ANALYTICAL

BIOCHEMISTRY

75, l-8 (1976)

A Simple and Sensitive Staining Method for the Detection of Cellulase lsozymes in Polyacrylamide Gels RAPHAELGORENAND Department

of Horticulture,

Hebrew

MOSHE HUBERMAN

University

of Jerusalem,

Rehovot,

Israel

Received May 5, 1975; accepted April 15, 1976 A simple and rapid staining procedure is described for qualitative and quantitative determination of the activity of plant (Citrus sinensis (L.) Osbeck cv. Shamouti) andfungal (Trichodermata viride) cellulases in polyacrylamide gels. The method is based on the incorporation of carboxymethyl cellulose, a cellulase substrate, into the gels. After electrophoresis of crude extracts the gels are incubated in sodium-potassium phosphate buffer for the cellulase reaction which is stopped at the desired time by acidification of the gels in 60% sulfuric acid. The gels are then exposed to 2.0% KI + 0.2% Iz. No color develops in areas containing cellulase activity. The experimental procedure is described, and its different aspects are discussed.

No techniques are yet available for visualization of cellulase isozyme activity in polyacrylamide gels. Therefore, when cellulases in a cell-free preparation are to be separated by disc electrophoresis, the acrylamide gels must first be sliced and the location of enzymic activity determined by viscosimetric or other analytical methods (3-6) after extraction of isozymes from the slices by buffer (1) or by a buffer-carboxymethyl cellulose (CMC) mixture (2). This procedure is time consuming, limits the number of replications that can be analysed simultaneously, and involves recovery problems. It is therefore highly probable that isozymes present in very small quantities will not be detected. The present communication describes a new and sensitive method for qualitative as well as quantitative determination of cellulase isozyme activities in gels, containing substrate in a way similar to determination of amylase isozymes by incorporation of starch into gels (7,8). The procedure is simple, avoids slicing and extraction phases, and can be used for detecting fungal as well as plant cellulase isoenzymes. MATERIALS

AND METHODS

Extraction of plant cellulase. Citrus leaf explants (Citrus sinensis (L.) Osbeck, cv. Shamouti) were incubated as previously described (5,9) until they reached 50 to 60% abscission. Tissue sections of 1 to 2 mm were sampled from both sides of the separation line and either deep-frozen (-20°C) or immediately homogenized (Q/15 ml of buffer) for 2 min by means of an Ultra Turrax homogenizer (24,000 rpm) in sodium-potassium

Copyright 0 1976 by Academic Press. Inc. All rights of reproduction in any form reserved

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phosphate buffer (pH 7.0,0.2 M, enriched with 1.O M NaCl) in the presence of 0.05% L-cysteine. The homogenate was filtered through four layers of gauze following a 30-min stirring in an ice bath. The remaining plant residue was resuspended in the same buffer and the procedure was repeated. After residual plant material was discarded, the combined filtrates were centrifuged (5000 rpm, 10 mitt; IEC ultracentrifuge, Model B-35, rotor A-147), and the supernatant was decanted and recentrifuged (12,000 rpm, 10 min). The extraction attained under such conditions was complete (5). Protein was precipitated from the supernatant by adding anhydrous ammonium sulfate to obtain 80% (w/v) saturation. Protein was collected about 2 h later by centrifugation (12,000 rpm, 10 min) and resuspended in the above-mentioned buffer. The protein solution was dialyzed overnight against the same buffer, diluted 10 times and then centrifuged (12,000 rpm, 10 min). All operations were performed at 4°C to prevent digestion of dialyzing tubes as well as protein breakdown. In preliminary experiments it was established that no loss in enzymic activity is caused by dialysis. Preparation offungal cellulase. Fourteen milligrams of a commercial preparation of fungi C, cellulase (Worthington Biochemical Corp., Freehold, N.J., EC 3.2.1.4 Trichodermutu viride cellulase), containing 1.7 cellulase units/mg, were dissolved in 1.4 ml of the above-mentioned buffer enriched also with 1 M NaCl. The enzyme solution was centrifuged (10,000 rpm, 10 min) and the supernatant was dialyzed for 2 h against the same buffer diluted lo-fold. Electrophoresis procedure. Aliquots (0.1 ml) of enzyme solutions containing 20% sucrose (w/v), and 200 to 300 pg of protein (plant) or 1.7 cellulase units (fungi) were subjected to electrophoresis (10,ll) in 8% polyacrylamide gels (0.6 x 10.2 cm) at 5 mA/tube and at 4°C in the dark for 105 min. The polyacrylamide gels contained CMC (Na-salt, BDH Chemicals, England) at the desired concentrations (Figs. 2,3, and 5) which was added to the water fraction used for polymerization of CMU67.5 ml of H20) of the gels. When using a slicing method (9) it was established that 60% of citrus cellulase is recovered from the gels. However, when using a modification (13) of Anker’s method (14) for solubilizing acrylamide gel slices, 95 to 100% of fungal cellulase activity was recovered. Staining procedure. After completing the electrophoretic separation, gels were transferred to 12-ml test tubes containing sodium-potassium buffer (pH 6.0,0.2 M), and the tubes were incubated for 10 min in a water bath at 37°C. At the end of this incubation period the buffer was decanted; tubes containing gels were stoppered and further incubated in the water bath for the desired time. This procedure was adopted since incubation of the gels in buffer for periods longer than 10 min caused diffusion of substrate and enzyme from the gels into the external buffer,

