Znt. J. Peptide Protein Res. 14, 1979, 1-4 Published by Munksgaard, Copenhagen, Denmark No part m a y be reproduced by any process without written permission from the author(s)
THERMAL ACTIVATION O F P E R O X I D A S E FROM TOBACCO L E A F MESOPHYLL CELL WALLS YUEN-MIN CHOY, YUM-SHING WONG, KIT-MAN LAU and KUN
Biochemistry Department and Biology Department, The Chinese Shatin, N.T., Hong Kong
Received 30 May, accepted for publication 9 Nove Cell wall-bound peroxidase isolated from tobacco leaf meso found to consist of two isoenzymes (PIand Pz). Each exi single subunit with the same molecular weight (35 000) as de dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. exhibited maximum activities at 7 0 ' . PI increased to 265% and P2 to 140% o f the activities assayed at 270; below this temperature the two isoenzymes had the same specific activity. On hydrolysis, PI showed a carbohydrate content o f 26.22% when the monosaccharides were analyzed by gas liquid chromatography; Pz gave 21.45%. Key words: carbohydrate contents of isoperoxidases; isoperoxidases; peroxidases. thermal activation; tobacco leaf isoperoxidases.
Like most chemical reactions, the rate of enzyme-catalyzed reactions generally increases with temperature within the temperature range in which the enzyme is stable. The exhibition of a temperature optimum of enzymatic reactions usually at around 45", and not at too high temperatures, somehow reflects thermal activation versus denaturation. However, some types of enzymes have higher maxima, probably because they are stable even at high temperatures. The stability might be the result of the presence of carbohydrate in the enzyme molecules. Pazur & Aronson (1972) in working with the enzyme from Asperginus niger, which contains 15% sugar by weight, found that extensive oxidation by periodate of the carbohydrate residues in the enzyme markedly reduced its stability upon storage even in the cold. According to Sharon (1975), the major role of the carbohydrate moieties of some glycoproteins might be to assist in the maintenance of a specific tertiary structure. Support for this suggested role of the carbohydrate in maintain0367-8377/79/050001-04
ing the tridimensional structure could be obtained also from the work on glucoamylases (Pazur &Aronson, 1972). in these glycoenzymes, many carbohydrate side chains are present on the surface of the molecule in such a way as to minimize molecular transformation. Accordingly, an unfolding of the polypeptide chain is hindered because of the presence of the carbohydrate residues, and the integrity of the glycoenzyme is thus preserved. Since tobacco leaf peroxidase isoenzymes exhibit higher activities at 70°, it is our object to investigate whether these enzymes contain carbohydrate and also to try to explain the difference in activities of these isoenzymes at high temperature. EXPERIMENTAL PROCEDURES Materials and methods for the isolation and purification of tobacco leaf peroxidase were described by Yung e l al. (1979). Molecular weight determination
The sodium dodecyl sulphate (SDS) electro-
.$02.00/0 0 1979 Munksgaard, Copenhagen
1
Y.-M. CHOY ET AL.
phoresis was performed as described by Dunker & Ruechert (1969) in 10% acrylamide gels.
