334
P. Aud) and A . Asselin
Electrophoresis 1992, 13, 334-337
RNA onto nylon sheets for Southern or Northern hybridization. Using the 254 nm UV light (NIS 8W GL-8 installed in the Stratalinker) instead of the commonly used 3 12 nm bulbs avoids the necessity of using BrdU in crosslinking. A piece of Parafilm is placed onto an ice bag and the DNAprotein mixture is added on top as a single drop. This assembly is carefully placed into the “Stratalinker”, leaving a distance of about 5 cm to the UV bulbs.Titration of the irradiation energy, required for complex formation, is routinely performed to minimize DNA degradation. Best results with ourprobes are obtained after exposure for 30 min (120000 pJ/cm2 X 30 s).
8 - 12% polyacrylamide gels according to Ausubel et al. [6]. As size markers, we use a I4C-labeled protein mixture (Amersham). After electrophoresis the gel is fixed in 10% methanol, 10% acetic acid for 1 h, dried and exposed overnight at -80” C using intensifying screens. Cutting off the unbound DNA (front, height of dye marker) from the gel before exposing will reduce signal noise.
(iii) Determination of the molecular weight of the crosslinked proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography. The short oligonucleotides (20 - 30 bp) require no digestion with nucleases prior to electrophoresis because their contribution to the electrophoretic mobility of the complex is quite negligible (the free probe migrates in this gel system with the Bromophenol Blue front). Transfer the drop (after irradiation) into a reaction tube, add 2 WLof 10 X SDS probe buffer (125 mM Tris-HC1, pH 6.8; 10% 6-mercaptoethanol; 0.05% Bromophenol Blue; 10% SDS) and 2 pL 86% gjycerol, boil for 5 min and separate in
References
Patrice Audy Main Asselin DCpartment de phytologie FacultC des sciences de l’agriculture et de I’alimentation UniversitC Laval, QuCbec
Supported in part by a DFG grant (B1166/3-3). Received December 3. 1991
111 Dooley, S., Radtke, J., Blin, N. and Unteregger, G., NucleicAcids Res. 1988, 16, 11893. [2] Markowitz, A., Biochem. Biophys. Acta 1972, 281, 522-534. [3] Chodosh, L. A., Carthew, R. W. and Sharp, P. A., Mol. Cell. Biol. 1986, 6, 4723-4733. [4] Lynn, S. Y. and Riggs, A. D., Proc. Natl. Acad. USA 1974,71,947-951. [5] Dooley, S., Welter C., Theisinger, B. and Blin, N. Genet. Anal. Techn. Appl. 1990, 7, 133-137. [6] Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidmann, J. G . , Smith, F. A. and Struhl, K., Current Protocols i n MolecularBiology 1987.
Gel electrophoretic analysis of chitosan hydrolysis products Enzymatic hydrolysis of commercial crustacean chitosan by barley chitosanases was analyzed by subjecting chitosan to electrophoresis in a 10% w/v polyacrylamide slab gel in the presence of 7 M urea and 5.5 O/o v/v acetic acid. Chitosan migrated as a polycation. Chitosan was stained with Coomassie Brilliant Blue R-250 or visualized by ultraviolet transillumination after staining with Calcofluor White M2R. Some chitosan molecules were retarded by gel electrophoresis while small chitosan molecules migrated at the bottom of a 10 O/o w/vpolyacrylamide gel. Such analysis revealed that 96 h were necessary to convert all chitosan to oligosaccharides under our assay conditions. Chitosan oligosaccharides generated by enzymatic or chemical hydrolysis were further analyzed by electrophoresis in a 33 Yo w/v polyacrylamide gel containing urea and acetic acid. Coomassie Brilliant Blue R-250 was found to be better than CalcofluorWhite M2Rfor staining chitosan oligosaccharides. Chitosan oligomers of four residues (tetramers) or more were easily resolved in such a polyacrylamide gel system.To our knowledge, this is the first report of a gel electrophoretic separation of chitosan and its oligosaccharides.