CELLULASE

STAINING

IN POLYACRYLAMIDE

GELS

3

and no cellulase activity could thereafter be detected after staining gels with 2.0% KI and 0.2% I2 in distilled water (w/v). When the period required for full development of color was less than 10 min, gels were incubated in buffer for the desired length of time and stained immediately after decantation of the buffer. When the required period of incubation was longer, the first 10 min of incubation in buffer was included in the total incubation period measured. The enzymic reaction was stopped by adding 60% H,SO, to the test tubes for 5 to 10 min. After removal of the HzS04, gels were washed with distilled water and the liquid was decanted immediately after. Staining solution was poured into the tubes which were kept for 30 to 60 min at room temperature; the duration of incubation in the staining solution determines the intensity of background color in areas with no enzymic activity. The color reaction of CMC with iodine is analogous to the well-known starchiodine reaction (12) and does not appear where CMC is degraded. Therefore, increasing values of percent transmission represent increases in enzymic activity. Faster staining can be obtained if tubes are incubated in a water bath at 37°C. At the end of the incubation period, the staining +

FIG. 1. Fungal (FU-C, 2Smin incubation: 0.1% CMC) and plant (PL-C, 18-hr incubation: 0.1% CMC) cellulase zymograms against control (CO) stained with 2.0% KI + 0.2% I, for 30 min following 5-min acidification in 60% sulfuric acid. F = running front.

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solution was decanted and any excess staining solution absorbed to the gel surface was washed off with distilled water. This was done to avoid masking of active regions by diffusion of excessive color from inactive regions. Gels were kept in rubber-stoppered tubes, and then either scanned at 612 nm in a Gilford Model 2400 spectrophotometer or photographed within the first hour after staining. When the test tubes are tightly sealed, scanning can be delayed, but restaining may be required. Viscasimetric cellulase assay. Fungal cellulase activity was tested as previously described (5). Changes in viscosity of the enzyme-substrate mixture (0.1 ml of enzyme preparation in 10 ml of 1.1% CMC in 0.02~ phosphate buffer pH 6.0) was determined by means of an Exca viscosimeter (size 300). Preparations boiled for 10 min were used as controls. RESULTS

While testing cellulase activity in citrus tissue homogenates by the viscosimetric method (5,9) we found that 1.1% CMC in sodium-potassium phosphate buffer (pH 6.0,0.2 M) and 18-hr incubation at 37°C were optimal conditions. In preliminary experiments we tried to incubate gels in the

FIG. 2. The effect on transmission at 612 nm of CMC conceqtration in 8% polyacrylamide gels and incubation time of the gels in sodium-potassium phosphate buffer (pH 6.0, 0.2 M) after electrophoresis (5 mA/tube; 105 min) of plant cellulase (200-300 pg of protein/gel). Arrows indicate running front.

