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Purification of ginger proteases by DEAE-Sepharose and isoelectric focusing
BB
ELSEVIER
Biochimicaet BiophysicaActa 1243 (1995) 181-184
etBiochi~ic~a BiophysicaAEta
Purification of ginger proteases by DEAE-Sepharose and isoelectric focusing Kozo Ohtsuki *, Kuniko Taguchi, Kenji Sato, Makoto Kawabata Department of Food ScienceandNutrition, KyotoPrefecturalUniversity,Nakaragi-cho,Shimogamo, Saltyo-ku,Kyoto606, Japan Received8 July 1994;accepted29 August 1994
Abstract
Ginger proteases in ginger rhizome (Zingiber officinale roscoe) were extracted from the ginger acetone powder and purified on DEAE-Sepharose and Sephadex G-75 columns. Before the purification, excess p-chloromercuribenzoate was added to the enzymes to prevent their autodigestion. The mercuribenzoate-proteases were further purified and fractionated by isoelectric focusing in Ampholine of pH 3-10 or pH 4-6. The proteases were fractionated into three components by the isoelectric focusing, having pl value of 4.5, 4.6 and 4.8 respectively. All these proteases had a molecular mass of 29000 as measured by SDS- polyacrylamide gel electrophoresis and by TSK G2000SW XL gel chromatography. The Ampholine in the purified enzymes can quickly be removed by the gel chromatography of TSK G2000SW. Some divalent metal ions, such as Hg 2÷, Cu2÷, Cd 2÷ and Zn2+, strongly inhibited these purified enzymes.
Keywords: Cysteineprotease; Gingerprotease; p-Chloromercuribenzoate;Isoelectricfocusing; SDS electrophoresis;TSK G2000 gel chromatography
1. Introduction
Ginger rhizome (Zingiber ofpcinale roscoe) has often been used as a peptic drug or the other drugs in Chinese medicine from olden times, and so it is of value to isolate several enzymes from the rhizome by chromatography. The protease in ginger rhizome was first reported by Michi et al. [1]. Ichikawa et al. separated the enzyme into two fractions, GP I and GP II, by DEAE-cellulose chromatography and reported the molecular mass of the enzymes to be 22500 by Sephadex G-100 gel-chromatography [2]. This value was too low in comparison with the value, 29000, which we got by Bio-Gel P-100 gel chromatography [5]. Some other properties of the enzyme were studied by several authors [3-8]. These studies indicate that the enzymes belong to the sulfhydryl proteases such as papain (EC 3.4.22.2) [9] and bromelins (EC 3.4.22.4) [14,151. In this report, the extracted ginger proteases were reacted with p-chloromercuribenzoate to get MB-proteases which do not autodigest and can be re-activated with some
Abbreviations: PCMB, p-chloromercuribenzoate; MB-protease, mercu-ribenzoate-protease;DTI', dithiothreitol;p/, isoelectricpoint; Mr, molecularmass; PMSF, phenylmethanesulfonylfluoride * Correspondingauthor. Fax: +81 75 7233503. 0304-4165/95/$09.50 © 1995 ElsevierScienceB.V. All rights reserved SSDI 0304-4165(94)00145-6
reducing reagents such as cysteine, 2-mercaptoethanol and DTT. The MB-proteases were purified by DEAE-Sepharose and Sephadex (G-25 and G-75) and then the enzymes were further purified and preparatively separated into several enzymes according to their pls by isoelectric focusing. The molecular masses of purified enzymes were then determined by SDS-gel electrophoresis and by gel chromatography with TSK G2000SW XL which is packed with a different material to Sephadex or Bio-Gel. The effect of some chemical reagents and metal ions on the activity of the purified protease was also investigated.
2. Materials and methods
2.1. Materials Ginger rhizome was obtained from Shimogamo municipal market. DEAE-Sepharose, Sephadex (G-75 and G-25) and Ampholine (pH 3-10 and pH 4-6) were obtained from Pharmacia LKB Co. The other chemicals (G.R. grade) were obtained from Nacalai Tesque Co.
