Detection of elastase activity with a zymogram method after isoelectric focusing in polyacrylamide gel

ANALYTICAL

BIOCHEMISTRY

Detection

14&472-4-?7

(1984)

of Elastase Activity with a Zymogram Isoelectric Focusing in Polyacrylamide CONCETTA

Method after Gel

GARDI AND GIUSEPPE LUNGARELLA

Istituto di Patologia Generale dell’Vniversitci di Siena. Via del Luterino 8, 53100 Siena, Italy Received December 2, 1983 A zymogram method for detecting elastase activity following isoelectric focusing in polyacrylamide gel is described. After enzyme activity has been visualized, the gel itself is available for protein staining and for analysis in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in second dimension. The zymogram method is suitable for detecting microgram amounts of elastase and has one step only. It can be used with the purified enzyme as well as with crude extracts of tissue containing elastases showing activity toward succinyl-(Ala)p-pnitroanilide. By this method a major component of elastase in both porcine and rat pancreas was detected. In addition, two forms of elastase with isoelectric points of 8.2 and 8.8, respectively, were identified in rat leukocyte extracts. KEY WORDS: elastase(s); isoelectric focusing; zymogram technique.

Elastases are a heterogeneous group of enzymes falling into all four classes of proteinases (aspartic, cysteine, serine, and metallo) and are able to solubilize fibrous elastin (1). In mammals elastinolytic proteases are present in a wide variety of cells (polymorphonuclear leukocytes, platelets, etc.) and tissues (pancreatic, aortic, skin, etc.) in which they play important roles in a diverse range of physiological processes characterized by limited proteolysis. The increasing interest in these enzymes is due to the fact that elastases have been implicated in several pathological processes characterized by uncontrolled destruction of structural proteins (2). In particular certain elastases (such as pancreatic or leukocyte), belonging to the serine proteinase group, are believed to be mediators of diseases like atherosclerosis ( 1), arteritis (3), pancreatitis-induced lung damage (4), inflammatory joint disease (l), and lung emphysema (5). Supportive evidence for such a view derives from several experimental findings which suggest that the release of elastolytic enzymes in extracellular fluids results in a variety of pathological conditions (6,7) and that the elastolytic power 0003-2697184 $3.00 Copyright &I 1984 by Academic Press, Inc. All rights of reproduction in any form resewed.

detected in such fluid is directly related to the degree of the observed lesions (8). Therefore the availability of a simple method for the demonstration and characterization, within complex mixtures, of proteins with the enzyme activity of elastases of the serine proteinase group should be of great interest to laboratories involved in the identification of the enzyme activities implicated in the pathological conditions mentioned above. This paper describes a rapid and simple contact print method for direct visualization in polyacrylamide gel of protein bands showing hydrolytic activity toward succinyl-(Ala)spnitroanilide (the most commonly used substrate for porcine pancreatic elastase (9)). Such bands are detected, after isoelectric focusing, in unsliced and unwashed gels by superimposition of agar gel plates containing the above-mentioned substrate. The technique described detects enzyme activity at levels down into the nanogram range. If required, the gel itself may also be stained for protein after the activity staining procedure. No supplementary destaining is necessary since the bands corresponding to 472

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enzyme activity disappear during the current fixing procedures employing acetic acid or trichloroacetic acid. MATERIALS

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All the preparations were extensively dialyzed against HZ0 at +4”C to remove salts which seemed to interfere with isoelectric focusing.

AND METHODS

Materials Acrylamide,N,N’-methylenebisacrylamide, ammonium persulfate, tetramethylenediamine (TEMED),’ sodium dodecyl sulfate (SDS), Coomassie blue R, and molecular weight markers were purchased from Bio-Rad Laboratories, Richmond, California. Ampholytes were from LKB-Produkter AB, Bromma, Sweden. Porcine pancreatic elastase (type III), agar (type IV), and N-succinyl-L-alanyl-Lalanyl-L-alanyl-pnitroanilide (SAPNA) were from Sigma Chemical Company, St. Louis, Missouri. Pancreatin was from Merck, Darmstadt, West Germany. Other reagents were of the highest quality available and were used without further purification. Solutions were prepared with deionized water of maximum conductivity of l-2 rmho . cmv3.