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IN POLYACRYLAMIDE

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FIG. 3. The effect of CMC concentration on the resolution of fungal cellulase activity. See legend to Fig. I for further experimental details.

substrate as suggested for polygalacturonase (15) for various periods after electrophoresis and to stain them thereafter. Some cellulase activity could be traced on the gels using this method, but results were not satisfactory. Consequently, in the following experiments we incorporated the substrate into the gels. An example of plant and fungal cellulase complex (PL-C and FU-C, respectively) separation on polyacrylamide gels containing this medium is presented in Fig. 1. No CMC breakdown could be detected in control (CO) gels during electrophoresis of gels without cellulase. Although we have established that 18 hr of incubation at 37°C did not cause any detectable enzyme degradation (5), probably due to the protecting presence of CMC (l), we tried several incubation periods in buffer following electrophoresis of plant cellulase complex. Since the amount of cellulase in the tissue is relatively small, at least 1.5 hr were required to detect a significant activity (left part of Fig. 2). After an incubation period of 1.5 hr, only one significant activity peak was detected at R, 0.38 and 0.025% CMC. By increasing the concentration of the substrate from 0.025 to0.05% we improved the resolution by promoting enzymic activity, whereas the

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GOREN AND HUBERMAN loo,

I

I

I

I

I

I

I

P4 ml reaction

a5 mixture)

a6

a7



I

‘r

60-

0

01

a2

a3 FU-C (units/X)

1.7

FIG. 4. Determination of fungal cellulase (FU-C) activity by the viscosimetric method. Reaction conditions: 0.1 ml of enzyme preparation was incubated in 10 ml of 1.1% CMC in 0.02 M sodium-phosphate buffer, pH 6.0, for 20 min at 37°C.

concentration of 0.1% CMC was less satisfactory. Results obtained at 18 hr of incubation (right part in Fig. 2) in buffer after electrophoresis definitely showed that at least three Citrus cellulase isozymes (at R, 0.05, 0.10, and 0.38) could be detected. Under such conditions (Fig. 2) 0.1% CMC yielded the best resolution. The number of isozymes was the same as reported when using the slicing procedure (9). The activity of the fungal cellulase preparation was much higher, and at least four isozymes (at R, 0.1, 0.5, 0.65, and 1.0) were detected (Fig. 3). Lewis et al. (16) found two cellulase isozymes in Trichodermata viride using an isoelectric focusing technique. Enzyme incubation periods ranged between 5 and 40 min (Fig. 3) because of the higher specific activity of the protein-enzyme commercial preparation. The enzyme preparation was run on gels containing 0.05 and 0.1% CMC. It is evident that both substrate concentrations were efficient and results were similar. Incubation for 5 min inbuffer following electrophoresis was sufficient to produce definite activity. An increase in incubation period made the contrast between activity zones more marked with 0.1% CMC than with 0.05%. To establish the sensitivity of the electrophoretic staining method and its possible use for quantitative research, we present (Figs. 4 and 5) data showing the relationship between total fungal cellulase activity determined by traditional viscosimetric method (13) and by the electrophoretic method suggested by us for detection of isozyme activity. It is clear (Fig. 4) that at the very low level of cellulase activity (0.013 U/gel), two significant peaks of cellulase isozymes can already be detected by the electrophoretic method (Fig. 5). By loading gels with cellulase units in increasing con-

CELLULASE

STAINING

IN POLYACRYLAMIDE

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centrations (0.013-0.425 U/gel) isozyme activity was significantly stepped up, and a gradual appearance of additional isozymes could be detected. Application of higher concentrations (0.85 and 1.7 U/gel) overloaded the gels and obscured the results. DISCUSSION

The present results clearly show that cellulase isozymes can be detected in polyacrylamide gels when CMC is incorporated into the gels before polymerization. The staining procedure is very efficient and provides a simple way for visualization of the enzymic activity which does not involve time-consuming procedures of slicing and extraction methods mentioned in the introduction. Densitometric scannings (Figs. 2, 3, and 5) show that the staining procedure provides an easy and reliable method for quantitative determination of isozyme activity along the gels. The appropriate incubation period and the optimal CMC concentration should be determined prior to adoption of the method for other plant systems to ensure optimal conditions and reduce as much as possible the incubation period in buffer prior to staining. When cellulase activity is low and a short incubation period is selected for the enzymic reaction, a relatively high CMC concentration may cause vague results (compare incubation period of 1.5 to 18 hr in gels containing 0.1% CMC, Fig. 2), because only a relatively small amount of substrate is degraded under such conditions. However, by reducing the concentration of substrate to 0.05%, improvement in resolution was obtained. Some crucial points need special attention when adopting this method.

oJ.25

0.850

4 -745 FIG. 5. Detection of fungal cellulase (FU-C) isozymes activity by the electrophoretic method. See Fig. 1 for further experimental details.