2.2. Preparation of ginger protease Ginger rhizome was homogenized with a juicer and quickly mixed with 5 parts ( v / v ) of acetone [3]. The
182
K. Ohtsuki et al. / Biochimica et Biophysica Acta 1243 (1995) 181-184
homogenate was filtered and washed with another 5 parts of acetone. The powder was dried in a draft chamber. The dried powder was homogenized in 20 parts of 0.1 M phosphate buffer (pH 6.0), containing 0.5 mM DTF and the extract was filtered with cotton cloth. The filtrate was centrifuged at 16000 X g for 30 min and the supernatant was reacted with 5 molar excess of PCMB over DqT for 30 min at 4°C. The reaction mixture was then applied on a column of Sephadex G-25 (3.0 x 30 cm) which was preequilibrated with 20 mM phosphate buffer (pH 7.0), and the MB-protease was collected. The protease was then absorbed on a column of DEAE-Sepharose (3.0 × 10 cm) which was pre-equilibrated with 0.02 M phosphate buffer (pH 7.0) and the column was washed with the same buffer. The washing did not contain any protease activity. The MB-protease fraction was eluted as a purified state from the column with the same buffer containing 0.25 M NaC1, and after this elution, neither MB-protease nor active protease was eluted with 1 M NaC1 as checked with the enzyme assay. The purified MB-protease was then gelchromatographed on a column of Sephadex G-75 (1.5 x 40 cm) with distilled water to remove some low molecular contaminants and the protease fraction was lyophilized. 2.3. Enzyme assay
Protease activity was determined as follows [10]. Active or MB-protease, 0.1 ml, was mixed with 1 mM DTT, 0.1 ml, for 5 min, and then mixed with 2 ml of 1% casein (Hammarsten, Merck) in 0.1 M phosphate buffer (pH 6.0), and incubated for 20 min at 50°C. The reaction was stopped by adding 3 ml of 5% trichloroacetic acid. The reaction mixture was centrifuged and the soluble peptide in the supernatant fraction was determined by measuring an absorbance at 280 nm. In the experiments to test the effect of some reagents and metal ions on the protease activity, the enzyme reaction was carried out in the absence of EDTA, DTT and phosphate buffer. Equal volumes of the active protease solution and 2 mM reagent were mixed for 10 min at 15°C, and then the activity was assayed as described above. To get the active enzymes, the purified MB-enzyme solution, fractionated by isoelectric focusing as described below, 1.5 ml, was activated with 0.5 mM DTF, and then MB, DTT, and the other excess reagents (sucrose, Ampholine etc.) were removed from the activated enzyme by TSK G2000SW gel chromatography, equilibrated with 0.1 M acetate buffer, pH 6.0. 2.4. Isoelectric focusing
6.0 mg of the enzymes, 0.75% Ampholine (pH 3 - 1 0 and pH 4 - 6 ) and 0 - 3 2 % sucrose concentration gradient. The isoelectric focusing was performed at 700 V, 2 mA and 4°C for 40 h. After the isoelectric focusing, 1.5 ml fractions were collected from the bottom of the column, monitoring the absorbance at 280 nm for protein concentration with a micro-flowcell and a Hitachi model 100-50 spectrophotometer. The protease activity of the fractions was assayed as described above and the pH was measured with a Radiometer model PHM-62 pH meter. 2.5. Molecular mass determination
Slab polyacrylamide gel electrophoresis with 0.1% SDS (Mini Slab, Atto Co., Tokyo) [12] was used for the molecular mass determination of the proteases fractionated by the isoelectric focusing. Ovalbumin ( M r = 45 000, Sigma), bovine chymotrypsinogen A (PMSF-treated, M r = 25 000, Sigma), papain (PCMB-treated, M r = 23 000, Sigma) and subtilisin BPN' [13] (PMSF-treated, M r = 27600, Nagase Biochemicals) were used as the molecular mass markers. For molecular mass determination by gel chromatography, a JASCO HPLC apparatus model Tri-rotor (Tokyo) and a column of TSK G2000SW XL (Tosoh Co., Tokyo) equilibrated with 0.15 M CH3COONH 4 were used. In this experiment, bovine serum albumin ( M r = 66000, Sigma), ovalbumin, soybean trypsin inhibitor ( M r = 20100, Sigma), horse heart cytochrome c ( M r -- 12 400, Sigma) and bovine lung aprotinin ( M r = 6500, Nacalai Tesque Chem. Co.) were used as the M r markers.
3. Results and discussion 3.1. Fractionation of MB-ginger protease
One skew peak with protease activity was found in the isoelectric focusing of the MB-protease at pH 3 - 1 0 (Fig. 1). This means that this protease peak must contain some 1.2-
-~
oJ
10oo
o
0.8.
o g
=-
0,66
vo I
o_
=o o , 2 2
The purified proteases by DEAE-Sepharose as described above were further refined and fractionated according to their p l s by isoelectric focusing [11], at the same time, the determination of the p l s of the ginger proteases. An isoelectric focusing column (120 ml) was loaded with
ol L°
-
-
-
..~
lb
Tube
number
Fig. 1. Isoelectricfocusing of MB-gingerrhizome proteasein Ampholine pH 3-10. Six mg of the proteasewere applied. --, absorbanceat 280 nm for protein concentration; • - - • , proteaseactivity; O O O O, pH.