Methods

Isoelectric focusing. Electrophoresis was conducted using an LKB Multiphor unit and associated gel-casting equipment. The procedure for casting thin layers of polyacrylamide was similar to that described in the LKB Application Note 250. Gel slabs (245 X 110 X 0.5 mm) were prepared as follows: 8.0 ml distilled water, 3.0 ml 50% (v/v) glycerol, 3.0 ml 20% (w/v) acrylamide plus 0.8% (w/v) bisacrylamide, 5.0 ~1 tetramethylenediamine, and 0.95 ml 40% (w/v) ampholytes of appropriate pH range (corresponding to an ampholyte concentration in the gel of about 2.5%) were introduced into a 25-ml Erlenmeyer flask. The mixture was degassed for 4 min under vacuum; 50.0 ~1 10% (w/v) ammonium persulfate was then added and the mixture was degassed again for Samples 1 min under vacuum. The mixture was imCrude extract of rat pancreas was prepared mediately pipetted into the gel mold and layin a I:5 (w/v) volume of 0.2 M NaHCOsered carefully with H20. Polymerization was NaOH buffer, pH 8.4, by centrifugation of complete in 20-30 min. Samples (5-30 ~1) homogenized pancreas for 30 min at 5OOOg. were applied as streaks or on small pieces of The procedure was carried out at 0-4’C. The Whatman 3MM paper (0.3 X 0.5 or 0.5 supematant was stored at -20°C and was X 1.0 cm), placed on top of the gel, and rethawed just before use. moved after 1 h of electrophoresis. Electrode The procedure for preparation of partially strips were soaked in the proper catholyte ( 1.O purified porcine elastase was essentially the M NaOH) or anolyte solution (1 .O M H3P04 same as that described by Lewis et al. (10) for for gel with pH range 3.5-9.5; 2.0% amphothe preparation of the “euglobin precipitate” lytes, pH 6.0-8.0, for gel with pH range from commercial preparations of acetone 7.0-10.0). powder of porcine pancreas. Samples were then focused at 25 W constant The preparation of rat granulocyte lyso- power (voltage and current at maximum) for somal fraction was performed according to 1.5-3 h at 4°C. The pH gradient in the slab the procedure described for rabbit granular gel was measured, after isoelectric focusing, (lysosomal) fraction (8). by cutting the gel into pieces (OS cm) which were homogenized with l-ml aliquots of ’ Abbreviations used: TEMED, tetramethylenediamine; deionized water. The gel pieces were obtained SDS, sodium dcdecyl sulfate; SAPNA, N-succinyl+akanylfrom a side strip of the gel that was cut at t-alanyl+alanyl-pnitroanilide; PPE, porcine pancreatic equal distances (0.5 cm) after isoelectric foelastase; SDS-PAGE, sodium dodecyl sulfate-polyacrylcusing. The gradient curve was constructed amide gel electrophoresis.

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by plotting pH values versus distances from the cathode. Agar substrate slab. The stock solution of SAPNA (125 mM) was prepared in N-methylpyrrolidone by heating at 60°C for 10 min and stirring. This solution was stable for at least 1 year if stored at 4“C in a dark bottle. Its concentration was checked by reading absorptivity at 3 15 nm (E = 14,600 mol-’ cm-‘) after dilution to 1.5 mM with 0.2 M Tris-HCl, pH 8.0. This substrate solution was prepared just before use. Agar substrate slabs were prepared as follows: agar (1.5% w/v) was suspended in 0.2 M Tris-HCl buffer, pH 8.0, heated to 100°C for 10 min to liquefy, and cooled at 60°C in a water bath. The substrate solution was then added to the agar mixture at a concentration of 10 &ml and stirred for 3 min at 6O’C. The agar solution containing the substrate was then carefully poured onto preheated (45°C) glass plates (245 X 110 X 0.75 mm) and allowed to solidify at room temperature for some minutes. When the gel had set, the glass plates were carefully removed and replaced by plastic sheets. The gel could be immediately used in the localization procedure or stored in an airtight bag at 4’C for several months. When used, the substrate slab was precut to size that would cover only the area where enzyme activity was expected. Detection of elastase activity following isoelectric focusing. After completion of the electrophoretic run, the agar substrate slab was gently applied on top of the electrophoretogram, ensuring that no air bubbles were trapped between the two slabs. The plastic sheet of the agar substrate slab was left on top to prevent drying. After an incubation period (varying from a few seconds to minutes) at room temperature or at 37°C in a humified box, the progress of the enzyme reaction could be assessed from time to time by viewing the yellow bands of reaction product against a clear background. Bands of yellow reaction product appeared almost immediately if in the presence of a few (0.5- 1.O) micrograms of elastase.

