Detection of chitinase activity after polyacrylamide gel electrophoresis

ANALYTICAL

BIOCHEMISTRY

178,362-366

(1989)

Detection of Chitinase Activity after Polyacrylamide Gel Electrophoresis Jean Trudel and Alain Asselin Dipartement de Phytologie, Faculte’desSciencesde 1’Agriculture et de I’Alimentation, Universite’Lava& Qu&bec,CanadaGl K 7P4 Received

November

29,1988

and Serratia marcescens chitinases and purified wheat germ WIA and hen egg white lysozymes were subjected to polyacrylamide gel electrophoresis under native conditions at pH 4.3. After electrophoresis, an overlay gel containing 0.01% (W/V) glycol chitin as substrate was incubated in contact with the separation gel. Lytic zones were revealed by uv illumination with a transilluminator after staining for 5 min with 0.01% (W/V) Calcofluor white M2R. As low as 500 ng of purified hen egg lysozyme could be detected after 1 h incubation at 37°C. One band was observed with WI A lysozyme and several bands with the commercial microbial chitinases. The same system was also used with native polyacrylamide gel electrophoresis at pH 8.9. Several bands were detected with the microbial chitinases. The same enzymes were also subjected to denaturing polyacrylamide gel electrophoresis in gradient gels containing 0.01% (W/ V) glycol chitin. After electrophoresis, enzymes were renatured in buffered 1% (V/V) purified Triton X- 100. Lytic zones were revealed by uv after staining with Calcofluor white M2R as for native gels. The molecular weights of chitinolytic enzymes could thus be directly estimated. In denaturing gels, as low as 10 ng of purified hen egg white lysozyme could be detected after 2 h incubation at 37°C. Estimated molecular weights of St. griseus and Se. marcescens were between 24,000 and 72,000 and between 40,500 and 73,000, respectively. Some microbial chitinases were only resistant to denaturation with sodium dodecyl sulfate while others were resistant to sodium dodecyl sulfate and &mercaptoethanol. 0 1989 Academic Press, Inc. Commercial

Streptomyces

griseus

Chitin, a linear polymer of p(1 + 4)-N-acetylglucosamine, is often considered to be the second most abundant polysaccharide in nature (1). It is present in diatoms (2), yeasts, fungi (3,4), protozoans (5), arachnids, insects, crustaceans, nematodes, and other invertebrates (6-8). It is also present in some tunicates (5). Chitinase (EC 3.2.1.14) activity has been found with 362

bacteria and Streptomycetes (g-12), fungi (13-16), plants (17-20), invertebrates (21-23), and vertebrates (24). Chitinase activities play a role in the molting process of insects and the digestion of chitinous food and could also serve as potential defense enzymes against chitin-bearing pathogens (20,25). Chitinolysis is assayed by viscosimetry, nephelometry, or endproduct analysis (26). Glycol chitin, a soluble modified form of chitin, has recently become a very useful substrate for study of several endochitinases and lysozymes like hen egg white lysozyme (27). Despite the importance of chitin metabolism in nature, there is still no method available for detection of chitinase activity after polyacrylamide gel electrophoresis under native or denaturing conditions. We report the detection of several chitinolytic enzymes representing microbial, plant, or animal chitinase activities after native or denaturing PAGE.’ These techniques are based on the affinity of Calcofluor white M2R for chitin (28). MATERIALS

AND

METHODS

Chemicals and enzymes. All chemicals for electrophoresis, analytical grade mixed bed resin AG 501-X8 (20-50 mesh), protein molecular weight markers, and protein assay dye reagent concentrate were from BioRad (Canada). Grade I hen egg white lysozyme (HEWL), turkey hen egg white lysozyme (TEWL), Serratia marcescens and Streptomyces griseus chitinases, bovine milk a-lactalbumin, glycol chitosan, Calcofluor white M2R (C.I. 40622), and Triton X-100 were from Sigma Chemical Co. (St. Louis, MO). Wheat germ WlA lysozyme was purified according to Audy et al. (29). PAGE under native conditions. Polyacrylamide gel electrophoresis under native conditions was performed at pH 4.3 according to Reisfeld et al. (30) and at pH 8.9 according to Davis (31) using 15% (W/V) polyacryl1 Abbreviations used: PAGE, polyacrylamide HEWL, hen egg white lysozyme; TEWL, turkey zyme; SDS, sodium dodecyl sulfate. Copyright 0 1989 All rights of reproduction

gel electrophoresis; hen egg white lyso0003-2697/89 $3.00 by Academic Press, Inc. in any form reserved.