Myoglobin, chymotrypsinogen A, ovalbumin and bovine serum albumin (Scwarr/Mann Co., U.S.A.) were used as calibration markers. Before electrophoresis, all samples and markers were incubated at 37" for 2 h in 0.01 M phosphate buffer, pH 7, containing 1% sodium dodecyl sulphate, 1% 2-mercaptoethanol and 4 M urea. Electrophoresis was carried out at a constant current of 7 mA per tube for 5 h. Enzyme assay
The peroxidase activity was assayed by the modified method of Chance & Maehly (1955). One unit of peroxidase activity was defined as that which brings about a 1% increase in the &dmin. Action of heat on peroxidase activity
The isoperoxidase activities at various temperatures were assayed by using the same concentration of enzyme and substrate as described above. Neutral sugars and amino sugars determinations
The neutral sugars and amino sugars of the enzyme preparations were determined qualitatively and quantitatively by gas liquid chromatography as their alditol acetate derivatives (Niedermeir, 1971 ; Niedermeir & Tomana, 1974). Samples were dried to constant weight in an evacuated dessicator. For neutral sugars determination, about 1 mg of enzyme preparation was hydrolyzed with 1 ml 1 N HU in a sealed and evacuated tube at 100" for 2 h (Niedermeir, 1971). For amino sugars, about 1 mg of the sample was hydrolyzed in 1 ml 2 N HC1 in a sealed and evacuated tube at 105" for 5 h (Bahl, 1969). After cooling, the tube was opened and 0.2ml internal standard solution containing 0.25 pmol standard was added. For amino sugars, the internal standard used was N-acetylmannosamine and for neutral sugars, it wasmyo-inositol. The hydrolyzate was then neutralized to pH6 with Dowex 1 HCO;. The resin was removed by filtration. The monosaccharides in the filtrate were then reduced by an excess amount of sodium borohydride. Following reduction for 12h at 4", excess sodium borohydride was 2
decomposed by the addition of 200pl 6 N HCl and the sample was evaporated to dryness by the use of a rotatory evaporator. Borate was removed as volatile trimethyl borate by the addition of five 5-mI portions of methanol with concentration to dryness by a rotatory evaporator after each addition. For acetylation, 0.4ml pyridine and 0.4ml acetic anhydride were added to the dry residue and the flask was then capped. The sample was heated at 100" for 20 min. After cooling, 5 ml distilled water was added. The pyridine together with water was co-distilled to dryness by a rotatory evaporator. This treatment was repeated five times. The dry acetylated mixture was dissolved in 2 0 f l chloroform. 1111 of this aliquot was injected into a Hewlett-Packard Model 402B gas chromatograph, equipped with dual-flame ionization detectors with a single channel electrometer and 6' U-shape 4'' diameter glass columns. To determine the amino sugars, the column packed with 3% Poly A-103 on 100/120 mesh Gas Chrom. Q was used, and the chromatographic temperature was 230". For neutral sugars determination, the column used was packed with 3% ECNSS-M on 100/120 mesh Gas Chrom. Q and the temperature was 200'. Standard alditol acetate derivatives for neutral sugars (containing equal molar amounts of fucose, arabinose, xylose, mannose, galactose, @ucose and myo-inositol) and for amino sugars (containing mannosamine, galactosamine and glucosamine) were used to determine the retention times and the molar responses of the detector to the sugars relative to its response to the internal standards. Sialic acid determination
Sialic acid was released from the enzyme preparations according to Codington et al. (1976). About 0.5 mg sample was hydrolyzed with 0.20ml 0.05 N H2S04in a centrifuge tube at 80' for 1 h. The liberated sialic acid was assayed by thiobarbituric method of Warren (1959). RESULTS Molecular weight determination
When the two isoenzymes were incubated in the presence and absence of mercaptolethanol
TOBACCO LEAF MESOPHYLL PEROXIDASE TABLE 1 Carbohydrate compositions of the peroxidase isoenzymes g/100 g dry samplea Carbohydrate
PI
Molar Response Factor
p,
Fucose Arabinose Xylose Mannose Galactose Glucose N-Acetylglucosamine N-Acetylgalactosamine Sialic acid
0.33 2.45 16.81 1.46 2.75 2.22 0.20 trace nil -
-
Total carbohydrate content
26.22
21.45
4.9
-
Carbohydrate compositions of peroxidase isoenzymes
3 L
f
-Y0
=
P 0
am4.4
-
4.P
-
1.17b 0.9gb 0.94b 1.Ogb 1.14b l.lSb 1.WC 0.93'
the activities assayed at 27" respectively. When the reaction temperature was increased to 90°, both isoenzymes were completely inhibited.
50 -
I'i
0.32 3.22 8.80 1.85 3.73 3.25 0.28 trace nil
The carbohydrate compositions of both enzymes are summarized in Table 1. It was found that PI had a higher content of carbohydrate (26.22%) than P2 (21.45%). The two enzyme preparations contained no sialic acid.
B\P
\
a
DISCUSSION 4-01
It has been found that isoenzymes of invertase become denatured at rates that decrease as their Rdativa Mobility carbohydrate contents increase (Arnold, 1969). Isoenzymes having a high content of carbohydrate are quite resistant to denaturation, FIGURE 1 Plot of the logarithm of the molecular weights of whereas isoenzymes low in carbohydrate are protein standards versus their relative mobilities on rapidly denatured. Our present findings support 10% polyacrylamide gel containing 0.1% SDS: A, the above contentions. The two isoenzymes of bovine serum albumin; B, ovalbumin; C, chymo- tobacco leaf peroxidase were found to have trypsinogen A; D, myoglobin; P,peroxidase isoenzyme. high carbohydrate contents thus conferring the 0
0.P
0.4
0s
0.9
1.0
3
Y.-M. CHOY ET AL.