183
K. Ohtsuki et al. /Biochimica et Biophysica Acta 1243 (1995) 181-184
•,•1C
o~o~oooooO~O~oOO~o °
o6 ~
5
o
i
0.4
Sub- - - ~ .
o
o
o ~ ° ~ t
c"
o 4 o
° oo°
"5 2 7 0
Pop ,
J
0.2
0.2
I
1
0.4
~
~ I
0.6
J
I
0.8
I
I
1.0
Relative mobility
8 ~
3 10
20
30
40
50-
- 60
Tube number
Fig. 2. Isoelectricfocusing of MB-gingerrhizome proteasein Ampholine pH 4-6. Six mg of the proteasewere applied. The symbolsare the same as those in Fig. 1.
proteases with slightly different pls. This type of the pattern was found in every triplicate run. Then, we tried the second isoelectric focusing of the MB-protease, using a narrower pH range Ampholine, pH 4-6. Fig. 2 was a typical pattern of the isoelectric focusing in triplicate runs at pH 4-6. MB-proteases were clearly separated into three main components, having p I values of 4.5, 4.6 and 4.8. The proteases obtained here may contain both GP I and GP II which were named by Ichikawa et al. [2]. They separated the ginger proteases into two enzymes by DEAE-cellulose chromatography in the presence of 2 mM cysteine, in the active state, but did not determine the p I values of the enzymes. The protease peaks with p I values of 4.8 in Fig. 2 may belong to GP I and the proteases found between pH 4.5 and pH 4.6 in Fig. 2 may be GP II, because GP I was more positively charged than GP II. We here separated GP I and GP II into three components, which can easily be freed from Ampholine by TSK G2000SW gel chromatography preparatively within 20 min. These purified proteases, thus prepared, can be used for the other exmeriments. Such multiple forms of sulfhydryl proteases were reported in the cases of bromelin [15] and asclepain [16]. 3.2. Molecular mass
To determine the molecular mass of the ginger proteases fractionated by the isoelectric focusing as shown in Fig. 2, SDS-polyacrylamide gel electrophoreses at two kinds of poly-acrylamide gel concentration, 10% and 12.5%, were performed. The relative mobility of each ginger protease fractionated in Fig. 2 was the value shown with the arrow in Fig. 3. In this electrophoresis, we used the inhibited proteases, such as MB-papain, a representative sulfhydryl protease, and PMSF-treated subtilisin BPN' ( M r = 27600) [13] as the molecular mass markers. The
Fig. 3. Molecular mass determination of MB-ginger proteases by SDSpolyacrylamidegel electrophoresis. OVA, ovalbumin;Sub, PMSF-treated subtilisin BPN'; CTg, chymotrypsinogen;Pap, PCMB-treatedpapain; GP and arrow, the relative mobilities of the three fractionated proteases in Fig. 2; -©-, 10% acrylamide-gel;-O-, 12.5% acrylamidegel. result shown in Fig. 3 clearly indicates that the molecular mass of the ginger proteases is not 22 500 [2], but 29 000 [5], larger than that of papain. To assure the value of the molecular mass of the enzymes, we also tried TSK G2000SW gel chromatography, using the same molecular mass marker, bovine serum albumin, ovalbumin and cytochrome c, as Ichikawa et al. [2] did. We additionally used soybean trypsin inhibitor and bovine lung aprotinin. From the result of Fig. 4, all the ginger proteases isolated by the isoelectric focusing had a molecular mass of 29 000, which agreed with the value obtained in Fig. 3, and was larger value than that obtained by Ichikawa et al. who used Sephadex G-100 [2]. Since anomalous molecular masses of some enzymes by Sephadex G-100 gel chromatography were reported in some cases [17,18], it is necessary to use a few different methods for a molecular-mass determination of a protein. 3.3. Effects o f reagents and metal ions
Some reagents and divalent metal ions were reacted with these purified proteases (Table 1). Reagents, SDS, 10! 8 BSAN~N~.