CHITINASE WlA

Sg

ACTIVITY

AFTER

HEWL

Sm

5

1

0.5

0.1

0.05

FIG. 1. Chitinase activity after electrophoresis in a 15% (W/V) polyacrylamide gel at pH 4.3. Purified wheat germ WlA lysozyme (WlA) (0.25 pg), commercial Streptomyces griseus (Sg) (0.1 unit) and Serratiu marcescens (Sm) (0.15 unit) chitinases, and purified hen egg white lysozyme (HEWL) (5,1,0.5,0.1, and 0.05 pg) were subjected to nondenaturing PAGE according to Reisfeld et al. (30). An overlay gel containing 0.01% (W/V) glycol chitin was incubated on top of the separating gel. After staining with Calcofluor white M2R, bands with lytic activity appeared as dark zones after uv illumination. One unit of chitinase is defined as the release of 1 mg of N-acetyl-D-glucosamine from chitin in 48 h at pH 6.0 at 25°C.

amide resolving gels (50 X 140 X 0.75 mm) and 5% (W/ V) polyacrylamide stacking gels (15 X 140 X 0.75 mm). Samples contained 15% (W/V) sucrose and 0.01% (W/ V) bromphenol blue (Davis system) or 0.01% (W/V) methylene blue (Reisfeld system). Electrophoresis was at room temperature for 3 h at 30 mA (Reisfeld system) or for 65 min at 20 mA (Davis system). SDS-PAGE. SDS-PAGE was performed in lo-15% (W/V) polyacrylamide gradient gels (50 X 140 X 0.75 mm) containing 0.01% (W/V) glycol chitin and 0.1% (W/V) SDS. Stacking gels (15 X 140 X 0.75 mm) were made of 5% (W/V) polyacrylamide containing 0.1% (W/ V) SDS. Samples were boiled for 5 min with 15% (W/V) sucrose and 2.5% (W/V) SDS in 125 mM Tris-HCl (pH 6.7) withor without 2% (V/V) P-mercaptoethanol. Bromphenol blue (O.Ol%), (W/V) was the tracking dye and gels were run as for the Davis system. Protein molecular weight markers were lysozyme (14,400), soybean trypsin inhibitor (21,500), carbonic anhydrase (31,000), ovalbumin (45,000)) bovine serum albumin (66,200)) and phosphorylase b (92,500). Detection of chitinase activity after PAGE under native conditions. Gels were incubated in 150 mM sodium acetate buffer (200 ml/gel) at pH 5.0 for 5 min. They were then put on a clean glass plate (80 X 170 mm) and covered with a 7.5% (W/V) polyacrylamide overlay gel (65 X 155 X 0.75 mm) containing 0.01% (W/V) glycol chitin in 100 mM sodium acetate buffer (pH 5.0). The liquid between the gels and the glass plate was eliminated by gently sliding a 12 X 75-mm test tube over the surface of the overlay gel. Gels were incubated at 37°C for 1 h in a plastic container under moist conditions. Following incubation, plastic spacers (Bio-Rad) (3 mm in thickness) were sealed with 1% (W/V) agarose on the overlay gel