stabilities of these isoenzymes at high tempera- had roughly the same specific activity. The ture. As a result, both isoenzymes exhibited increase of activity can be due to thermal actimaximum activities at 70'. PI increased to 265% vation as predicted by the kinetic theory. The and Pz to 140% of the activities assayed at 27'; higher activity of PI is probably due to the below this temperature the two isoenzymes different degree of denaturation of the two isoenzymes as proved by the fact that PI has a higher carbohydrate content (26.22%) than Pz (21.45%). ACKNOWLEDGEMENTS We thank Mr. Lee Wing Tat, Mr. KO Fook Son and the Takshing Investment Co., Ltd., Hong Kong for financial support. REFERENCES Arnold, W.N. (1969) Bioehim. Biophys. Aeta 178, 347-353 Bahl, O.P. (1969)J. BioL Chem. 244,567-574 Chance, B. & Maehly, A.C. (1955) Methods Enzymol. 2,764-775 Codington, J.F., Linsley, K.B. & Silber, C. (1976) Methods Carbohyd. Chem. 7,226-232 Dunker, A.K. & Ruechert, R.R. (1969)J. Biol. Chem. 244,5074-5080 Niedermeir, W. (1971) A n d . Biochem 40,465-475 Niedermeir, W. & Tomana, M. (1974) Anal. Biochem. 57,363-368 Pazur, J.H.& Aronson, N.N. (1972) Adv. Carbohyd. Chem Biochem. 27,301-341 Sharon, N. (1975) in Complex Carbohydrate, pp. 177- 192, Addison-Wesley, U.S.A. Warren, L. (1959)J. Biol. Chem. 234,1971-1975 Yung, K.H.,Wong, Y.S. & Choy, Y.M. (1979)Int. J. Peptide Protein Res., 14.5-11 FIGURE 2 Effect of temperature on the activity of peroxidase isoenzymes PI (.-*-.) and P2 (0-0-0). Duration of incubation: 15s. Activity at 27" was designed at 100%.
4
Address:
Dr.Kung-Hing Yung Department of Biology The Chinese University of Hong Kong Shatin, N.T., Hong Kong
THERMAL ACTIVATION O F P E R O X I D A S E FROM TOBACCO L E A F MESOPHYLL CELL WALLS YUEN-MIN CHOY, YUM-SHING WONG, KIT-MAN LAU and KUN
Biochemistry Department and Biology Department, The Chinese Shatin, N.T., Hong Kong
Received 30 May, accepted for publication 9 Nove Cell wall-bound peroxidase isolated from tobacco leaf meso found to consist of two isoenzymes (PIand Pz). Each exi single subunit with the same molecular weight (35 000) as de dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. exhibited maximum activities at 7 0 ' . PI increased to 265% and P2 to 140% o f the activities assayed at 270; below this temperature the two isoenzymes had the same specific activity. On hydrolysis, PI showed a carbohydrate content o f 26.22% when the monosaccharides were analyzed by gas liquid chromatography; Pz gave 21.45%. Key words: carbohydrate contents of isoperoxidases; isoperoxidases; peroxidases. thermal activation; tobacco leaf isoperoxidases.
Like most chemical reactions, the rate of enzyme-catalyzed reactions generally increases with temperature within the temperature range in which the enzyme is stable. The exhibition of a temperature optimum of enzymatic reactions usually at around 45", and not at too high temperatures, somehow reflects thermal activation versus denaturation. However, some types of enzymes have higher maxima, probably because they are stable even at high temperatures. The stability might be the result of the presence of carbohydrate in the enzyme molecules. Pazur & Aronson (1972) in working with the enzyme from Asperginus niger, which contains 15% sugar by weight, found that extensive oxidation by periodate of the carbohydrate residues in the enzyme markedly reduced its stability upon storage even in the cold. According to Sharon (1975), the major role of the carbohydrate moieties of some glycoproteins might be to assist in the maintenance of a specific tertiary structure. Support for this suggested role of the carbohydrate in maintain0367-8377/79/050001-04
ing the tridimensional structure could be obtained also from the work on glucoamylases (Pazur &Aronson, 1972). in these glycoenzymes, many carbohydrate side chains are present on the surface of the molecule in such a way as to minimize molecular transformation. Accordingly, an unfolding of the polypeptide chain is hindered because of the presence of the carbohydrate residues, and the integrity of the glycoenzyme is thus preserved. Since tobacco leaf peroxidase isoenzymes exhibit higher activities at 70°, it is our object to investigate whether these enzymes contain carbohydrate and also to try to explain the difference in activities of these isoenzymes at high temperature. EXPERIMENTAL PROCEDURES Materials and methods for the isolation and purification of tobacco leaf peroxidase were described by Yung e l al. (1979). Molecular weight determination
The sodium dodecyl sulphate (SDS) electro-
.$02.00/0 0 1979 Munksgaard, Copenhagen
1
Y.-M. CHOY ET AL.