4"
GP
Lo x .E ce
6
b g5 O.
i
8
L
9
I
10
Apro i
11
I
12
E l u t i o n volume (ml)
Fig. 4. Molecular mass determination of MB-ginger proteases by TSK G2000SW XL gel chromatography.BSA, bovine serum albumin; OVA, ovalbumin; STI, soybean trypsin inhibitor; Cyt, cytochrome c; Apro, bovine lung aprotinin; GP and arrow, the elution positions of the three fractionated proteases in Fig. 2.
184
K. Ohtsuki et al. / Biochimica et Biophysica Acta 1243 (1995) 181-184
Table 1 Effect of various reagents on the activity of ginger proteases
References
Reagents (1 mM)
[1] Michi, K., Osawa, H., Nakahama, N. and Sakurai, S. (1968) Kaseigaku Zasshi 19, 167-169. [2] Ichikawa, Y., Sasa, H. and Michi, K. (1973) Eiyo To Shokuryo 26, 377-383. [3] Thompson, E.H., Wolf, I.D. and Allen, C.E. (1973) J. Food Sci. 38, 652-655. [4] Ohtsuki, K., Kawabata, M. and Taguchi, K. (1975) Seikagaku 47, 470. [5] Ohtsuki, K., Kawabata, M. and Taguchi, K. (1978) Sci. Rep. Kyoto Pref. Univ. 29, 33-40. [6] Ohtsuki, K., Kawabata, M. and Taguchi, K. (1978) Seikagaku 50, 890. [7] Mega, A., Mitsuhashi, T., Fujiki, S. and Arakawa, N. (1983) Kaseigaku Zasshi 34, 79-82. [8] Lee, Y.B., Sehnert, D.J. and Ashmore, C.R. (1986) J. Food Sci. 51, 1558-1559. [9] Glazer, A.N. and Smith, E.L. (1971) in The Enzymes (Boyer, A.D., ed.), Vol. 3, pp. 501-546, Academic Press, New York. [10] Anson, M.L. (1938) J. Gen. Physiol. 22, 79. [11] Vesterberg, O. (1971) in Methods in Enzymology (Jakoby, W.B., ed.), Vol. 22, pp. 389-412, Academic Press, New York. [12] Laemmli, U.K. (1970) Nature 227, 680-685. [13] Matsubara, H., Kasper, C.B., Brown, D.M. and Smith, E.L. (1965) J. Biol. Chem. 240, 1125-1130. [14] Murachi, T. (1970) in Methods in Enzymology (Perlmann, G.E. and Lorand, L., eds.), Vol. 19, pp. 273-284, Academic Press, New York. [15] Rowan, A.D., Buttle, D.J. and Barrett, A.J. (1990) Biochem. J. 266, 869-875. [16] Lynn, K.R., Brockband, W.J. and Clevette, N.A. (1980) Biochim. Biophys. Acta 612, 119-125. [17] Voordouw, G., Gaucher, M. and Roche, R. (1974) Biochem. Biophys. Res. Commun. 58, 8-12. [18] McDowall, M.A. (1970) Eur. J. Biochem. 14, 214-221. [19] Sluyterman, L.A. (1967) Biochim. Biophys. Acta 139, 430-438.
None DTT EDTA PCMB SDS Ca2+ Zn2+ Cd2+ Hg 2+ Cu2+
Relative activity ( % of control) GP-pl 4.8
GP-pl 4.6
GP-pl 4.5
100 123 119 0 112 112 82.3 48.9 0 0
100 125 1t6 0 115 110 81.5 49.1 0 0
100 120 115 0 110 111 78.9 53.6 0 0
Enzyme activity was assayed with 1% casein in 0.1 M acetate buffer, pH 6.0, in the absence of DTT and EDTA
EDTA and Ca 2+ did not inhibit the activities of the three enzymes, while DTT activated the activities to about 1.2fold over the control. PCMB, Hg 2÷ and Cu 2÷ completely inhibited the enzymes, and Zn 2+ or Cd 2÷ also inhibited the enzymes. These inhibitions were prevented by adding EDTA and DTF. The three purified proteases thus showed an alike nature to the reagents. Sluyterman showed that papain, a typical cysteine protease, is inhibited by some divalent metal ions [19]. In conclusion, we have separated the ginger proteases, which were thought to be two [2], into three proteases (pI values of 4.5, 4.6 and 4.8) by isoelectric focusing. These enzymes are thought to be the forms of the original proteases in ginger rhizome, since these MB-enzymes do not autodigest during the purification and fractionation. The three proteases have similar features to some sulfhydryl-reacting reagents and metal ions.