GEL

363

ELECTROPHORESIS

around the area of the separating gel standing below the overlay gel. Care must be taken to seal not only the spacers to the gel but also the overlay gel to the glass plate. The area between the spacers was filled (about 20 ml) with freshly prepared 0.01% (W/V) Calcofluor white M2R in 500 mM Tris-HCl (pH 8.9) (Calcofluor white is light sensitive in dilute solution). After 5 min, the brightener solution was removed and the gels were incubated for about 1 h at room temperature in distilled water. Lytic zones were visualized by placing the gels on a Chromato-Vue C-62 transilluminator (UV Products). Lytic zones in gels were photographed with Polaroid type 55 film with uv-haze and 02 orange filters. The exposure time varied from 1 to 5 min with a 127 mm lens at f 4.7. Overlay gels must be photographed in the hours following the staining procedure. Detection of chitinase activity after SDS-PAGE. After electrophoresis, gels were incubated for 2 h at 37°C with reciprocal shaking in 100 mM sodium acetate buffer (pH 5.0) containing 1% (V/V) Triton X-100 purified through a mixed-bed resin deionizing column (AG 501X8) (32). Gels were then stained and destained and lytic zones were photographed as for native gels. Glycol chitin synthesis. Glycol chitin was obtained by acetylation of glycol chitosan by a modification of the method of Molano et al. (18). Five grams of glycol chitosan was dissolved in 100 ml of 10% acetic acid by grinding in a mortar. The viscous solution was allowed to stand overnight at 22°C. Methanol (450 ml) was slowly added and the solution was vacuum filtered through a Whatman No. 4 filter paper. The filtrate was transferred into a beaker and 7.5 ml of acetic anhydride was added with magnetic stirring. The resulting gel was allowed to stand for 30 min at room temperature and then cut into small pieces. The liquid extruding from the gel pieces was discarded. Gel pieces were transferred to a Waring Blendor, covered with methanol, and homogenized for 4 min at top speed. This suspension was centrifuged at

-A--

B --

L

Sg

Sm

L

Sg

CSm

L

Sg

Sm

FIG. 2. Chitinase activity after electrophoresis in a 15% (W/V) polyacrylamide gel at pH 8.9. Commercial Streptomyces griseus (Sg) (0.1 unit) and Serrutia marcescens (Sm) (0.15 unit) chitinases were subjected to nondenaturing PAGE according to Davis (31). Purified (20 pg) bovine milk ol-lactalbumin (L) was included as a control protein with no chitinolytic activity. Detection and units of chitinase activity (A) were as in Fig. 1. Gel sections were stained with Coomassie blue R-250 (B) or silver nitrate (C).

364

TRUDEL

AND ASSELIN

27,000g for 15 min at 4°C. The gelatinous pellet was resuspended in about 1 vol of methanol, homogenized, and centrifuged as in the preceding step. The pellet was resuspended in distilled water (500 ml) containing 0.02% (W/V) sodium azide and homogenized for 4 min. This was the final 1% (W/V) stock solution of glycol chitin. Protein determination and staining of polyacrylamide gels. Protein concentration was determined by the BioRad protein microassay procedure using purified hen egg white lysozyme as reference protein. In some cases, gels were stained with Coomassie blue R-250 or aqueous silver nitrate as previously described (29). RESULTS

AND

DISCUSSION

We recently developed techniques for detection of lysozyme activity after native (29) or denaturing (32,33) polyacrylamide gel electrophoresis by incorporating the substrate of lysozymes into gels. The same approach was tested for the detection of chitinase activity in gels. Experiments with various substrates (colloidal chitin, chitin azure) indicated that only soluble glycol chitin could be uniformly incorporated into gels. Glycol chitin is a well-defined substrate for various chitinolytic enzymes (27). Enzymes with chitinase activity were then chosen to represent microbial (commercial St. griseus and Se. marcescens chitinases), plant (wheat germ WlA lysozyme which also acts as a chitinase (29)), and animal (HEWL and TEWL lysozymes with chitinase activity) proteins. Enzymes were then separated in native and denaturing PAGE as previously described for various lysozymes (29,32,33). After digestion of the substrate, intact glycol chitin was stained with a fluorescent brightener (Calcofluor white M2R) because of its high affinity for chitin (28). Lysis zones were then visualized by uv illumination as nonfluorescent dark bands in contrast to the fluorescent intact glycol chitin. Chitinase Activity

in Native Gels

Two native PAGE systems were used. The Reisfeld system (30) is run at pH 4.3 and is designed to separate basic proteins. The Davis system works at alkaline pH (8.9) and is used for neutral or acidic proteins. In both systems, detection of chitinase activity was based on the use of an overlay gel containing 0.01% (W/V) glycol chitin as substrate. After incubation, the overlay gel was stained with Calcofluor white M2R (0.01% W/V) while still on top of the separating gel. After long-wave uv illumination, lysis zones can be marked by piercing small holes (tip of a Pasteur pipet) through both gels. The separating gel can then be stained (Coomassie blue, silver) to identify protein bands corresponding to lysis zones in the overlay gel. With this technique, a very precise identification of protein bands with chitinase activity is feasible. This is not the case when the substrate is directly incorporated into the native gels because of the presence of smears instead of well-defined bands.

kDa

T

H

WlA

-Sg--Sm-

-

+

-

+

FIG. 3.

