Studies on the Myrosinase in Mustard Seed. Part I. The Chromatographic Behaviors of the Myrosinase Some of its Characteristics*

Transcription

1 [Agr. Biol. Chem., Vol. 31, No. 1, p. 1826, 1967] Studies on the Myrosinase in Mustard Seed Part I. The Chromatographic Behaviors of the Myrosinase and Some of its Characteristics* By Isao TsuRUO**, Mizuho YOSHIDA and Tadao HATA Research Institute for Food Science, Kyoto University, Kyoto **Technical Research Laboratory, Asahi Chemical Industry Co., Ltd., Tokyo Received July 4, 1966 In order to clarify whether the myrosinase containes only thioglucosidase, or thioglu cosidase and sulfatase, chromatographic behaviors of the myrosinase on cellulose ion exchanger were studied. Myrosinase showed two peaks by the chromatography on TEAEcellulose at ph 8.5, but the fractionation of the two enzymes was not accomplished. It was concluded that the two peaks was not ascribable to an artifact but the two closely resembled species of myrosinase existing in the sample solution. Chromatographic separa tion of the two enzymes on DEAE-cellulose resulted in failure. These results confirmed that the myrosinase was a single Ĉ-thioglucosidase originally. INTRODUCTION Previously, Gadamer1) assigned the structure (I) for mustard oil glucoside. This glucoside is hydrolysed by an enzyme, myrosinase, contained in the plants under Cruciferae family generating mustard oil (namely isothiocyanate), glucose and sulfuric acid. Neuberg et al." reported the separation of thioglucosidase and sulfatase which hydrolysed thioglucoside linkage and sulfuric acid ester linkage of (I) respectively. Thereafter, it had believed generally" that the myrosinase in plants was the mixture of the thioglucosidase and the sulfatase. * Parts of this work were presented at the Annual Meetings of the Agricultural Chemical Society of Japan, Sapporo, July 1964 and Tokyo, April ) J. Gadamer, Arch. Pharm., 235, 44 (1897). 2) C. Neuberg and 0. Schoenebeck, Biochem. Z., 265, 223 (1933); Naturwissenschaften, 21, 404 (1933). 3) "The For example; J. B. Sumner and K. Myrback, Enzymes", 1 st ed., vol. 1, part 1, Academic press, New York, Ettlinger et al.,') however, corrected the structure (I) to the structure (II) and suggested that the liberation of sulfate would occur by the Lossen rearrangement after the cleavage of the thioglucoside linkage. Nagashima et al." also supported the mechanism of Lossen rearrangement, and concluded that the myrosinase was not a mixture of the two enzymes but a single Ĉ-thioglucosidase and the liberation of sulfate would occur nonenzymatically. At that time Virtanen et al." found an enzymatic production of thiocyanate beside isothiocyanate from the mustard oil glucoside in some species of the plants exhibiting myrosinase activity. From this fact, some uncertainty occurred in the theory of Lossen rearrangement of the glucoside. Therefore, 4) M. G. Ettlinger and A. Lundeen, J. Am. Chem. Soc., 78, 41,72 (1956); 79, 1764 (1957). 5) Z. Nagashima and M. Uchiyama, J. Agr. Chem. Soc. Japan, 33, 1144 (1959); This journal, 23, 555 (1959). 6) R. Gmelin and A. Virtanen, Acta Chem. Scand., 13, 1474 (1959).