ELSEVIER
Biochimicaet BiophysicaActa 1243 (1995) 181-184
etBiochi~ic~a BiophysicaAEta
Purification of ginger proteases by DEAE-Sepharose and isoelectric focusing Kozo Ohtsuki *, Kuniko Taguchi, Kenji Sato, Makoto Kawabata Department of Food ScienceandNutrition, KyotoPrefecturalUniversity,Nakaragi-cho,Shimogamo, Saltyo-ku,Kyoto606, Japan Received8 July 1994;accepted29 August 1994
Abstract
Ginger proteases in ginger rhizome (Zingiber officinale roscoe) were extracted from the ginger acetone powder and purified on DEAE-Sepharose and Sephadex G-75 columns. Before the purification, excess p-chloromercuribenzoate was added to the enzymes to prevent their autodigestion. The mercuribenzoate-proteases were further purified and fractionated by isoelectric focusing in Ampholine of pH 3-10 or pH 4-6. The proteases were fractionated into three components by the isoelectric focusing, having pl value of 4.5, 4.6 and 4.8 respectively. All these proteases had a molecular mass of 29000 as measured by SDS- polyacrylamide gel electrophoresis and by TSK G2000SW XL gel chromatography. The Ampholine in the purified enzymes can quickly be removed by the gel chromatography of TSK G2000SW. Some divalent metal ions, such as Hg 2÷, Cu2÷, Cd 2÷ and Zn2+, strongly inhibited these purified enzymes.
Keywords: Cysteineprotease; Gingerprotease; p-Chloromercuribenzoate;Isoelectricfocusing; SDS electrophoresis;TSK G2000 gel chromatography
1. Introduction
Ginger rhizome (Zingiber ofpcinale roscoe) has often been used as a peptic drug or the other drugs in Chinese medicine from olden times, and so it is of value to isolate several enzymes from the rhizome by chromatography. The protease in ginger rhizome was first reported by Michi et al. [1]. Ichikawa et al. separated the enzyme into two fractions, GP I and GP II, by DEAE-cellulose chromatography and reported the molecular mass of the enzymes to be 22500 by Sephadex G-100 gel-chromatography [2]. This value was too low in comparison with the value, 29000, which we got by Bio-Gel P-100 gel chromatography [5]. Some other properties of the enzyme were studied by several authors [3-8]. These studies indicate that the enzymes belong to the sulfhydryl proteases such as papain (EC 3.4.22.2) [9] and bromelins (EC 3.4.22.4) [14,151. In this report, the extracted ginger proteases were reacted with p-chloromercuribenzoate to get MB-proteases which do not autodigest and can be re-activated with some
Abbreviations: PCMB, p-chloromercuribenzoate; MB-protease, mercu-ribenzoate-protease;DTI', dithiothreitol;p/, isoelectricpoint; Mr, molecularmass; PMSF, phenylmethanesulfonylfluoride * Correspondingauthor. Fax: +81 75 7233503. 0304-4165/95/$09.50 © 1995 ElsevierScienceB.V. All rights reserved SSDI 0304-4165(94)00145-6
reducing reagents such as cysteine, 2-mercaptoethanol and DTT. The MB-proteases were purified by DEAE-Sepharose and Sephadex (G-25 and G-75) and then the enzymes were further purified and preparatively separated into several enzymes according to their pls by isoelectric focusing. The molecular masses of purified enzymes were then determined by SDS-gel electrophoresis and by gel chromatography with TSK G2000SW XL which is packed with a different material to Sephadex or Bio-Gel. The effect of some chemical reagents and metal ions on the activity of the purified protease was also investigated.
2. Materials and methods
2.1. Materials Ginger rhizome was obtained from Shimogamo municipal market. DEAE-Sepharose, Sephadex (G-75 and G-25) and Ampholine (pH 3-10 and pH 4-6) were obtained from Pharmacia LKB Co. The other chemicals (G.R. grade) were obtained from Nacalai Tesque Co.