Chitin is the most abundant aminopolysaccharide in nature [l]. It is an unbranched polymer of P-1,4-linked anhydro-2-acetamido-~-glucosefound in fungi [2], insects [3] and some marine invertebrates [4]. Chitosan consists of ~~
Correspondence: Dr. A. Asselin, Departement de phytologie, F.S.A.A., Universite Laval, Quebec, Canada G1K 7P4
Abbreviations: PAGE, polyacrylamide gel electrophoresis; TLC, thinlayer chromatography
‘0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992
N-deacetylated derivatives of chitin. In nature, chitosan is prevalent in the walls of some fungi [2]. Chitosan is also commercially produced by chemical deacelylation of crustacean chitin [5]. Chitosan is a cationic polyelectrolyte with several applications in the fields of food science [6], medicine [7,8] and process biochemistry [9,10]. Moreover, chitosan can induce various defense reactions in higher plants [ll-151 and some chitosan derivatives can exhibit antimicrobial properties [ 161. Chitosan can be chemically or enzymatically degraded into chitosan oligosaccharides. Such oli0173-0835/92/0505-0334 $3.50+.25/0
Electrophoresis 1992, 13. 334-331
gosaccharides can also be of value because of their bioactivity [17]. Chitosan and its oligosaccharides are thus a growing source of potentially useful substances. The analysis of such chemicals is often limited to an estimate of the degree of polymerization in addition to the degree of deacelylation. In many cases, these values apply to mixtures of components. Because of the need to define these components more precisely, we investigated the use of polyacrylamide gel electrophoresis to analyze chitosan subjected to enzymatic or chemical hydrolysis. All chemicals for polyacrylamide gel electrophoresis (PAGE) and Coomassie Brilliant Blue R-250 were from Bio-Rad (Mississauga, Ontario, Canada). Calcofluor White M2R (C.I. 40622),was from Sigma Chemical Co. (St. Louis, MO). Barley chitosanases were stimulated with silver nitrate in barley leaves and the intercellular fluid (IF) was extracted as previously described [18].Nine volumes of chitosan (Fluka, low molecular weight, No. 22741; 5 yg per yL) were incubated at 37°C in 50 mM sodium acetate (pH 5.0) with 1 volume of intercellular fluid extract from barley leaves [18]. Incubation was up to 96h and hydrolysis was stopped by boiling for 10 min. Samples were kept frozen. Chitosan (2.5 g; Fluka, low molecular weight, No. 22741) was also incubated for 24 h at 53 "C in 50 mL of 10 N HC1 [19].The hydrolysate was centrifuged at 5000 g for 5 min and evaporated to dryness with a Speed Vac concentrator (Savant). It was resuspended in distilled water (100 mg per mL) and kept frozen until use. Chitosan oligosaccharides generated by chemical degradation of chitosan were separated by preparative thin-layer chromatography (TLC) on silica plates (Sigma, T6270) using propanol-30 O/o ammonia (2:1, v/v) [20]. Chitosan oligomers were detected after spraying with ninhydrin [21]and eluted (dimer to hexamer) in water. The eluate was clarified at 16000 g for 5 min at room temperature and concentrated with a Speed Vac concentrator. The samples, dissolved in 0.5% v/v acetic acid, 2 M urea and 15O/o w/v sucrose, were boiled for 3 min just before loading for electrophoresis. Electrophoresis was in 10% or 33 O/o w/v polyacrylamide gels containing 7 M urea and 5.5 O/o v/v acetic acid [22].Electrophoresis was performed at 30 mA for 2.5 h or at 15 mA for 3.5 h at room temperature using 5.5 O/o acetic acid as electrode buffer. Gels were stained for 20 min with freshly prepared 0.01 Yo w/v Calcofluor White M2R in 0.5 M Tris-HC1, pH 8.9, [23] and destained in distilled water for at least 4 h [23]. Chitosan molecules were visualized by placing the gel on a chromato-Vue C-62 long wave UVtransilluminator (UV Products) [23]. Gels were also stained in 0.2% w/v Coomassie Brilliant Blue R-250 in methanolwater-acetic acid (50:40:10; v/v/v) for 20 min, destained, and kept in 7% v/v acetic acid. Because of its polycationic nature, several electrophoretic systems, such as the Reisfeld etal. [24] and the Panyim and Chalkley [22] system, designed to separate basic proteins, were tested with chitosan. The Panyim and Chalkley system was the only one allowing separation of chitosan (Fig. 1, lane 1). The electrophoretic profiles revealed heterodispersity of the chitosan samples. This is quite normal for chitosan prepared underrelatively harsh conditions (acids and bases). Chitosan was revealed by staining with Coomassie Brilliant Blue R-250, which had previously been shown to be efficient for staining chitosan [18].The same profile was
335
Electrophoresis of chitosan hydrolysis products