When the reaction had proceeded adequately, the gels could be photographed immediately following development of sufficient color since the yellow bands increased progressively in intensity and the color spread over the agar gel in the long run. Protein staining. After the activity-staining procedures, the isoelectric-focusing gel was fixed for 30 min in an aqueous solution containing 3.5% sulfosalicylic acid and 11% trichloroacetic acid to remove ampholytes, washed in destaining solution (25% methanol, 8% acetic acid) for 10 min, and then stained in the same solution containing 0.1% Coomassie brilliant blue R for 15 min at 55°C. Destaining was carried out by washing in several changes of destaining solution (containing 0.005% Coomassie brilliant blue R to prevent total destaining) until a clear background was obtained. Molecular-weight determination of the bands showing elastase activity. The molecular weight of the bands showing elastase activity was determined with the aid of electrophoresis of protein-SDS complexes in a discontinuous buffer system ( 11). In particular, after electrofocusing the slab gels were fixed, stained with Coomassie blue R, and destained as reported above. The individual bands were then cut and excised using a sharp scalpel and equilibrated prior to electrophoresis in the second dimension. For this end, the strips were immersed in distilled water for 10 min at room temperature to remove acetic acid and methanol and incubated in 1 ml of equilibration buffer (0.625 M Tris-HCl, 2.3% SDS (w/v), 5% 2-mercaptoethanol, 10% glycerol, pH 6.5) for 2 min at room temperature. After incubation the excess equilibration buffer was removed by placing the gel strips on a paper tissue for 5 min. The gel strips were then loaded onto sample wells preformed in the stacking gel polymerized on the top of an 8 or a 12% polyacrylamide resolving gel. Slab gels (160 X 140 X 0.75 mm) were preformed in a cooled vertical apparatus (Protean Cell, Bio-Rad) and run at a constant current (15 mA for 0.75-mm slab gel) until the dye -

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front (bromphenol blue 0.005%) had moved 9.5 cm into the separation gel. Gels were then stained with Coomassie brilliant blue R-250 (12).

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RESULTS

The results obtained after isoelectric focusing of porcine pancreatic elastase (PPE, lane l), of the “euglobin precipitate” from porcine pancreas (lane 2), and of the crude extracts of rat pancreas (lane 3) are shown in Fig. 1. Each gel lane was split longitudinally; one half was used for protein staining (Coomassie blue, Fig. 1A) and the other one was used for activity staining (Fig. 1B). The correspondence between the protein bands in Coomassie-stained gels and the yellow bands revealing the enzyme activity was established by measuring the distances between the bands and the origin of the acrylamide gel. Only a single component of “euglobin precipitate” (lane A2) and crude pancreatic extract (lane A3) showed enzyme activity (lanes B2, B3). The band seen in the gel lane of “euglobin precipitate” by activity staining (lane B2) is located in the same pH region (8.5) in which PPE (lane Bl) is focused. The band corresponding to enzyme activity from B

A c-3

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12

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Pi

FIG. 1. Comparison of protein and activity stains of elastase atier isoelectric focusing. (A) Isoelectric focusing of porcine pancreatic elastase (lane 1), “euglobin precip itate” from porcine pancreas (lane 2), and crude extract of rat pancreas (lane 3). The gel was stained with Coomassie blue. (B) The enzyme activity shown as dark bands was revealed in agar by the zymogram method. Lanes 1, 2, and 3 represent the enzyme activity detected in agar from the other half of the gel (lanes 1, 2, and 3 of (A)).

21 kFIG. 2. Analytical SDS-PAGE of protein bands associated with elastase activity. Lane a, porcine pancreatic elastase; lane b, rat pancreatic elastase; lane c, “euglobin precipitate” from porcine pancreas; lanes d and e, the two nonactive cathodic bands with isoelectric points of 8.4 (lane d) and 8.15 (lane e) isolated in isoelectric focusing from crude extract of rat pancreas. The slab gel, containing 8% polyacrylamide, was stained with Coomassie brilliant blue as reported under Materials and Methods.

crude extracts of pancreas (lane B3) showed an isoelectric point (8.75) different from that shown by PPE. Analytical SDS-PAGE of protein bands associated with enzyme activity (Fig. 2) revealed the same mobility for PPE (lane a) and the major band observed in the “euglobin precipitate” (lane c). These bands showed a different mobility than that showed by the active band from crude pancreatic extracts (lane b). The latter showed a “relative mobility” corresponding to AI, 23,600, whereas in PPE and the major band of “euglobin precipitate” the corresponding figure was 24,700. The SDSPAGE bands present in lanes (d) and (e) are illustrated in Fig. 2. The results obtained with rat granulocyte lysosomal extracts are shown in Fig. 3, where protein (a) and enzyme stains (b) are traced. The appearance of two active components in these extracts (b) suggests the occurrence, in rat leukocytes, of two active forms of elastase, which were focused in the region of 8.8 and 8.2, respectively. The band focused at pH 8.2 showed a slower reactivity in the activitystaining test. The occurrence of two components in leukocyte elastase has also been observed in human leukocytes (13). The analytical SDS-PAGE of protein bands

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a I

(3 b FIG. 3. Comparison of protein and activity stains of elastase after isoelectric focusing. (a) Isoelectric focusing of rat granulocyte lysosomal fraction. The gel was stained with Coomassie blue. (b) Enzyme activity revealed in agar by the zymogram method on the other half of the gel lane of (a).