Chitinase activity after SDS-PAGE in a gel containing 0.01% (W/V) glycol chitin as substrate. Purified turkey hen egg white lysozyme (T) (50 ng), hen egg white lysozyme (H) (50 ng), wheat germ WlA lysozyme (WlA) (2.5 fig), and commercial Streptomyces griseus (Sg) and Serratia marcescens (Sm) chitinases were separated in a lo15% (W/V) gradient polyacrylamide gel containing 0.1% (W/V) SDS. Samples were denatured with 2.5% (W/V) SDS (-) or 2.5% (W/V) SDS plus 2% (V/V) @-mercaptoethanol (+). S. grkeus samples contained 0.005 unit (-) or 0.1 unit (+) and both S. marcescens samples contained 0.1 unit of chitinase activity. Renaturation of enzymes was in buffered Triton X-100 at 1% (V/V). Detection and units of chitinase activity were as in Fig. 1. Numbers on the left refer to molecular mass markers (kilodaltons).

Chitinase Activity

in the Reisfeld System

Commercial St. griseus and Se. marcescenschitinases, wheat germ WlA lysozyme, and HEWL were separated in a 15% (W/V) polyacrylamide gel in the Reisfeld system. As expected, HEWL migrated as a highly charged basic protein near the bottom of the gel (29) (Fig. 1). Wheat germ WlA lysozyme had a relative electrophoretie mobility value of less than half that of HEWL (29) (Fig. 1). Four and two bands of low electrophoretic mobility were observed with St. griseus and Se. marcescens extracts, respectively (Fig. 1). This indicated that these commercial extracts contained several electrophoretic forms of chitinases. Sensitivity of Detection in the Reisfeld System The sensitivity of detection of chitinase activity after PAGE in the Reisfeld system was evaluated with purified HEWL. This enzyme was chosen because it is highly purified and readily available. Moreover, it has been used for determining the level of sensitivity of a similar technique for determination of lysozyme activity (33). Results in Fig. 1 showed that 0.5 pg (500 ng) of HEWL could be detected after 1 h incubation at 37°C. This level of sensitivity is good, especially if we take into account that HEWL is much more efficient as a lysozyme than as a chitinase (34). Moreover, the hydrolytic activity of HEWL upon chitin is at least 600 times lower than that of Streptomyces chitinase (35). Chitinase Activity

in the Davis System

Results in Fig. 2 showed that St. griseus and Se. marcescenschitinases yielded several bands in the Davis sys-

CHITINASE

tern in a 15% polyacrylamide

ACTIVITY

gel. As expected,

AFTER

HEWL

GEL

365

ELECTROPHORESIS

kDa

HEWL

and wheat germ WlA lysozyme did not migrate in that system.In the caseof St. griseus, two activities had relatively high electrophoretic mobility (Fig. 2). The staining of chitinase activity (Fig. 2A) was also compared with the electrophoretic profile of proteins after Coomassie blue R-250 staining (Fig. 2B) and silver staining (Fig. 2C). Purified a-lactalbumin was included as a control for monitoring the effect of a protein without chitinase activity. As expected, large amounts of cu-lactalbumin (20 pg per sample) did not show chitinolysis. Bands with chitinase activity from microbial extracts corresponded to bands at the limit of detection after silver staining of proteins. Activity staining is thus rather sensitive. Chitinase

Activity

1

5

10

50

100

FIG. 4. Chitinase activity with varying amounts of purified HEWL after SDS-PAGE in a gel containing 0.01% (W/V) glycol chitin as substrate. HEWL (1, 5, 10, 50, 100 ng) was separated as in Fig. 3. Samples were denatured with 2.5% (W/V) SDS. Renaturation of enzymes and molecular weight markers were as in Fig. 3. Detection of chitinase activity was as in Fig. 1.