2 Studies on the Myrosinase in Mustard Seed. Part I 19 Gaines et al."' carried out further investiga tions on the homogeneity of the myrosinase, and successfully separated thioglucosidase and sulfatase. Furthermore, Ettlinger et al." suggested that there may exist two additional enzymes, one of which is activated by L-ascorbic acid. As outlined above, there are two fully opposing conceptions about the myrosinase. Considering about the above mentioned points, the authors have investigated the chro matographic behaviors of the myrosinase from yellow mustard seed with respect to the glucosidase and the sulfatase activities, and the effect of ascorbic acid on each activity; especially the strange behavior in the chromato graphy on TEAE-cellulose at ph 8.5. Some enzymatic chatacteristics of the myrosinase were also clarified with the sample which had been highly purified by the chromatography on TEAE-cellulose. From these experimental results, the propriety of the above stated hypotheses about the original nature of the myrosinase were discussed. MATERIALS AND METHODS Substrate. Sinigrin was prepared by the method of Nagashima et allot from commercial yellow mustard (Brassica juncea) seed, purchased from Takii Seed and Seedling Co., Kyoto. Enzyme preparation. The enzyme solution was prepared from commercial yellow mustard (Brassica juncea) seed, purchased from Takii Seed and Seedling Co., Kyoto. The extract from mustard seed by 0.5M NaCl, 0.01M 2-mercaptoethanol solution was frac tionated by ammonium sulfate at ph 7.0. The fraction precipitated from 0.5 to 0.7 saturation of ammonium sulfate was dialyzed against the same NaCI solution. The enzyme was purified ap proximately 12 fold and was stable for several 7) R. D. Gaines and K. J. Goering, lzoctzem. Biophys. Res. Comn., 2, 207 (1960). 8) R. D. Gaines and K. J. Goering, Arch. Biochem. Biophys., 96, 13 (1962). 9) M. G. Ettlinger, Harrison, T. J. Mabry G. P. Dateo, Jr., and C. P. Thompson, B. W. Proc. Natl. Acad. Sci., 47, 1875 (1961). 10) Z. Nagashima and M. Uchiyama, J. Agr. Chem. Soc. Japan, 33, 478 (1959). months at 5 C. The activity of this enzyme was lost by irreversible precipitation in the storage under 0 C. Ettlinger et al.9) stored their enzyme solution at 5 C. Because myrosinase of mustard seed often was accompanied with globlin-like protein and easily precipitated at low ionic strength, the above solution was used for the extraction of the enzyme. Assay of protein. Protein concentrations were determined by the method of Lowry et al.") FIG. 1. Chromatograms of Partially Purified Myrosi nase on SE-Cellulose. Protein (30mg) was charged on a 10ml SE-cellulose column equilibrated with 0.05M acetate buffer, ph 5.0. The chromatogram was developed by the same buffer containing increasing concentration of NaCl by factors 0.2M stepwise to 1.0M. Each fraction was 20 ml. The enzymatic activity was tested with (0) and without (/) 10-3M ascorbic acid. 11) 0. H. Lowry, N.J. Rosenbrough, A. L. Forr and R. T. Randall, J. Biol. Chem., 193, 265 (1951).

3 20 Isao TSURUO, Mizuho YOSHIDA and Tadao HATA Enzyme assay. Enzymatic reactions were carried out in the system containing 2.5 Đmoles of the substrate, 0.5 mmole of NaCI and 1 Đmole of 2- mercaptoethanol in 1 ml of water. The system for thioglucosidase assay contained 0.2 mmole of acetate (ph 5.2) additionally. In the case of measuring the activity in the presence of ascorbic acid, 1 Đmole of L-ascorbic acid was added to the system. The reactions were carried out at 37 C and ph 5.2 unless otherwise mentioned. Thioglucosidase activity and sulfatase activity were represented as the liberation of glucose and sulfate respectively. Glucose was determined by the following modifica tion of the Sumner's dinitrosalicylic acid method12). Five ml of dinitrosalicylic acid reagent (containing sodium bicarbonate instead of sodium bisulfite of the Sumner's original reagent) and 1 ml of solution of glucose (usually 1.0! mole/ml, and 0.5 Đmole/ml in the presence of ascorbic acid in reaction mixture) were added to the reaction mixture, I ml. The glucose solution was added in order to make the standard curve of glucose cross the origin. Then, the mixture was heated in boiling water for ten minutes, and immediately cooled by running water. Optical density of this mixture was measured at 530 mp. In this treatment, the substrate, sinigrin produced some colored materials, therefore the difference of the optical density with zero hour reaction was corrected by multiplying the factor, 1.25*. Sulfate was determined by titration with 0.01N NaOH, using a recording ph-stat, Radiometer Model SBR2/SBU1/TTT1 Autotitrator at ph 5.2. RESULTS 1. Column chromatography on cellulose ion exchanger 1) Chromatography on SE-cellulose. A column of 10 ml SE-cellulose (1 cm i. d.) was equili brated with 0.05 M acetate, ph 5.2. Myrosinase solution containing 30 mg of protein, dialyzed against the same buffer, was charged on the column and the protein was eluted by the same buffer containing increasing concentra tion of NaCl by factors 0.2M stepwise to 1.0M. Distinguishable behavior was not observed FIG. 2. Chromatography of the Partially Purified Myrosinase on TEAE - Cellulose in 0.02 M Veronal Buffer, 0.01 M 2-Mercaptoethanol, ph 8.5. Protein (3.5g) was contained in the sample solution. A linear gradient f rom 0 to 0.2 M NaCl was applied for elution of the orotein. A fraction was 2 0 ml. The fractions within the horizontal arrows were combined separatedly. The thioglucosidase activity in the presence (*) and absence (0) of 10-8 M ascorbic acid and the sulfatase activity in the presence (A) and absence (A) of 10-3 M ascorbic acid were tested and shown in the graph. 12) J. B. Sumner, J. Biol. Chem., 65, 393 (1925). * See the addendum.

4 Studies on the Myrosinase in Mustard Seed. Part I 21 between the thioglucosidase activity and the sulfatase activity (Fig. 1). 2) Chromatography on TEAE-cellulose. Myro sinase solution containing 3.5 g of protein was charged on a 150 ml TEAE-cellulose column (2 cm i. d.) equilibrated with 0.02 M veronal buffer, and 0.01 M 2-mercaptoethanol, ph 8.5. Ninety two per cent of the total protein broke through the column, though complete absorption of the enzymatic activity was observed. A linear gradient from 0 to 0.2 M NaCl was applied for elution of the protein (Fig. 2). Two separated peaks of the myrosinase activity appeared on the elution pattern, but the separation of the thioglu cosidase activity and the sulfatase activity could not be observed, and the ratio of the thioglucosidase activity against the sulfatase activity in these two peaks were almost equal, i.e. it was 76% in the former eluted peak and was 77% in the latter eluted peak (Calculated with the data presented in Table 1, step C). Furthermore, the position on the FIG. 3. Comparison of the First and the Rechromato graphy of the Myrosinase. A. Chromatogram of the partially purified myro sinase (same as Fig. 2). B. Chromatogram of the fraction I of Fig. 2. C. Chromatogram of the fraction II of Fig. 2. A linear gradient from 0 to 0.2 M NaCl in 0.02 M veronal buffer, 0.01 M 2-mercaptoethanol, ph 8.5 was applied for elution of the protein. In Figs. 3B and 3C, a fraction is 5 ml. The myrosinase activity was represented as the liberation of sulfate. In each chromatogram, the upper curve is the activity measured in the presence of 10-3M ascorbic acid. The fractions within the horizontal arrows in Figs. 3B and 3C were combined. FIG. 4. Chromatograms of the Myrosinase on UbAh- Cellulose. Protein (4.4 mg) was applied to a DEAF-cellulose column equilibrated with 0.02 M citrate buffer, ph 6.2. The chromatogram was developed with each 20 ml of the same buffer containing increasing con centration of NaCl by factors 0.2 M stepwise to 1.0 M. The enzymatic activity was tested in the presence (0) and absence (M) of 10-3M ascorbic acid.