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The duration of incubation in sulfuric acid determines the staining efficiency by the iodine solution. Care should also be taken not to incubate gels for too long in the staining solution. It would therefore be advisable to follow development of the background color during staining and to terminate it when the color intensity seems to be satisfactory. Iodine evaporates from gel edges very easily; therefore, it is important to perform the scanning as rapidly as possible. The best procedure is to scan the gels within the first 1 or 2 hr after staining. Since evaporation starts from the edges, isozyme activity (especially that remaining close to the application edge) may be lost due to background bleaching. When gels are kept in stoppered tubes, the inner atmosphere may become immediately saturated with iodine vapor and prevent bleaching. Thus gels can be stored for a few days with no major changes in color. If it becomes necessary to restain the gels before scanning due to partial bleaching, gels should be reacidified by short immersion in sulfuric acid before proceeding as described above. Restaining, however, is not always successful; if gels are not removed from sulfuric acid at the appropriate time, irreversible disappearance of isozyme bands may occur. It should be noted that the same staining procedure can be successfully used with polyacrylamide plate electrophoresis. ACKNOWLEDGMENTS The authors wish to express their gratitude to Dr. R. C. Hall, University of Wisconsin, for valuable correspondence and especially for his suggestion to incorporate CMC into the gels and use the iodine solution for staining; to Dr. Barbara Siegel, University of Hawaii at Manoa, for stimulating and helpful suggestions made while she visited Israel, and to Prof. Yehudith Birk, Department of Agricultural Biochemistry, for her cooperation and critical reading of the manuscript.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Lew, F. T., and Lewis, L. N. (1974) Phytochemistry 13, 1359- 1366. Ferrari, T. E., and Amison, P. G. (1974) Plant Physiol. 54, 487-493. Maclachlan, G. A., and Perrault, J. (1964) Nature (London) 204, 81-82. Horton, R. F., and Osborne, D. J. (1967) Nature (London) 214, 1086- 1088. Ratner, A., Goren, R., and Monselise, S. P. (1969) Plunr Physiol. 44, 1717-1723. Li, L. H., and King, K. W. (1963) Appl. Microbial. 11, 320. McCown, B. H., Hall, T. C., and Beck, G. E. (1969) Plant Physiol. 44,210-216. Rudolph, K., and Stahmann, M. A. (1966) PIant Physiol. 41, 389-394. Huberman, M., Goren, R., and Birk, Y. (1975) Plant Physiol. 55, 941-945. Omstein, L. (1964) Ann. N. Y. Acad. Sci. 121, 321-349. Davis, B. J. (1965) Ann. N. Y. Acad. Sci. 121, 404-427. Meyer, K. H., and Gibbons, G. C. (1951) Advun. Enzymol. 12, 341-377. Huberman, M. (1973) Characteristics and Site of Action of Cellulase in Abscission Zones of Citrus Leaves; M.Sc. thesis submitted to the Hebrew University of Jerusalem (in Hebrew), 120 pp., Jerusalem. 14. Anker, H. S. (1970) FESS Left. 7, 293. 15. Lisker, N., and Retig, Nira (1974) J. Chromatogr. 96, 245-249. 16. Lewis, L. N., Linkins, A. E., O’Sullivan, S., and Reid. P. D. (1974) in Plant Growth Substances 1973, pp. 708-718, Hirokawa Publishing Company Tokyo.

A simple and sensitive staining method for the detection of cellulase isozymes in polyacrylamide gels.

ANALYTICAL BIOCHEMISTRY 75, l-8 (1976) A Simple and Sensitive Staining Method for the Detection of Cellulase lsozymes in Polyacrylamide Gels RAPHAE...
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