also revealed by CalcofluorWhite (not shown) but with less sensitivity. It was important to boil samples in at least 2 M urea prior to electrophoresis to allow for good electrophoretic separation of chitosan. Some chemicals such as anions (phosphates, for example) should be avoided because they precipitate chitosan dissolved in acetic acid. Chitosan is only soluble at acidic pH and is usually dissolved in acetic acid [5,10].Basic solutions should thus be avoided. Despite the absence of specific molecular mass markers applying to the separation of chitosan, the electrophoretic profiles of chitosan can be useful to assess the relative importance of various chitosan molecules in a complex chitosan solution. Small molecules will migrate as a sharp band at the bottom of the gel while large molecules will stay at the top. The mobility of such large chitosan molecules (top of the gel) was not influenced by various treatments designed to separate chitosan aggregated by proteins (sodium dodecyl sulfate, phenol, chloroform). Barley (Hordeum vulgare L. cv. Leger) was recently found to be a good source of six chitosanases accumulating in the intercellular fluid of stressed leaf tissue [18].This mixture of chitosanases was incubated with commercial chitosan for the electrophoretic analysis of hydrolysis products generated over time (Fig. 1, lanes 2-8). Results showed that 96 h were required for complete disappearance of chitosan retarded in a 10% polyacrylamide gel (Fig. 1,lane 8 versus the other lanes). The initial sample (Fig. 1, lane 1) migrated at the bottom and top of the gel in addition to chitosan migrating in the upper half of the gel. After 1h, chitosan was already affected, as detected by the electrophoretic profile (Fig. 1, lane 2). Note that all material at the top of the gel disappeared after 96 h (Fig. 1, lane 8). An identical profile was detected after Calcofluor White M2R staining (not shown).
1
2
3
4
5
6
7
8
Figure 1. Electrophoretic pattern of crustacean chitosan digested with barley chitosanases. Nine volumes of commercial chitosan (5 bg/pL in 0.5 Yo acetic acid) were incubated with one volume of barley intercellular fluid prepared as described in the text. Incubation at 37 "Cwas in 50 mM sodium acetate (pH 5.0) for 0, I, 2,6,10,20,48 and 96 h (lanes 1-8, respectively). The reaction was stopped by boiling for 10 min. Electrophoresis was in a 10% w/v polyacrylamide slab gel containing 7 M urea and 5.5% v/v acetic acid. Each sample contained 45 pg of chitosan. Samples were dissolved in 0.5% v/v acetic acid containing 2 M urea and 15% w/v sucrose. After boiling for 3 min, samples were electrophoresed at 30 mA for 2.5 h at room temperature using 5.5% v/v acetic acid as the electrode buffer. Chitosan migrated from the top of the gel (anode) to the bottom (cathode). The gel was stained with 0.2% w/v Coomassie Brilliant Blue R-250 as described in the text.
336
P. ,\tidy .and 2 . Asselin
Elr~riroghoi’cci~1992. 13, 331-337
Coomassie Brilliant Blue R-250 staining appears more sensitive than Calcofluor staining for smaller inolecules (not shown). Controls were run where chitinases (such as purified hen egg white lysozyme) could not generate such hydrolysis patterns over time (not shown). Chitosanases, distinct from chitinases, are thus responsible for this hydrolysis [18]. To our knowledge, this is the first report dealing with the electrophoretic separation of chitosan and of its products. However, PAGE has previously been used to characterize other charged polysaccharides [26-281. Chitosan oligosaccharides generated by enzymatic hydrolysis or acid hydrolysis were analyzed by gel electrophoresis in a 33 n/n w/v polqacrylamide gel. These oligosaccharides were compared in mobility with a series of purified chitosan oligosaccharides (tetramer, pentamer and hexamer; Fig. 2, lanes 1,2,3, respectively). Such purified oligosaccharides were obtained after preparative TLC separation. Chitosan oligosaccharides generated by 24 h of enzymatic hydrolysis (Fig. 2, lane 4) migrated as a series of bands between the pentamer and oligomers of20 residues and more. This was the same for chitosan oligosaccharides generated by acid hydrolysis (Fig. 2, lane 5). This technique also allowed the conclusion that barley chitosanases act as endochitosanases because of the series of hydrolysis products. Exochitosanases would only generate monomers or diiners depending on the mode of action. Note that Coomassie Brilliant Blue R-250 should be used for analysis of the fastest migrating oligosaccharides. Calcofluor White M2R is only suitable to stain decamers and oligomers of higher molecular mass. It is also noteworthy that D-glucosamine (monomer) and it:
P. Aud) and A . Asselin
Electrophoresis 1992, 13, 334-337
RNA onto nylon sheets for Southern or Northern hybridization. Using the 254 nm UV light (NIS 8W GL-8 installed in the Stratalinker) instead of the commonly used 3 12 nm bulbs avoids the necessity of using BrdU in crosslinking. A piece of Parafilm is placed onto an ice bag and the DNAprotein mixture is added on top as a single drop. This assembly is carefully placed into the “Stratalinker”, leaving a distance of about 5 cm to the UV bulbs.Titration of the irradiation energy, required for complex formation, is routinely performed to minimize DNA degradation. Best results with ourprobes are obtained after exposure for 30 min (120000 pJ/cm2 X 30 s).