5 22 Isao TSURUO, Mizuho YOSHIDA and Tadao HATA FIG. 5. Comparison of the Two Species of the Myrosinase. The upper graphs represent the enzymatic characteristics of the fraction I of Fig. 2 and the lower graphs represent those of the fraction II of Fig. 2. The enzymatic activity was measured by the liberation of sulfate with (A) and without (0) 10-3M ascorbic acid. A. PH-activity curves. In alkaline side of ph, the measurement of the enzymatic activity was carried out in stream of N2 gas. B. ph-stability curves. The sample solution was incubated in M citratephosphate buffer (below ph 7.0) or M tris buffer (above ph 7.5) at 37 C for 2 hours before measurement of the enzymatic activity. Each buffers for preincubation contained 0.5 M NaCl and 0.01M 2-mercaptoethanol. C. Temperature-stability curves. The sample solution were incubated in M citrate buffer, 0.5 M NaCl, 0.01M 2-mercaptoethanol, ph 6.0 for 10 min. at each temperature before measurement of the enzymatic activity. elution pattern of the activity peaks in the presence of 10-3m ascorbic acid coincided with those in the absence of ascorbic acid. A higher activity of sulfatase than that of thioglucosidase was observed when the enzymatic activity was tested in the presence of 10-3m ascorbic acid, but this disagreement of the observed two enzymatic activities would be interpreted as the effect of con centration of salts in the presence of ascorbic acid. This effect of salts will be explained in the following paper."' 3) Rechromatography on TEAE-cellulose. There occurred some doubts about the results showed in Fig. 2 whether the appearance of the two peaks was ascribable to an artifact 13) 1. Tsuruo and T. Hata, Part III of this study, in preparation. or not. Therefore, the two peaks of the enzymatic activity in Fig. 2 were combined separatedly, then each combined solution, after concentration by precipitation with ammonium sulfate, was rechromatographed on TEAE-cellulose under the same developing conditions as in the preceding chromato graphy (Fig. 3). In Fig. 3, the myrosinase activity was represented as the liberation of sulfate, but the same result was obtained when the enzymatic activity was presented as the liberation of glucose. This result shows that the two peaks of the myrosinase activity in Fig. 2 were not that of an artifact formed during the chromatographic procedure, but that the two entities of the myrosinase molecule really existed in the crude extract of yellow mustard seeds.

6 Studies on the Myrosinase in Mustard Seed. Part I 23 4) Chromatography on DEAE-cellulose. Gaines et al."" reported the separation of the thioglucosidase activity and the sulfatase activity of the white mustard myrosinase by chromatography on DEAE-cellulose at ph 6.2. Reinvestigation of their chromatographic experiment under the possibly same condi tions8) was carried out as follows. Myrosinase solution containing 4.4 mg of protein was dialyzed against 0.02M citrate buffer, ph 6.2. The dialyzate was charged on the column of 10 ml DEAE-cellulose (1 cm i. d.) equilibrated FIG. 6. Comparison of Lineweaver-Burk Plots of the Two Fractions of the Myrosinase in Fig. 2. The enzymatic activity was tested by the libera tion of sulfate. A: Fraction I, B: Fraction II. The lines (1) and (2) corresponds to the tests of enzymatic activity in the absence and presence of 10-3M ascorbic acid respectively. with the same buffer, then the protein was eluted with 0.02M citrate, ph 6.2 containing increasing concentration of NaCl by factors 0.2M stepwise to 1.0M. Both the thioglucosidase activity and the sulfatase activity passed the column with little absorption (Fig. 4). Thus the result of Gaines et al. could not be reproduced by the present authors. 2. On the two activity peaks appeared in the column chromatography on TEAE-cellulose As shown above, the two enzymatically active peaks in the chromatography on TEAEcellulose at ph 8.5 were not assumed as those of artifacts, therefore, the general charac teristics of each fraction were compared (Figs. 5 & 6). There were little differences between the two fractions on the curves of ph-activity, ph-stability and temperature-stability of the myrosinase. In Fig. 5, the myrosinase activity was represented as the liberation of sulfate, and the similar results were obtainable when the activity was given as the liberation of glucose. Nagashima et al.") had reported the accordance between the thioglucosidase activity and the sulfatase activity in these three curves in the absence of ascorbic acid. Nearly equal Michaelis' constants were obtained from their Lineweaver-Burk plots. The K,, values of them were calculated as 9.2x10-4M in the presence of 10-3 M ascorbic acid, 1.9x10-4M in the absence of ascorbic acid for the fraction I, and similarly 9.3x10-4M and 1.8x10-8M for the fraction II. These K.. values were calculated for the sul fatase activity but the equal K. values for each fraction were obtained by calculation for the thioglucosidase activity. These distinctions of the two fractions indicate that they are almost indistinguishable as the enzyme proteins except their chromato graphic behaviors. 14) Z. Nagashima and M. Uchiyama, J. Agr. Chem. Soc. Japan, 33, 484 (1959).