phoresis was performed as described by Dunker & Ruechert (1969) in 10% acrylamide gels.
Myoglobin, chymotrypsinogen A, ovalbumin and bovine serum albumin (Scwarr/Mann Co., U.S.A.) were used as calibration markers. Before electrophoresis, all samples and markers were incubated at 37" for 2 h in 0.01 M phosphate buffer, pH 7, containing 1% sodium dodecyl sulphate, 1% 2-mercaptoethanol and 4 M urea. Electrophoresis was carried out at a constant current of 7 mA per tube for 5 h. Enzyme assay
The peroxidase activity was assayed by the modified method of Chance & Maehly (1955). One unit of peroxidase activity was defined as that which brings about a 1% increase in the &dmin. Action of heat on peroxidase activity
The isoperoxidase activities at various temperatures were assayed by using the same concentration of enzyme and substrate as described above. Neutral sugars and amino sugars determinations
The neutral sugars and amino sugars of the enzyme preparations were determined qualitatively and quantitatively by gas liquid chromatography as their alditol acetate derivatives (Niedermeir, 1971 ; Niedermeir & Tomana, 1974). Samples were dried to constant weight in an evacuated dessicator. For neutral sugars determination, about 1 mg of enzyme preparation was hydrolyzed with 1 ml 1 N HU in a sealed and evacuated tube at 100" for 2 h (Niedermeir, 1971). For amino sugars, about 1 mg of the sample was hydrolyzed in 1 ml 2 N HC1 in a sealed and evacuated tube at 105" for 5 h (Bahl, 1969). After cooling, the tube was opened and 0.2ml internal standard solution containing 0.25 pmol standard was added. For amino sugars, the internal standard used was N-acetylmannosamine and for neutral sugars, it wasmyo-inositol. The hydrolyzate was then neutralized to pH6 with Dowex 1 HCO;. The resin was removed by filtration. The monosaccharides in the filtrate were then reduced by an excess amount of sodium borohydride. Following reduction for 12h at 4", excess sodium borohydride was 2
decomposed by the addition of 200pl 6 N HCl and the sample was evaporated to dryness by the use of a rotatory evaporator. Borate was removed as volatile trimethyl borate by the addition of five 5-mI portions of methanol with concentration to dryness by a rotatory evaporator after each addition. For acetylation, 0.4ml pyridine and 0.4ml acetic anhydride were added to the dry residue and the flask was then capped. The sample was heated at 100" for 20 min. After cooling, 5 ml distilled water was added. The pyridine together with water was co-distilled to dryness by a rotatory evaporator. This treatment was repeated five times. The dry acetylated mixture was dissolved in 2 0 f l chloroform. 1111 of this aliquot was injected into a Hewlett-Packard Model 402B gas chromatograph, equipped with dual-flame ionization detectors with a single channel electrometer and 6' U-shape 4'' diameter glass columns. To determine the amino sugars, the column packed with 3% Poly A-103 on 100/120 mesh Gas Chrom. Q was used, and the chromatographic temperature was 230". For neutral sugars determination, the column used was packed with 3% ECNSS-M on 100/120 mesh Gas Chrom. Q and the temperature was 200'. Standard alditol acetate derivatives for neutral sugars (containing equal molar amounts of fucose, arabinose, xylose, mannose, galactose, @ucose and myo-inositol) and for amino sugars (containing mannosamine, galactosamine and glucosamine) were used to determine the retention times and the molar responses of the detector to the sugars relative to its response to the internal standards. Sialic acid determination
Sialic acid was released from the enzyme preparations according to Codington et al. (1976). About 0.5 mg sample was hydrolyzed with 0.20ml 0.05 N H2S04in a centrifuge tube at 80' for 1 h. The liberated sialic acid was assayed by thiobarbituric method of Warren (1959). RESULTS Molecular weight determination
When the two isoenzymes were incubated in the presence and absence of mercaptolethanol
TOBACCO LEAF MESOPHYLL PEROXIDASE TABLE 1 Carbohydrate compositions of the peroxidase isoenzymes g/100 g dry samplea Carbohydrate
PI
Molar Response Factor
p,
Fucose Arabinose Xylose Mannose Galactose Glucose N-Acetylglucosamine N-Acetylgalactosamine Sialic acid
0.33 2.45 16.81 1.46 2.75 2.22 0.20 trace nil -
-
Total carbohydrate content
26.22
21.45
4.9
-
Carbohydrate compositions of peroxidase isoenzymes
3 L
f
-Y0
=
P 0
am4.4
-
4.P
-
1.17b 0.9gb 0.94b 1.Ogb 1.14b l.lSb 1.WC 0.93'
the activities assayed at 27" respectively. When the reaction temperature was increased to 90°, both isoenzymes were completely inhibited.