8 - 12% polyacrylamide gels according to Ausubel et al. [6]. As size markers, we use a I4C-labeled protein mixture (Amersham). After electrophoresis the gel is fixed in 10% methanol, 10% acetic acid for 1 h, dried and exposed overnight at -80” C using intensifying screens. Cutting off the unbound DNA (front, height of dye marker) from the gel before exposing will reduce signal noise.
(iii) Determination of the molecular weight of the crosslinked proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography. The short oligonucleotides (20 - 30 bp) require no digestion with nucleases prior to electrophoresis because their contribution to the electrophoretic mobility of the complex is quite negligible (the free probe migrates in this gel system with the Bromophenol Blue front). Transfer the drop (after irradiation) into a reaction tube, add 2 WLof 10 X SDS probe buffer (125 mM Tris-HC1, pH 6.8; 10% 6-mercaptoethanol; 0.05% Bromophenol Blue; 10% SDS) and 2 pL 86% gjycerol, boil for 5 min and separate in
References
Patrice Audy Main Asselin DCpartment de phytologie FacultC des sciences de l’agriculture et de I’alimentation UniversitC Laval, QuCbec
Supported in part by a DFG grant (B1166/3-3). Received December 3. 1991
111 Dooley, S., Radtke, J., Blin, N. and Unteregger, G., NucleicAcids Res. 1988, 16, 11893. [2] Markowitz, A., Biochem. Biophys. Acta 1972, 281, 522-534. [3] Chodosh, L. A., Carthew, R. W. and Sharp, P. A., Mol. Cell. Biol. 1986, 6, 4723-4733. [4] Lynn, S. Y. and Riggs, A. D., Proc. Natl. Acad. USA 1974,71,947-951. [5] Dooley, S., Welter C., Theisinger, B. and Blin, N. Genet. Anal. Techn. Appl. 1990, 7, 133-137. [6] Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidmann, J. G . , Smith, F. A. and Struhl, K., Current Protocols i n MolecularBiology 1987.
Gel electrophoretic analysis of chitosan hydrolysis products Enzymatic hydrolysis of commercial crustacean chitosan by barley chitosanases was analyzed by subjecting chitosan to electrophoresis in a 10% w/v polyacrylamide slab gel in the presence of 7 M urea and 5.5 O/o v/v acetic acid. Chitosan migrated as a polycation. Chitosan was stained with Coomassie Brilliant Blue R-250 or visualized by ultraviolet transillumination after staining with Calcofluor White M2R. Some chitosan molecules were retarded by gel electrophoresis while small chitosan molecules migrated at the bottom of a 10 O/o w/vpolyacrylamide gel. Such analysis revealed that 96 h were necessary to convert all chitosan to oligosaccharides under our assay conditions. Chitosan oligosaccharides generated by enzymatic or chemical hydrolysis were further analyzed by electrophoresis in a 33 Yo w/v polyacrylamide gel containing urea and acetic acid. Coomassie Brilliant Blue R-250 was found to be better than CalcofluorWhite M2Rfor staining chitosan oligosaccharides. Chitosan oligomers of four residues (tetramers) or more were easily resolved in such a polyacrylamide gel system.To our knowledge, this is the first report of a gel electrophoretic separation of chitosan and its oligosaccharides.