7 24 Isao TSURUO, Mizuho YOSHIDA and Tadao HATA TABLE 1. SUMMARY OF PURIFICATION OF MYROSINASE Tg-ase:- Thioglucosidase, S-ase:- Sulfatase 1) I and II of the steps C, D and E correspond to the separatedly combined fraction in the peaks I and II in Fig. 2 respectively. 2) Activities tested in the presence (+) and absence (-) of 10-3 M ascorbic acid were presented here. 3) The specific activity and the yield are calculated from the activity tested with 10-3M ascorbic acid. 3. Results of purification by column chromato graphy on TEAE-cellulose It was demonstrated that the column chro matography on TEAE-cellulose was effective for purification of the myrosinase in the above experiments. The typical results of the purification of the myrosinase of yellow mustard are presented in Table I. The enzyme was purified approximately 100 fold by this procedure. 4. Paper electrophoresis Nagashima et al."' applied paper electro phoresis for the myrosinase sample of white mustard and reported that the separation of the thioglucosidase activity and the sulfatase activity was not observed. On the contrary, Gaines et al." reported the separation of the both activities by boundary electrophoresis with the myrosinase sample from white mustard. Therefore, the reinvestigation of the behavior of the myrosinase in electro phoresis had become necessary. The paper electrophoresis of the myrosinase sample was carried out and the results were presented in Fig. 7. The experimental results similar to that of Nagashima et al. were obtained. In addition, the distribution of protein on the filter paper was tested by staining with brom cresol green. Two peaks were found and one of them accorded with the peak of the enzymatic activity. DISCUSSION The behaviors of the myrosinase in column chromatography on the several kinds of cellulose ion exchanger were investigated. It was elucidated that the separation of the thioglucosidase activity and the sulfatase activity was impossible with SE-cellulose, TEAE-cellulose and even with DEAE-cellulose at ph 6.2. Furthermore, the results of the paper electrophoretic experiment of the enzyme was the same with that of Nagashima et al. Therefore, the results of Gaines et al. on the chromatographic behavior of the enzyme, could not be confirmed by the present authors. Reexamination of their boundary electrophoretic experiments is also advisable. Nevertheless, the myrosinase was separated to the closely resembled two components by the chromatography on TEAE-cellulose at

8 Studies on the Myrosinase in Mustard Seed. Part I 25 FIG. 7. Paper Electrophoretical Patterns of the Myrosinase. One tenth ml of the partially purified enzyme solution containing 1 mg protein dialyzed against 0.1 M acetate buffer, ph 5.0 was spread upon Toyo filter paper, No. 51 with 6 cm width. Electro phoretical experiment carried out in the same buffer at 5 C with 200V, 20 ma/cm for 20 hours. After electrophoresis, the filter paper was cut off at each 1 cm length along the direction of migration and extracted by each 1 ml of 0.5 M NaCl solution. The enzymatic activity was determined in the presence of 10-3M ascorbic acid. ph 8.5. Such separation in chromatography on a cellulose ion exchanger was described by Neurath et ally ' about procarboxypeptidase B with DEAE-cellulose. They concluded that the appearence of two peaks on the chromato gram was due to an artifact. But the results of the present authors by the rechromato graphy showed that two peaks were resulted from two different species of the enzyme. That the thioglucosidase activity and the sul fatase activity could not be separated chro matographically on TEAE-cellulose in spite of such possible separation of the two closely resembled myrosinase species suggests a serious doubt about the postulate that the myrosinase is consisted of the two components, thioglu considase and sulfatase. Schwimmerls' attempted an explanation for the reason of inconsistency between the results 15) E. Wintersberger, ll.j. Cox and 171. Neurath, Biochem., 1, 1069 (1962). 