50 -
I'i
0.32 3.22 8.80 1.85 3.73 3.25 0.28 trace nil
The carbohydrate compositions of both enzymes are summarized in Table 1. It was found that PI had a higher content of carbohydrate (26.22%) than P2 (21.45%). The two enzyme preparations contained no sialic acid.
B\P
\
a
DISCUSSION 4-01
It has been found that isoenzymes of invertase become denatured at rates that decrease as their Rdativa Mobility carbohydrate contents increase (Arnold, 1969). Isoenzymes having a high content of carbohydrate are quite resistant to denaturation, FIGURE 1 Plot of the logarithm of the molecular weights of whereas isoenzymes low in carbohydrate are protein standards versus their relative mobilities on rapidly denatured. Our present findings support 10% polyacrylamide gel containing 0.1% SDS: A, the above contentions. The two isoenzymes of bovine serum albumin; B, ovalbumin; C, chymo- tobacco leaf peroxidase were found to have trypsinogen A; D, myoglobin; P,peroxidase isoenzyme. high carbohydrate contents thus conferring the 0
0.P
0.4
0s
0.9
1.0
3
Y.-M. CHOY ET AL.
stabilities of these isoenzymes at high tempera- had roughly the same specific activity. The ture. As a result, both isoenzymes exhibited increase of activity can be due to thermal actimaximum activities at 70'. PI increased to 265% vation as predicted by the kinetic theory. The and Pz to 140% of the activities assayed at 27'; higher activity of PI is probably due to the below this temperature the two isoenzymes different degree of denaturation of the two isoenzymes as proved by the fact that PI has a higher carbohydrate content (26.22%) than Pz (21.45%). ACKNOWLEDGEMENTS We thank Mr. Lee Wing Tat, Mr. KO Fook Son and the Takshing Investment Co., Ltd., Hong Kong for financial support. REFERENCES Arnold, W.N. (1969) Bioehim. Biophys. Aeta 178, 347-353 Bahl, O.P. (1969)J. BioL Chem. 244,567-574 Chance, B. & Maehly, A.C. (1955) Methods Enzymol. 2,764-775 Codington, J.F., Linsley, K.B. & Silber, C. (1976) Methods Carbohyd. Chem. 7,226-232 Dunker, A.K. & Ruechert, R.R. (1969)J. Biol. Chem. 244,5074-5080 Niedermeir, W. (1971) A n d . Biochem 40,465-475 Niedermeir, W. & Tomana, M. (1974) Anal. Biochem. 57,363-368 Pazur, J.H.& Aronson, N.N. (1972) Adv. Carbohyd. Chem Biochem. 27,301-341 Sharon, N. (1975) in Complex Carbohydrate, pp. 177- 192, Addison-Wesley, U.S.A. Warren, L. (1959)J. Biol. Chem. 234,1971-1975 Yung, K.H.,Wong, Y.S. & Choy, Y.M. (1979)Int. J. Peptide Protein Res., 14.5-11 FIGURE 2 Effect of temperature on the activity of peroxidase isoenzymes PI (.-*-.) and P2 (0-0-0). Duration of incubation: 15s. Activity at 27" was designed at 100%.
4
Address:
Dr.Kung-Hing Yung Department of Biology The Chinese University of Hong Kong Shatin, N.T., Hong Kong