Chitin is the most abundant aminopolysaccharide in nature [l]. It is an unbranched polymer of P-1,4-linked anhydro-2-acetamido-~-glucosefound in fungi [2], insects [3] and some marine invertebrates [4]. Chitosan consists of ~~
Correspondence: Dr. A. Asselin, Departement de phytologie, F.S.A.A., Universite Laval, Quebec, Canada G1K 7P4
Abbreviations: PAGE, polyacrylamide gel electrophoresis; TLC, thinlayer chromatography
‘0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992
N-deacetylated derivatives of chitin. In nature, chitosan is prevalent in the walls of some fungi [2]. Chitosan is also commercially produced by chemical deacelylation of crustacean chitin [5]. Chitosan is a cationic polyelectrolyte with several applications in the fields of food science [6], medicine [7,8] and process biochemistry [9,10]. Moreover, chitosan can induce various defense reactions in higher plants [ll-151 and some chitosan derivatives can exhibit antimicrobial properties [ 161. Chitosan can be chemically or enzymatically degraded into chitosan oligosaccharides. Such oli0173-0835/92/0505-0334 $3.50+.25/0
Electrophoresis 1992, 13. 334-331
gosaccharides can also be of value because of their bioactivity [17]. Chitosan and its oligosaccharides are thus a growing source of potentially useful substances. The analysis of such chemicals is often limited to an estimate of the degree of polymerization in addition to the degree of deacelylation. In many cases, these values apply to mixtures of components. Because of the need to define these components more precisely, we investigated the use of polyacrylamide gel electrophoresis to analyze chitosan subjected to enzymatic or chemical hydrolysis. All chemicals for polyacrylamide gel electrophoresis (PAGE) and Coomassie Brilliant Blue R-250 were from Bio-Rad (Mississauga, Ontario, Canada). Calcofluor White M2R (C.I. 40622),was from Sigma Chemical Co. (St. Louis, MO). Barley chitosanases were stimulated with silver nitrate in barley leaves and the intercellular fluid (IF) was extracted as previously described [18].Nine volumes of chitosan (Fluka, low molecular weight, No. 22741; 5 yg per yL) were incubated at 37°C in 50 mM sodium acetate (pH 5.0) with 1 volume of intercellular fluid extract from barley leaves [18]. Incubation was up to 96h and hydrolysis was stopped by boiling for 10 min. Samples were kept frozen. Chitosan (2.5 g; Fluka, low molecular weight, No. 22741) was also incubated for 24 h at 53 "C in 50 mL of 10 N HC1 [19].The hydrolysate was centrifuged at 5000 g for 5 min and evaporated to dryness with a Speed Vac concentrator (Savant). It was resuspended in distilled water (100 mg per mL) and kept frozen until use. Chitosan oligosaccharides generated by chemical degradation of chitosan were separated by preparative thin-layer chromatography (TLC) on silica plates (Sigma, T6270) using propanol-30 O/o ammonia (2:1, v/v) [20]. Chitosan oligomers were detected after spraying with ninhydrin [21]and eluted (dimer to hexamer) in water. The eluate was clarified at 16000 g for 5 min at room temperature and concentrated with a Speed Vac concentrator. The samples, dissolved in 0.5% v/v acetic acid, 2 M urea and 15O/o w/v sucrose, were boiled for 3 min just before loading for electrophoresis. Electrophoresis was in 10% or 33 O/o w/v polyacrylamide gels containing 7 M urea and 5.5 O/o v/v acetic acid [22].Electrophoresis was performed at 30 mA for 2.5 h or at 15 mA for 3.5 h at room temperature using 5.5 O/o acetic acid as electrode buffer. Gels were stained for 20 min with freshly prepared 0.01 Yo w/v Calcofluor White M2R in 0.5 M Tris-HC1, pH 8.9, [23] and destained in distilled water for at least 4 h [23]. Chitosan molecules were visualized by placing the gel on a chromato-Vue C-62 long wave UVtransilluminator (UV Products) [23]. Gels were also stained in 0.2% w/v Coomassie Brilliant Blue R-250 in methanolwater-acetic acid (50:40:10; v/v/v) for 20 min, destained, and kept in 7% v/v acetic acid. Because of its polycationic nature, several electrophoretic systems, such as the Reisfeld etal. [24] and the Panyim and Chalkley [22] system, designed to separate basic proteins, were tested with chitosan. The Panyim and Chalkley system was the only one allowing separation of chitosan (Fig. 1, lane 1). The electrophoretic profiles revealed heterodispersity of the chitosan samples. This is quite normal for chitosan prepared underrelatively harsh conditions (acids and bases). Chitosan was revealed by staining with Coomassie Brilliant Blue R-250, which had previously been shown to be efficient for staining chitosan [18].The same profile was
335
Electrophoresis of chitosan hydrolysis products