after SDS-PAGE

In the case of SDS-PAGE, glycol chitin could be directly incorporated into the gel without any interference during migration. Glycol chitin did not migrate in the gels and there was no interaction with the denatured enzymes during electrophoresis. The procedure for renaturation of chitinases was the same as previously described for lysozymes (32,33) involving the use of buffered purified Triton X-100 which efficiently removes SDS from the enzymes. Preliminary experiments indicated that 0.1 M sodium acetate buffer at pH 5.0 was the most efficient buffer for renaturation of chitinases. Molecular weights of chitinases could be directly estimated in denaturing gels. Effect of Denaturation P-Mercaptoethanol

with SDS Alone and SDS plus

It is well known that some enzymes cannot ciently renatured if denatured in the presence

be effiof SDS

and a reducing agent, such as @-mercaptoethanol (33,36). Some enzymes (chymotrypsin, Pronase, trypsin, subtilisin, some lysozymes) can be renatured (as determined by activity staining in gels) only if denatured in the presence of SDS alone (33,36). We thus compared the effect of denaturation with SDS alone and of SDS plus ,&mercaptoethanol on renaturation of chitinases. Wheat germ WlA lysozyme, HEWL, and TEWL could not be efficiently renatured after SDS and P-mercaptoethanol denaturation (data not shown). Dithiothreitol had the same effect as P-mercaptoethanol. Several microbial chitinase bands were revealed after denaturation with SDS alone (Fig. 3). Moreover, some microbial chitinase bands could also be detected (Fig. 3) after denaturation with SDS and P-mercaptoethanol. Estimation of Molecular Weights of Chitinases after SDS-PAGE

HEWL and TEWL migrated as proteins of n/r, 14,400 in agreement with their known molecular weights (Fig. Estimated Molecular Weights of Commercial Chitinases 3). Wheat germ WlA lysozyme had an estimated molecfrom Streptomyces griseus (Sg) and Serrutia marcescens (Sm) ular weight of 28,000. This corresponds well to the pubafter SDS-PAGE asin Fig. 3 lished value of 25,400 (29), especially if we take into account that the molecular weight of carbonic anhydrase Sm sg was set at 31,000 instead of 29,000 in the previous publiDenaturation Denaturation cation (29). Table I lists the estimated molecular weights of St. griseus and Se. marcescens chitinases after denawith SDS and with SDS and Denaturation P-mercaptoDenaturation P-mercaptoturation with SDS alone or SDS plus P-mercaptoethawith SDS ethanol with SDS ethanol nol. In many cases, there is good agreement between our estimates and published molecular weights (9,lO) of 72,000 73,000 Streptomyces and Serratia chitinases. For example, the 57,500 60,500 60,500 most abundant chitinase of Se. marcescens with an esti55,000 54,000 54,000 51,000 40,500 40,500 mated M, 57,000 (10) probably corresponds to the chiti45,000 nase activity at 60,500. TABLE

41,000 39,500 36,500 25,000 24,000

41,000 -

1

-

-

Sensitivity of Detection after SDS-PAGE The level of sensitivity after SDS-PAGE was studied with HEWL. As low as 10 ng of purified HEWL could

TRUDEL AND ASSELIN be detected after a 2 h incubation at 37°C (Fig. 4). It is thus more sensitive than the Reisfeld system probably because the enzyme is in direct contact with the substrate after renaturation. In the case of native gels, the enzymes have to diffuse out into the overlay gel. This seems to greatly reduce the level of sensitivity. In the case of SDS-PAGE, this technique is obviously limited to monomeric enzymes. Overall, several chitinolytic enzymes could be easily detected in different gel systems with the use of glycol chitin and Calcofluor white M2R. It will be interesting to assess the value of this technique to the array of enzymes involved in chitin metabolism. Very few chitinases seem unable to digest glycol chitin (18). With enzymes exhibiting lysozyme and chitinase activity, it is now feasible to easily compare both activities after PAGE under native or denaturing conditions (29,32,33).

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ACKNOWLEDGMENTS This work was partly supported by grants from the Conseil des recherches en p&he et agro-alimentaire du Quebec (CORPAQ) and from the Natural Sciences and Engineering Research Council of Canada (NSERC) to A. Asselin. The authors thank Lucette Bilanger for typing the manuscript and Dr. Patrice Audy for his collaboration.

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