from rat leukocyte extracts associated with enzyme activity is reported in Fig. 4. The most cathodally focusing component seen in rat leukocyte extracts (lane a) showed a “relative mobility” corresponding to A& 26,000. The band with slower activity staining and with an isoelectric point of 8.2 gives rise in SDSPAGE to two fractions (lane c) with a “relative mobility” corresponding to -26,000 and 24,000, respectively. The SDS-PAGE bands present in lanes b, d, e, f, and g are illustrated in the legend to Fig. 4. The specificity of the zymogram method described here was checked on polyacrylamide gel in which various proteases such as trypsin, chymotrypsin, and collagenase had been focused. No active bands developed in the presence of these enzymes. The minimum amount of PPE detectable within 2 h by the method described here is 0.1 pm. However, the method can be improved for detecting smaller amounts of elastase down in the nanogram range if the time of incubation is increased and the thickness of the overlayer agar substrate slab is decreased. DISCUSSION

The technique described here provides a good tool for the identification in biological materials of individual forms of elastase belonging to the serine proteinase group. In the present study, application of the procedure to the crude extracts of rat pancreas and leu-

kocytes has revealed previously uncharacterized forms of elastase. Although esterolytic activity of elastase or elastase-like enzymes has been visualized in polyacrylamide gels by using an azo dye to stain the hydrolyzed substrate (14,15), it has been demonstrated that, in addition to elastase, trypsin and chymotrypsin also show activity toward this synthetic substrate ( 16). Recently other authors reported a zymogram technique for detecting elastase activity in polyacrylamide gels by placing the latter directly on agarose containing natural solid elastin (17) or elastin labeled with a chromophore, i.e., elastin-orcein (17) or elastinCongo red (17,18). Although in our hands these procedures had proven to be highly specific for detecting elastase activity in PAGE, their versatility is lacking in the area of SDSPAGE and electrofocusing (17), which, as is known, are more powerful than other electrophoresis procedures as analytical tools for protein separation. In addition, the general applicability of these techniques appears still

a

bed

e

f

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FIG. 4. Analytical SDS-PAGE of protein bands associated with elastase activity. Lanes a and c, the active bands from rat leukocyte extract, focused at pH 8.8 and 8.2, respectively. Lane b, nonactive band from rat leukocyte extract with isoelectric point of 8.35; lane d, molecular-weight markers (from the top: carbonic anhydrase 31K, soybean trypsin inhibitor 2 lSK, and lysozyme 14.4K). Lane e, whole lysosomal fraction from rat leukocyte extract. Lanes f and g, porcine pancreatic elastases from purified commercial preparation and from “euglobin precipitate,” respectively. The slab gel, containing 12% polyacrylamide, was stained with Coomassie brilliant blue.

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more limited by the relatively poor sensitivity to the detection of a small amount of enzyme within an acceptable time. The considerable time necessary for developing the zymogram in these procedures enhances the potential risk that enzyme zones focused closely mix with each other because of their diffusion. The same risk is enhanced, furthermore, by the high protein concentration necessary in these systems for developing the zymogram. The elastin-orcein method, in fact, which was found to give better results than either elastin-Congo red or the natural elastin method has a detection limit of 0.5 pg of PPE after an incubation at 37°C overnight (17). The same amount of PPE can be detected with our proposed zymogram technique within few minutes from the start of the incubation. Although the procedure described has proven to be very successful in detecting individual forms of elastases belonging to the serine proteinase group, it is worthwhile to emphasize that such a technique cannot be assumed to be a general means of assay for elastases belonging to the different protemase classes. It is well known, in fact, that certain elastases (particularly those belonging to the metallo proteinase class) are not able to cleave substrates derived from (Ala)3 (2,19). Also, these elastases may reveal an apparent loss of enzyme activity owing to the chelation properties of ampholytes. The technique described in the present report has given reproducible results to the characterization of individual forms of elastases, which show hydrolytic activity toward SAPNA, within complex mixtures containing one or several proteases. Moreover, it can be suitably used to characterize elastase activity under those particular in viva conditions in which the combination of nonelastolytic enzymes (i.e., trypsin and chymotrypsin) results in the acquirement of the ability to degrade elastin (2,19). Although some elastase-like esterases cleaving SAPNA but not elastin may occur in certain biological fluids (i.e., human synovial fluids) and increase the “total” elastase activity biochemically determined, the present method

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offers a tool for distinguishing these elastaselike enzymes from true elastases by means of their isoelectric points or molecular weights. The reported technique, which greatly facilitates the isolation and characterization of elastases belonging to the serine proteinase group, may lead to further insight into the pathogenetic role of these enzymes in various diseases. ACKNOWLEDGMENT The authors are grateful to M. Comporti, Chairman of the Institute of General Pathology, University of Siena, for his help in the preparation of the manuscript and for his useful advice during the study.

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