16) S. Schwimmer, Acta Chem. Scand., 14, 1439 (1960). of Nagashima et al. and that of Gaines et al. from the points of the purity of the enzyme preparation from the viewpoint of the latter. But his explanation also can not be acceptable in comparison with the experi mental results by chromatography described in this paper. Kojima et al."' gave a different interpretation to the experiment which had formed the basis of the Schwim mer's explanation. The structure (II) was further confirmed by the X-ray diffraction study of sinigrin crystal by Waser et al.,"' by chemical synthesis applied nitrile oxide by Benn19) and by the study of biosynthesis in plants by Underhill.20) Uchiyama"' supported the reaction mechanism proposed by Ettlinger et al. and Nagashima et al. from his studies of the enzymatic and non-enzymatic degradation of the compound in which the sulfonic acid group of the structure (II) was substituted by tosyl group. Furthermore, Virtanen22) reported that the production of thiocyanate from mus tard oil glucoside was not the result of the direct action of the myrosinase. Therefore, it would be concluded that the generation of thiocyanate from mustard oil glucoside could not deny the occurrence of Lossen re arrangement in the enzymatic hydrolysis of the glucoside. These reports above mentioned are regarded to support the hypothesis of single enzyme about the myrosinase and the results obtained by the present authors also support it strongly. The attempts to distinguish the two 17) M. Kojima and K. Tamiya, J. Vitaminol. (Japan) 10, 44 (1964); Vitamins (in Japanese), 28, 380 (1963). 18) J. Waser and W. 11. Watson, Nature, 198, 1297 (1963). 19) M. H. Benn, Can. J. Chem., 41, 2836 (1963). 20) E. W. Underhill, Can. J. Biochem., 43, 179, 189 (1965). 21) M. Uchiyama, J. Agr. Chem. Soc. Japan, 37, 543 (1963). 22) A. I. Virtanen, Arch. Biochem. Biophys. Supple ment 1, 200 (1962).

9 26 Isao TSURUO, Mizuho YOSHIDA and Tadao HATA myrosinase species which had been suggested by Ettlinger et al. (one of which was activated by ascorbic acid) were not accomplished by the present authors. About the Michaelis' constant of the myrosinase, Schwimmer23) had reported it in the absence of ascorbic acid at 25 C and Ettlinger et al." in the presence of the acid at the same temperature. In this paper, the change of the K value in the presence or absence of 10-3M ascorbic acid at 37 C was described and it was found that both the K. and Vmax value of this enzyme were increased with the activation by ascorbic acid. This fact seems to be an important characteristics of the myrosinase. But the details of the activation of this enzyme by ascorbic acid will be represented in the following paper.24) ADDENDUM In the colorimetry of glucose, the multiplying factor for correction, 1.25, was induced as follows. When the optical densities corresponded s moles of the substrate and p moles of the product are S and P respectively, from law of Lambert and Beer 23) S. Schwimmer, Acta Chem. Scand., 15, 535 (1961). 24) I Tsuruo and T. Hata, This, journal, (1967). S=kss, P=kpp (ks and kp are constants) (I ) are obtained; and when A is the optical the reaction mixture after t hour, density of A=Pt+St (2) (suffixes of P, S and the optical densities corresponded to them represent the reaction time) is obtained. From the above definitions St=ksst=ks(s0-pt)=S0-ksPt/kp (3) is obtained. Then, from the equations (2) and (3), A is represented as A=So+(I-ks/kp)Pt (4) The following equation is yielded from the equations (1) and (4) Pt=Pt/kp=(A-S0)/kp(I-ks/kp) (5) If we regard the optical density A corresponds only to the product Papp. moles, then is obtained. a as A-So=kPPapp. (6) When, we take the correcting coefficient Pt=aPapp. (7 ) Then, from the equations (5) and (6), a is given as a=pt/papp.=1/(1-ks/kp) (8) The observed value of the actual measurement ks=0.101, kp=0.506 From these values, a is calculated (8) as follows: a=1.25 was by the equation