2.2. Preparation of ginger protease Ginger rhizome was homogenized with a juicer and quickly mixed with 5 parts ( v / v ) of acetone [3]. The
182
K. Ohtsuki et al. / Biochimica et Biophysica Acta 1243 (1995) 181-184
homogenate was filtered and washed with another 5 parts of acetone. The powder was dried in a draft chamber. The dried powder was homogenized in 20 parts of 0.1 M phosphate buffer (pH 6.0), containing 0.5 mM DTF and the extract was filtered with cotton cloth. The filtrate was centrifuged at 16000 X g for 30 min and the supernatant was reacted with 5 molar excess of PCMB over DqT for 30 min at 4°C. The reaction mixture was then applied on a column of Sephadex G-25 (3.0 x 30 cm) which was preequilibrated with 20 mM phosphate buffer (pH 7.0), and the MB-protease was collected. The protease was then absorbed on a column of DEAE-Sepharose (3.0 × 10 cm) which was pre-equilibrated with 0.02 M phosphate buffer (pH 7.0) and the column was washed with the same buffer. The washing did not contain any protease activity. The MB-protease fraction was eluted as a purified state from the column with the same buffer containing 0.25 M NaC1, and after this elution, neither MB-protease nor active protease was eluted with 1 M NaC1 as checked with the enzyme assay. The purified MB-protease was then gelchromatographed on a column of Sephadex G-75 (1.5 x 40 cm) with distilled water to remove some low molecular contaminants and the protease fraction was lyophilized. 2.3. Enzyme assay
Protease activity was determined as follows [10]. Active or MB-protease, 0.1 ml, was mixed with 1 mM DTT, 0.1 ml, for 5 min, and then mixed with 2 ml of 1% casein (Hammarsten, Merck) in 0.1 M phosphate buffer (pH 6.0), and incubated for 20 min at 50°C. The reaction was stopped by adding 3 ml of 5% trichloroacetic acid. The reaction mixture was centrifuged and the soluble peptide in the supernatant fraction was determined by measuring an absorbance at 280 nm. In the experiments to test the effect of some reagents and metal ions on the protease activity, the enzyme reaction was carried out in the absence of EDTA, DTT and phosphate buffer. Equal volumes of the active protease solution and 2 mM reagent were mixed for 10 min at 15°C, and then the activity was assayed as described above. To get the active enzymes, the purified MB-enzyme solution, fractionated by isoelectric focusing as described below, 1.5 ml, was activated with 0.5 mM DTF, and then MB, DTT, and the other excess reagents (sucrose, Ampholine etc.) were removed from the activated enzyme by TSK G2000SW gel chromatography, equilibrated with 0.1 M acetate buffer, pH 6.0. 2.4. Isoelectric focusing
6.0 mg of the enzymes, 0.75% Ampholine (pH 3 - 1 0 and pH 4 - 6 ) and 0 - 3 2 % sucrose concentration gradient. The isoelectric focusing was performed at 700 V, 2 mA and 4°C for 40 h. After the isoelectric focusing, 1.5 ml fractions were collected from the bottom of the column, monitoring the absorbance at 280 nm for protein concentration with a micro-flowcell and a Hitachi model 100-50 spectrophotometer. The protease activity of the fractions was assayed as described above and the pH was measured with a Radiometer model PHM-62 pH meter. 2.5. Molecular mass determination
Slab polyacrylamide gel electrophoresis with 0.1% SDS (Mini Slab, Atto Co., Tokyo) [12] was used for the molecular mass determination of the proteases fractionated by the isoelectric focusing. Ovalbumin ( M r = 45 000, Sigma), bovine chymotrypsinogen A (PMSF-treated, M r = 25 000, Sigma), papain (PCMB-treated, M r = 23 000, Sigma) and subtilisin BPN' [13] (PMSF-treated, M r = 27600, Nagase Biochemicals) were used as the molecular mass markers. For molecular mass determination by gel chromatography, a JASCO HPLC apparatus model Tri-rotor (Tokyo) and a column of TSK G2000SW XL (Tosoh Co., Tokyo) equilibrated with 0.15 M CH3COONH 4 were used. In this experiment, bovine serum albumin ( M r = 66000, Sigma), ovalbumin, soybean trypsin inhibitor ( M r = 20100, Sigma), horse heart cytochrome c ( M r -- 12 400, Sigma) and bovine lung aprotinin ( M r = 6500, Nacalai Tesque Chem. Co.) were used as the M r markers.
3. Results and discussion 3.1. Fractionation of MB-ginger protease
One skew peak with protease activity was found in the isoelectric focusing of the MB-protease at pH 3 - 1 0 (Fig. 1). This means that this protease peak must contain some 1.2-
-~
oJ
10oo
o
0.8.
o g
=-
0,66
vo I
o_
=o o , 2 2
The purified proteases by DEAE-Sepharose as described above were further refined and fractionated according to their p l s by isoelectric focusing [11], at the same time, the determination of the p l s of the ginger proteases. An isoelectric focusing column (120 ml) was loaded with
ol L°
-
-
-
..~
lb
Tube
number
Fig. 1. Isoelectricfocusing of MB-gingerrhizome proteasein Ampholine pH 3-10. Six mg of the proteasewere applied. --, absorbanceat 280 nm for protein concentration; • - - • , proteaseactivity; O O O O, pH.
183
K. Ohtsuki et al. /Biochimica et Biophysica Acta 1243 (1995) 181-184
•,•1C
o~o~oooooO~O~oOO~o °
o6 ~
5
o
i
0.4
Sub- - - ~ .
o
o
o ~ ° ~ t
c"
o 4 o
° oo°
"5 2 7 0
Pop ,
J
0.2
0.2
I
1
0.4
~
~ I
0.6
J
I
0.8
I
I
1.0
Relative mobility
8 ~
3 10
20
30
40
50-
- 60
Tube number
Fig. 2. Isoelectricfocusing of MB-gingerrhizome proteasein Ampholine pH 4-6. Six mg of the proteasewere applied. The symbolsare the same as those in Fig. 1.
proteases with slightly different pls. This type of the pattern was found in every triplicate run. Then, we tried the second isoelectric focusing of the MB-protease, using a narrower pH range Ampholine, pH 4-6. Fig. 2 was a typical pattern of the isoelectric focusing in triplicate runs at pH 4-6. MB-proteases were clearly separated into three main components, having p I values of 4.5, 4.6 and 4.8. The proteases obtained here may contain both GP I and GP II which were named by Ichikawa et al. [2]. They separated the ginger proteases into two enzymes by DEAE-cellulose chromatography in the presence of 2 mM cysteine, in the active state, but did not determine the p I values of the enzymes. The protease peaks with p I values of 4.8 in Fig. 2 may belong to GP I and the proteases found between pH 4.5 and pH 4.6 in Fig. 2 may be GP II, because GP I was more positively charged than GP II. We here separated GP I and GP II into three components, which can easily be freed from Ampholine by TSK G2000SW gel chromatography preparatively within 20 min. These purified proteases, thus prepared, can be used for the other exmeriments. Such multiple forms of sulfhydryl proteases were reported in the cases of bromelin [15] and asclepain [16]. 3.2. Molecular mass
To determine the molecular mass of the ginger proteases fractionated by the isoelectric focusing as shown in Fig. 2, SDS-polyacrylamide gel electrophoreses at two kinds of poly-acrylamide gel concentration, 10% and 12.5%, were performed. The relative mobility of each ginger protease fractionated in Fig. 2 was the value shown with the arrow in Fig. 3. In this electrophoresis, we used the inhibited proteases, such as MB-papain, a representative sulfhydryl protease, and PMSF-treated subtilisin BPN' ( M r = 27600) [13] as the molecular mass markers. The
Fig. 3. Molecular mass determination of MB-ginger proteases by SDSpolyacrylamidegel electrophoresis. OVA, ovalbumin;Sub, PMSF-treated subtilisin BPN'; CTg, chymotrypsinogen;Pap, PCMB-treatedpapain; GP and arrow, the relative mobilities of the three fractionated proteases in Fig. 2; -©-, 10% acrylamide-gel;-O-, 12.5% acrylamidegel. result shown in Fig. 3 clearly indicates that the molecular mass of the ginger proteases is not 22 500 [2], but 29 000 [5], larger than that of papain. To assure the value of the molecular mass of the enzymes, we also tried TSK G2000SW gel chromatography, using the same molecular mass marker, bovine serum albumin, ovalbumin and cytochrome c, as Ichikawa et al. [2] did. We additionally used soybean trypsin inhibitor and bovine lung aprotinin. From the result of Fig. 4, all the ginger proteases isolated by the isoelectric focusing had a molecular mass of 29 000, which agreed with the value obtained in Fig. 3, and was larger value than that obtained by Ichikawa et al. who used Sephadex G-100 [2]. Since anomalous molecular masses of some enzymes by Sephadex G-100 gel chromatography were reported in some cases [17,18], it is necessary to use a few different methods for a molecular-mass determination of a protein. 3.3. Effects o f reagents and metal ions
Some reagents and divalent metal ions were reacted with these purified proteases (Table 1). Reagents, SDS, 10! 8 BSAN~N~.
4"
GP
Lo x .E ce
6
b g5 O.
i
8
L
9
I
10
Apro i
11
I
12
E l u t i o n volume (ml)
Fig. 4. Molecular mass determination of MB-ginger proteases by TSK G2000SW XL gel chromatography.BSA, bovine serum albumin; OVA, ovalbumin; STI, soybean trypsin inhibitor; Cyt, cytochrome c; Apro, bovine lung aprotinin; GP and arrow, the elution positions of the three fractionated proteases in Fig. 2.
184
K. Ohtsuki et al. / Biochimica et Biophysica Acta 1243 (1995) 181-184
Table 1 Effect of various reagents on the activity of ginger proteases
References
Reagents (1 mM)
[1] Michi, K., Osawa, H., Nakahama, N. and Sakurai, S. (1968) Kaseigaku Zasshi 19, 167-169. [2] Ichikawa, Y., Sasa, H. and Michi, K. (1973) Eiyo To Shokuryo 26, 377-383. [3] Thompson, E.H., Wolf, I.D. and Allen, C.E. (1973) J. Food Sci. 38, 652-655. [4] Ohtsuki, K., Kawabata, M. and Taguchi, K. (1975) Seikagaku 47, 470. [5] Ohtsuki, K., Kawabata, M. and Taguchi, K. (1978) Sci. Rep. Kyoto Pref. Univ. 29, 33-40. [6] Ohtsuki, K., Kawabata, M. and Taguchi, K. (1978) Seikagaku 50, 890. [7] Mega, A., Mitsuhashi, T., Fujiki, S. and Arakawa, N. (1983) Kaseigaku Zasshi 34, 79-82. [8] Lee, Y.B., Sehnert, D.J. and Ashmore, C.R. (1986) J. Food Sci. 51, 1558-1559. [9] Glazer, A.N. and Smith, E.L. (1971) in The Enzymes (Boyer, A.D., ed.), Vol. 3, pp. 501-546, Academic Press, New York. [10] Anson, M.L. (1938) J. Gen. Physiol. 22, 79. [11] Vesterberg, O. (1971) in Methods in Enzymology (Jakoby, W.B., ed.), Vol. 22, pp. 389-412, Academic Press, New York. [12] Laemmli, U.K. (1970) Nature 227, 680-685. [13] Matsubara, H., Kasper, C.B., Brown, D.M. and Smith, E.L. (1965) J. Biol. Chem. 240, 1125-1130. [14] Murachi, T. (1970) in Methods in Enzymology (Perlmann, G.E. and Lorand, L., eds.), Vol. 19, pp. 273-284, Academic Press, New York. [15] Rowan, A.D., Buttle, D.J. and Barrett, A.J. (1990) Biochem. J. 266, 869-875. [16] Lynn, K.R., Brockband, W.J. and Clevette, N.A. (1980) Biochim. Biophys. Acta 612, 119-125. [17] Voordouw, G., Gaucher, M. and Roche, R. (1974) Biochem. Biophys. Res. Commun. 58, 8-12. [18] McDowall, M.A. (1970) Eur. J. Biochem. 14, 214-221. [19] Sluyterman, L.A. (1967) Biochim. Biophys. Acta 139, 430-438.
None DTT EDTA PCMB SDS Ca2+ Zn2+ Cd2+ Hg 2+ Cu2+
Relative activity ( % of control) GP-pl 4.8
GP-pl 4.6
GP-pl 4.5
100 123 119 0 112 112 82.3 48.9 0 0
100 125 1t6 0 115 110 81.5 49.1 0 0
100 120 115 0 110 111 78.9 53.6 0 0
Enzyme activity was assayed with 1% casein in 0.1 M acetate buffer, pH 6.0, in the absence of DTT and EDTA
EDTA and Ca 2+ did not inhibit the activities of the three enzymes, while DTT activated the activities to about 1.2fold over the control. PCMB, Hg 2÷ and Cu 2÷ completely inhibited the enzymes, and Zn 2+ or Cd 2÷ also inhibited the enzymes. These inhibitions were prevented by adding EDTA and DTF. The three purified proteases thus showed an alike nature to the reagents. Sluyterman showed that papain, a typical cysteine protease, is inhibited by some divalent metal ions [19]. In conclusion, we have separated the ginger proteases, which were thought to be two [2], into three proteases (pI values of 4.5, 4.6 and 4.8) by isoelectric focusing. These enzymes are thought to be the forms of the original proteases in ginger rhizome, since these MB-enzymes do not autodigest during the purification and fractionation. The three proteases have similar features to some sulfhydryl-reacting reagents and metal ions.