Worthington 369,

3. Kerjan, P., Keryer, them. 98, 353-362. 4. Worthington Enzyme Freehold, NJ. 5. Schlage, W. K. (1988)

Biochemical, 357-363.

Hoppe-Seykr

6. Welker, B., Bernstein, G. S., Diedrich, K., Nakamura, R. M., and Krebs, D. (1988) Hum. Reprod. 3, 75-80. 7. Piening, C., and Riederer Henderson, M. A. (1989) J. Orthop. Res. 7, 228-234. 8. Rucklidge, G. J., and Milne, G. (1990) Anal. &o&m.

185, 265-

269.
9. Reimerdes, H. E., and Klostermeyer, Enzymology (Lorand, L., Ed.), Vol. Press, San Diego, CA. H. (1976) in Methods in 45, pp. 26-28, Academic