also revealed by CalcofluorWhite (not shown) but with less sensitivity. It was important to boil samples in at least 2 M urea prior to electrophoresis to allow for good electrophoretic separation of chitosan. Some chemicals such as anions (phosphates, for example) should be avoided because they precipitate chitosan dissolved in acetic acid. Chitosan is only soluble at acidic pH and is usually dissolved in acetic acid [5,10].Basic solutions should thus be avoided. Despite the absence of specific molecular mass markers applying to the separation of chitosan, the electrophoretic profiles of chitosan can be useful to assess the relative importance of various chitosan molecules in a complex chitosan solution. Small molecules will migrate as a sharp band at the bottom of the gel while large molecules will stay at the top. The mobility of such large chitosan molecules (top of the gel) was not influenced by various treatments designed to separate chitosan aggregated by proteins (sodium dodecyl sulfate, phenol, chloroform). Barley (Hordeum vulgare L. cv. Leger) was recently found to be a good source of six chitosanases accumulating in the intercellular fluid of stressed leaf tissue [18].This mixture of chitosanases was incubated with commercial chitosan for the electrophoretic analysis of hydrolysis products generated over time (Fig. 1, lanes 2-8). Results showed that 96 h were required for complete disappearance of chitosan retarded in a 10% polyacrylamide gel (Fig. 1,lane 8 versus the other lanes). The initial sample (Fig. 1, lane 1) migrated at the bottom and top of the gel in addition to chitosan migrating in the upper half of the gel. After 1h, chitosan was already affected, as detected by the electrophoretic profile (Fig. 1, lane 2). Note that all material at the top of the gel disappeared after 96 h (Fig. 1, lane 8). An identical profile was detected after Calcofluor White M2R staining (not shown).
1
2
3
4
5
6
7
8
Figure 1. Electrophoretic pattern of crustacean chitosan digested with barley chitosanases. Nine volumes of commercial chitosan (5 bg/pL in 0.5 Yo acetic acid) were incubated with one volume of barley intercellular fluid prepared as described in the text. Incubation at 37 "Cwas in 50 mM sodium acetate (pH 5.0) for 0, I, 2,6,10,20,48 and 96 h (lanes 1-8, respectively). The reaction was stopped by boiling for 10 min. Electrophoresis was in a 10% w/v polyacrylamide slab gel containing 7 M urea and 5.5% v/v acetic acid. Each sample contained 45 pg of chitosan. Samples were dissolved in 0.5% v/v acetic acid containing 2 M urea and 15% w/v sucrose. After boiling for 3 min, samples were electrophoresed at 30 mA for 2.5 h at room temperature using 5.5% v/v acetic acid as the electrode buffer. Chitosan migrated from the top of the gel (anode) to the bottom (cathode). The gel was stained with 0.2% w/v Coomassie Brilliant Blue R-250 as described in the text.
336
P. ,\tidy .and 2 . Asselin
Elr~riroghoi’cci~1992. 13, 331-337
Coomassie Brilliant Blue R-250 staining appears more sensitive than Calcofluor staining for smaller inolecules (not shown). Controls were run where chitinases (such as purified hen egg white lysozyme) could not generate such hydrolysis patterns over time (not shown). Chitosanases, distinct from chitinases, are thus responsible for this hydrolysis [18]. To our knowledge, this is the first report dealing with the electrophoretic separation of chitosan and of its products. However, PAGE has previously been used to characterize other charged polysaccharides [26-281. Chitosan oligosaccharides generated by enzymatic hydrolysis or acid hydrolysis were analyzed by gel electrophoresis in a 33 n/n w/v polqacrylamide gel. These oligosaccharides were compared in mobility with a series of purified chitosan oligosaccharides (tetramer, pentamer and hexamer; Fig. 2, lanes 1,2,3, respectively). Such purified oligosaccharides were obtained after preparative TLC separation. Chitosan oligosaccharides generated by 24 h of enzymatic hydrolysis (Fig. 2, lane 4) migrated as a series of bands between the pentamer and oligomers of20 residues and more. This was the same for chitosan oligosaccharides generated by acid hydrolysis (Fig. 2, lane 5). This technique also allowed the conclusion that barley chitosanases act as endochitosanases because of the series of hydrolysis products. Exochitosanases would only generate monomers or diiners depending on the mode of action. Note that Coomassie Brilliant Blue R-250 should be used for analysis of the fastest migrating oligosaccharides. Calcofluor White M2R is only suitable to stain decamers and oligomers of higher molecular mass. It is also noteworthy that D-glucosamine (monomer) and it:
