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In situ observation of the generation of isothiocyanates from sinigrin in horseradish and wasabi
Biochimica et Biophysica Acta 1527 (2001) 156^160
www.bba-direct.com
In situ observation of the generation of isothiocyanates from sinigrin in horseradish and wasabi Eileen Y. Yu a , Ingrid J. Pickering a , Graham N. George a
a;
*, Roger C. Prince
b
Stanford Synchrotron Radiation Laboratory, Stanford Linear Accelerator Center, 2575 Sand Hill Road, Stanford, CA 94025, USA b ExxonMobil Research and Engineering Company, Route 22 East, Annandale, NJ 08801, USA Received 29 January 2001; received in revised form 25 May 2001; accepted 25 May 2001
Abstract Sulfur K-edge X-ray absorption spectroscopy has been used to determine the chemical identity of the sulfur-containing species in horseradish (Armoracia lapthifolia) and wasabi (Wasabia japonica) in situ, before and after cell disruption. The major sulfur-containing species in the intact root is sinigrin (1-thio-L-D-glucopyranose 1-N-(sulfoxy)-3-buteneimidate) and related congeners. Disrupting the cells by applying local pressure allowed the conversion of the sulfur moieties in sinigrin to isothiocyanates and sulfate in approximately equimolar amounts. In contrast to previous suggestions, no detectable thiocyanates were formed, but an unusual thio intermediate may have been identified for the first time. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : X-ray absorption spectroscopy ; Isothiocyanate; Sinigrin; Myrosinase
1. Introduction Glucosinolates, or more accurately L-D-S-glucosides (Fig. 1, compound I), are widely distributed in cruciferous plants (Brassicaceae) ; they form a large family that di¡er in the substituent R (Fig. 1). The plants also produce a L-thioglucoside glucohydrolase (EC 3.2.3.1), commonly known as myrosinase, that catalyzes their hydrolysis. Glucosinolates probably play roles in resistance to fungi, nematodes and other plant pathogens, and herbivores [1^3]. They may also serve to store inactive precursors of plant hormones such as 3-indolylacetic acid [4], and be important in the storage and inactivation of ascorbic acid [5]. Glucosinolates are also receiving attention because of possible bene¢cial e¡ects in the human diet [6,7]. The glucosinolate known as sinigrin (1-thio-L-D-glucopyranose 1-N(sulfoxy)-3-buteneimidate; Fig. 1) is particularly abundant in horseradish (Armoracia lapthifolia) and wasabi (Wasabia japonica) [8,9]. In the intact cell, glucosinolates are compartmentalized in subcellular organelles, quite likely the vacuole [10]. When the cell is disrupted, the glucosinolate substrates mix with the membrane-associated cytosolic myrosinases
* Corresponding author. Fax: +1-650-926-4604. E-mail address : [email protected] (G.N. George).
[10,11]. Sinigrin itself is unpleasantly bitter, with no discernable odor, but its hydrolysis by myrosinases releases intensely lachrymatory (and £avorful) compounds. In both horseradish and wasabi, a series of isothiocyanates is produced as a result of cell damage [12], and the most abundant volatile compound released is allyl isothiocyanate [12] (Fig. 1, V), about 130 mg/100 g wet weight `root'. Allyl isothiocyanate is volatile (boiling point 150³C), and so intensely lachrymatory that it was used experimentally as a war gas by the German army in the First World War [13]. On the milder side, the more abundant larger compounds (4-pentenyl- and 6-methylsul¢nylhexyl- in wasabi, L-phenylethyl- in horseradish [12]) are perhaps important in the subtle culinary di¡erences between the two plants. Although the breakdown of sinigrin by myrosinase shown in Fig. 1 is thought to be a hydrolysis followed by a (non-enzymatically catalyzed) Lossen rearrangement [14,15], there have been suggestions that thiocyanates and nitriles are also produced [16]. Sulfur K-edge X-ray absorption spectroscopy can be used to identify the chemical forms of sulfur species in situ [17^19] and we have therefore addressed this possibility using X-ray absorption spectroscopy at the sulfur K-edge. We ¢nd no evidence for the generation of thiocyanates, but do see an essentially quantitative conversion of sinigrin to isothiocyanates and sulfate when cells are damaged. Furthermore, we have found evidence of the chemical precursor (IV) to the iso-
0304-4165 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 0 1 ) 0 0 1 6 1 - 1
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Fig. 1. Schematic of the degradation of L-D-S-glucosides (I) by myrosinase (L-thioglucoside glucohydrolase) and related biochemistry. The initial hydrolysis of (I) gives rise to glucose (II) and (III), which will be in equilibrium with (IV). (IV) is then thought to undergo a Lossen rearrangement to produce the isothiocyanate (V) and sulfate.
thiocyanate, which possesses the unusual thio ( s C = S) motif. 2. Materials and methods Horseradish (A. lapthifolia) and wasabi (W. japonica) were purchased fresh at local markets, and the former was also harvested at Stony Brook Apiary, NJ. The parts of the plant typically used for culinary purposes and available commercially are technically the root of horseradish and the stem of wasabi, but we will follow common parlance and refer to both as roots in this paper. Thin sections were prepared and mounted under 10 Wm thick polypropylene ¢lm to retard desiccation during exposure to the X-ray beam. Toluene extracts were prepared with an electrical food `juicer', the juice delivered directly into toluene to trap volatile organic compounds. No signi¢cant di¡erences were seen between freshly harvested (2 days between harvest and spectroscopy) and commercially obtained horseradish roots. Model compounds were purchased from Aldrich. Sulfur K-edge X-ray absorption spectra were collected on beamline 6-2 at the Stanford Synchrotron Radiation Laboratory using a Si(111) double crystal monochromator and a downstream Ni-coated harmonic rejection mirror. Energy resolution was optimized by decreasing the vertical aperture upstream from the monochromator until no further sharpening of features of the near-edge spectrum of a sodium thiosulfate (Na2 S2 O3 W5H2 O) standard was detected. The optimized energy resolution was quantitatively determined to be 0.51 eV by measuring the width of the 2471.4 eV 1sCZ*(3b1 ) transition of gaseous SO2 (using pseudo-Voigt peak deconvolution), which corresponds to a transition to a single orbital, rather than to a band of orbitals which can be the case with solid standards [20]. The incident X-ray intensity was monitored using a helium-¢lled ion chamber [17], and the X-ray absorption spectra were monitored in £uorescence using a Stern^Heald^ Lytle detector. The energy scale was calibrated with refer-
ence to the lowest energy peak of a sodium thiosulfate standard, assumed to be 2469.2 eV [21], and was reproducible to less than 0.1 eV. All spectra were recorded at room temperature, and samples had less than 10 min of exposure to the X-ray beam. More prolonged exposure to the X-ray beam (approx. 20 min) resulted in no signi¢cant
Fig. 2. Sulfur K-edge X-ray absorption spectra of compounds likely to be present in the roots of horseradish and wasabi. Allyl isothiocyanate (a), ethyl thiocyanate (b), cysteine (c) (a representative thiol) and methionine (d) (a representative thioether), sinigrin (e), and sulfate (f). Allyl isothiocyanate and ethyl thiocyanate were in toluene solution, and all other compounds were in aqueous solutions at neutral pH. The spectra are normalized to the sulfur K-edge jump, which is equivalent to total sulfur concentration (note that sinigrin (e) contains two di¡erent sulfurs). The spectrum of sulfate is very sharp, so it is displayed reduced by a factor of 3.
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changes in the spectra, indicating that X-ray photoreduction of the sample is probably insigni¢cant. Data were collected using the program XAS-Collect [22], and data analysis was carried out using the EXAFSPAK suite of programs (http://ssrl.slac.stanford.edu/exafspak.html). Quantitative speciation of the chemical forms of sulfur present in biological samples was achieved by least-squares ¢tting of sums of model compound spectra [23] using the EXAFSPAK program DATFIT. 3. Results Fig. 2 shows the sulfur K-edge X-ray absorption spectra of a range of relevant model compounds. All can be clearly distinguished under ideal conditions, even cysteine and methionine, as previously discussed [17]. The spectra of ethyl isothiocyanate and ethyl thiocyanate are clearly distinct, but the spectra of ethyl and allyl isothiocyanates were essentially indistinguishable. This is not surprising since the immediate environment of the sulfur atom is so similar in the two compounds, and likewise we do not expect to be able to distinguish allyl or ethyl isothiocyanate from larger alkylated forms (4-pentenyl- and 6-methylsul¢nylhexyl- in wasabi, L-phenylethyl- in horseradish [12]), although signi¢cant amounts of the methylsul¢nylsubstituted forms [12] would be expected to give rise to features attributable to thioether-like species. Fig. 3 shows a comparison of the spectra of sections of horseradish (a) and wasabi (b) both before and after bruis-
Fig. 3. Sulfur K-edge X-ray absorption spectra of horseradish and wasabi. Horseradish (a) and wasabi (b) are compared before and after bruising by rubbing with a polypropylene spatula (solid and dashed lines, respectively). Also shown are toluene extracts of horseradish (c) and wasabi (d) and a toluene solution of allyl isothiocyanate (e).
Fig. 4. Results of curve-¢tting analyses of sulfur K-edge X-ray absorption spectra of a section of horseradish root. Spectra are shown before (upper) and after (lower) disruption of the cells by bruising. The data are shown as ¢lled circles, the best ¢t as a solid line, and some of the model compounds of Fig. 2, scaled according to their ¢tted contributions to the horseradish spectra, as broken lines (sini., sinigrin; RNCS, allyl isothiocyanate ; RSR, methionine ; SO23 4 , sulfate). The residual (observed minus calculated) spectrum is shown beneath. The asterisk indicates a minor component present only in the bruised data set that was not modeled by the curve-¢tting analysis.
ing by rubbing with a polypropylene coated spatula. Sinigrin and sulfate are the major sulfur-containing species in both plants, accounting for approx. 50% of the total sulfur, and more than 70% of the organic sulfur. The di¡erences after bruising can be attributed to the loss of sinigrin and the generation of isothiocyanates and sulfate. Fig. 3 also shows a comparison of the spectra of the toluene extracts of horseradish and wasabi with that of a solution of allyl isothiocyanate in toluene. All three spectra (Fig. 3c^e) are essentially identical, indicating that isothiocyanates are indeed the major toluene-soluble compounds released when horseradish is ground. Etoh et al. [12] reported that 78% of the isothiocyanates in horseradish root and 82.5% of the isothiocyanates in wasabi root were allyl isothiocyanates, and our data are fully consistent with this. They also reported that about 11% of the isothiocyanates in wasabi root were methylsul¢nylalkyl derivatives, but curve-¢tting analyses (not illustrated) indicate essentially 100% isothiocyanate, and would not tolerate addition of a thioether (methionine) component. Fig. 4 shows a curve-¢tting analysis of the horseradish tissue sections. The ¢ts are very good, suggesting that the chosen model compounds are indeed appropriate for the bulk of the sulfur present. Similar quality ¢ts were obtained for wasabi (not illustrated). Table 1 shows the dis-
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Table 1 E¡ects of bruising on the distribution of sulfur-containing species in horseradish and wasabi root Horseradish Sinigrin Sulfate Isothiocyanate Thioether
Wasabi
Intact
Bruised
v% per S
Intact
Bruised
v% per S
53 (4) 31 (1) 13 (8) 3 (12)
15 47 35 2
338 16 22 31
45 29 13 14
7 49 31 12
338 20 18 32
(5) (1) (6) (7)
(2) (1) (3) (3)
(2) (1) (3) (5)
The table shows the curve-¢tting results for spectra of tissue samples both before and after bruising. The values given are in terms of percentage of total sulfur and the values in parentheses are 95% con¢dence limits, which were estimated from the diagonal elements of the covariance matrix.
tribution of sulfur-containing species before and after the bruising for the two plant materials, and it is clear that the disappearance of sinigrin is accompanied by an essentially equimolar appearance of sulfate and isothiocyanates. There is no evidence for the generation of thiocyanates, and the generation of nitriles [16] is unlikely to be occurring on a signi¢cant scale since this would probably result in the generation of additional sulfate. The concentrations of sinigrin and its breakdown products are so high in these samples that we were unable to distinguish between cysteine and methionine in the remainder [16]. As shown in Fig. 2, the di¡erences between the spectra of these two species are small. 4. Discussion Sulfur plays many essential roles in biochemistry, but tools for studying this element in vivo are not yet widely available. Previous work addressing the sulfur biochemistry of horseradish and wasabi has, of necessity, relied on extraction with organic solvents and gas chromatography. While this has provided much useful information, it cannot be used to probe intact tissues or without concerns that the extraction may cause chemical changes. Sulfur K-edge X-ray absorption spectroscopy provides a useful tool for determining the chemical forms of sulfur in intact biological samples. We have previously used it to determine the cellular levels of thiols, thioethers, organic disul¢des and other forms in erythrocytes and plasma [17]. Here we employ the technique to follow the metabolic conversion of sinigrin to its products in two cruciferous plants. Sinigrin and its congeners are clearly the most abundant sulfur-containing species in the roots of both horseradish and wasabi, accounting for approx. 50% of the total sulfur, and 70% of the organic sulfur in our measurements. The presence of organic isothiocyanates in the cut sections, before deliberate attempts to disrupt the cellular structure by bruising, probably re£ects some damage during sample preparation. If this is correct, and all the organic isothiocyanate comes from sinigrin in initially undamaged cells, then sinigrin accounted for approx. 95% of the organic sulfur in horseradish root, and more than 85% of the organic sulfur in wasabi root! Sinigrin contains two sulfur atoms, and their local
chemical environments are quite distinct (Fig. 1). This is re£ected in the sulfur K-edge absorption spectrum (Fig. 2). The spectral feature near 2471 eV arises from the thioether that bridges the glucose and nitrogen-containing portions of the molecule (Fig. 1, I) and is dominated by a (S)1sC(S-C)c* transition at 2471.0 eV. The features near 2480 eV are attributable to the formally hexavalent sulfur of the C = N-OSO3 3 group (see Fig. 1). PseudoVoigt deconvolution indicates that this is comprised of three major peaks, at 2477.6, 2478.5 and 2480.3 eV. In general, the R-OSO3 3 group is expected to give two major transitions, which for idealized C3v symmetry can be assigned as 1sCa1 and 1sCe. However, the symmetry at sulfur will actually be lower than C3v due to the in£uence of the nitrogen (the S-O-N angle is expected to be about 121³), and the 1sCe peak might therefore split, giving rise to the transitions at 2477.6 and 2478.5 eV. Alternatively, two di¡erent conformations might be present (in approximately equal amounts) which could give rise to two di¡erent 1sCa1 peak energies (2477.6 and 2478.5 eV). As expected, the spectra of organic thiocyanates and isothiocyanates are substantially di¡erent. The spectra of
Fig. 5. Spectroscopic identi¢cation of a species with novel sulfur coordination in bruised horseradish. A close-up of the spectrum of the bruised horseradish sample is shown in (a), clearly indicating a small peak of unknown origin at about 2468 eV. Pseudo-Voigt peak deconvolution of this spectrum yielded a peak energy of 2468.2 eV (aP). The spectrum of the thioketone, 2,2,4,4-tetramethyl-1,3-cyclobutanedithione, is compared in (b) (peak energy is 2467.5 eV).
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organic thiocyanates (R-S-C = N) resemble those of thioethers, with the major low energy feature arising from a (S)1sC(S-C)c* transition at 2471.1 eV. The organic isothiocyanates (R-N = C = S) are quite di¡erent, with lower energy features attributable to nearby (S)1sC(N = C = S)Z* and (S)1sC(N = C = S)c* transitions at 2469.2 and 2470.5 eV, respectively. As discussed above, the enzyme that hydrolyzes sinigrin to yield isothiocyanates is known as myrosinase [10,11,24]. Our work conclusively shows that no signi¢cant quantities of organic thiocyanates are produced during degradation of sinigrin upon cellular disruption, as has been suggested by Stoewesand [16]. Similarly it seems unlikely that the generation of nitriles [16] is a signi¢cant pathway, since this would be expected to release sulfate rather than isothiocyanate, and we do not see signi¢cant excess of sulfate over isothiocyanate in either plant (Fig. 5). Enzyme catalysis in vivo thus follows the scheme of Fig. 1. The spectrum of both the bruised horseradish and the bruised wasabi showed a peak at low energy (Fig. 3) that was not accounted for in the curve-¢tting analysis (see Fig. 4). Features in this region are characteristic of compounds containing a thio group, and an example of a spectrum from such a compound is shown in Fig. 5 (the thioketone, 2,2,4,4-tetramethyl-1,3-cyclobutanedithione [25]). This low energy peak at 2467.5 eV is attributable to the (S)1sC(C = S)Z* transition, associated with the Z-bonding of the C = S linkage, and the energy of this transition is expected to decrease with decreasing bond length (increasing covalency). Peak deconvolution of the low energy part of the plant spectrum indicates a peak energy of 2468.2 eV, some 0.7 eV higher than that of the thioketone. The intensity of this peak also suggests that it arises from at most 2% of the total sulfur. Organic thio compounds are very unusual in biological systems, and it seems likely that this peak arises from the thio-acid amide sulfate (IV), which is thought to be unstable, spontaneously undergoing a Lossen rearrangement to form the isothiocyanate (V) (Fig. 1). Ab initio calculations provide some support for this assignment. Hartree Fock calculations, using the STO-3G basis set, were performed upon the thioketone 2,2,4,4-tetramethyl-1,3-cyclobutanedithione and upon the thio-acid amide sulfate (IV) (Fig. 1). Geometry optimizations were ¢rst performed, and these gave C = S bond î , which is within lengths for the thioketone of 1.58 A î of the crystallographically determined value [25] 0.02 A î ) and gave an estimated C = S bond length for (1.60 A î . Examination of the orbital energies indi(IV) of 1.61 A cated that the lowest unoccupied molecular orbital Z* level of (IV) lies at 0.5 eV higher energy than that of the thioketone. We note that although these calculations were of the ground state, rather than the X-ray excited state (i.e. they did not include a 1s core hole), the relative orbital energy di¡erences are likely to be propagated to the excited states. Thus, sulfur K near-edge spectra provide strong evidence of the presence of the postulated intermediate compound (IV) in the bruised tissue samples.
Acknowledgements Part of this work was funded by the National Institutes of Health, award GM57375. The Stanford Synchrotron Radiation Laboratory is funded by the Department of Energy, O¤ces of Basic Energy Sciences and Biological and Environmental Research ; the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences. References [1] R. Menard, J.-P. Larue, D. Silue, D. Thouvenot, Phytochemistry 52 (1999) 29^35. [2] B.G. Shofran, S.T. Purrington, F. Breidt, H.P. Fleming, J. Food Sci. 63 (1998) 621^624. [3] R. Tsao, Q. Yu, I. Friesen, J. Potter, M. Chiba, J. Agric. Food Chem. 48 (2000) 1898^2002. [4] A. M Bones, J. Rossiter, Physiol. Plant. 97 (1996) 1194^1208. [5] S. Cottaz, B. Henrissat, H. Drigues, Biochemistry 35 (1996) 15256^ 15259. [6] T.A. Shapiro, J.W. Fahey, K.L. Wade, K.K. Stephenson, P. Talalay, Cancer Epidemiol. Biomarkers Prev. 7 (1998) 1091^1100. [7] T.K. Smith, E.K. Lund, I.T. Johnson, Carcinogenesis 19 (1998) 267^ 273. [8] B. Tokarska, K. Karwowska, Nahrung 27 (1983) 443^447. [9] H.E. Van Doorn, G.C. Van Der Kruk, G. Van Holst, N.C.M.E. Raaijmakers-Ruijs, E. Postma, B. Groeneweg, W.H.F. Jongen, J. Sci. Food Agric. 78 (1998) 30^38. [10] B. Lu«thy, P. Matile, Biochem. Physiol. P£anzen. 179 (1984) 5^12. [11] M. Ohtsuru, H. Kawatani, Agric. Biol. Chem. 43 (1979) 2249^ 2256. [12] H. Etoh, A. Nishimura, R. Takasawa, A. Yagi, K. Saito, K. Sakata, I. Kishima, K. Ina, Agric. Biol. Chem. 54 (1990) 1587^1589. [13] M.W. Ireland, The Medical Department of the United States Army in the World War, XIV Medical Aspects of Gas Warfare, Government Printing O¤ce, Washington, DC, 1926. [14] M.G. Ettlinger, A.J. Lundeen, J. Am. Chem. Soc. 78 (1956) 4172^ 4173. [15] M.G. Ettlinger, A.J. Lundeen, J. Am. Chem. Soc. 79 (1957) 1764^ 1765. [16] G.S. Stoewsand, Food Chem. Toxicol. 33 (1995) 537^543. [17] I.J. Pickering, R.C. Prince, T. Divers, G.N. George, FEBS Lett. 441 (1998) 11^14. [18] A. Rompel, R.M. Cinco, M.J. Latimer, A.E. McDermott, R.D. Guiles, A. Quintanilha, R.M. Krauss, K. Sauer, V.K. Yachandra, M.P. Klein, Proc. Natl. Acad. Sci. USA 95 (1998) 6122^6127. [19] I.J. Pickering, R.C. Prince, G.N. George, W.E. Rauser, W.A. Wickramasinghe, A.A. Watson, C.T. Dameron, I.G. Dance, D.P. Fairlie, D.E. Salt, Biochim. Biophys. Acta 1429 (1999) 351^364. [20] I. Song, B. Rickett, P. Janavicius, J.H. Payer, M.R. Antonio, Nucl. Instrum. Method. A360 (1995) 634^641. [21] H. Sekiyama, N. Kosugi, H. Kuroda, T. Ohta, Bull. Chem. Soc. Jpn. 59 (1986) 575^579. [22] M.J. George, J. Synchrotron Radiat. 7 (2000) 283^286. [23] G.N. George, M.L. Gorbaty, S.R. Kelemen, M. Sansone, Energy Fuels 5 (1991) 93^97. [24] W.P. Burmeister, S. Cottaz, P. Rollin, A. Vasella, B. Henrissat, J. Biol. Chem. 275 (2000) 39385^39393. [25] C.D. Shirrell, D.E. Williams, Acta Crystallogr. B 29 (1973) 1648^ 1653.
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In situ observation of the generation of isothiocyanates from sinigrin in horseradish and wasabi Eileen Y. Yu a , Ingrid J. Pickering a , Graham N. George a
a;
*, Roger C. Prince
b
Stanford Synchrotron Radiation Laboratory, Stanford Linear Accelerator Center, 2575 Sand Hill Road, Stanford, CA 94025, USA b ExxonMobil Research and Engineering Company, Route 22 East, Annandale, NJ 08801, USA Received 29 January 2001; received in revised form 25 May 2001; accepted 25 May 2001
Abstract Sulfur K-edge X-ray absorption spectroscopy has been used to determine the chemical identity of the sulfur-containing species in horseradish (Armoracia lapthifolia) and wasabi (Wasabia japonica) in situ, before and after cell disruption. The major sulfur-containing species in the intact root is sinigrin (1-thio-L-D-glucopyranose 1-N-(sulfoxy)-3-buteneimidate) and related congeners. Disrupting the cells by applying local pressure allowed the conversion of the sulfur moieties in sinigrin to isothiocyanates and sulfate in approximately equimolar amounts. In contrast to previous suggestions, no detectable thiocyanates were formed, but an unusual thio intermediate may have been identified for the first time. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : X-ray absorption spectroscopy ; Isothiocyanate; Sinigrin; Myrosinase
1. Introduction Glucosinolates, or more accurately L-D-S-glucosides (Fig. 1, compound I), are widely distributed in cruciferous plants (Brassicaceae) ; they form a large family that di¡er in the substituent R (Fig. 1). The plants also produce a L-thioglucoside glucohydrolase (EC 3.2.3.1), commonly known as myrosinase, that catalyzes their hydrolysis. Glucosinolates probably play roles in resistance to fungi, nematodes and other plant pathogens, and herbivores [1^3]. They may also serve to store inactive precursors of plant hormones such as 3-indolylacetic acid [4], and be important in the storage and inactivation of ascorbic acid [5]. Glucosinolates are also receiving attention because of possible bene¢cial e¡ects in the human diet [6,7]. The glucosinolate known as sinigrin (1-thio-L-D-glucopyranose 1-N(sulfoxy)-3-buteneimidate; Fig. 1) is particularly abundant in horseradish (Armoracia lapthifolia) and wasabi (Wasabia japonica) [8,9]. In the intact cell, glucosinolates are compartmentalized in subcellular organelles, quite likely the vacuole [10]. When the cell is disrupted, the glucosinolate substrates mix with the membrane-associated cytosolic myrosinases
* Corresponding author. Fax: +1-650-926-4604. E-mail address : [email protected] (G.N. George).
[10,11]. Sinigrin itself is unpleasantly bitter, with no discernable odor, but its hydrolysis by myrosinases releases intensely lachrymatory (and £avorful) compounds. In both horseradish and wasabi, a series of isothiocyanates is produced as a result of cell damage [12], and the most abundant volatile compound released is allyl isothiocyanate [12] (Fig. 1, V), about 130 mg/100 g wet weight `root'. Allyl isothiocyanate is volatile (boiling point 150³C), and so intensely lachrymatory that it was used experimentally as a war gas by the German army in the First World War [13]. On the milder side, the more abundant larger compounds (4-pentenyl- and 6-methylsul¢nylhexyl- in wasabi, L-phenylethyl- in horseradish [12]) are perhaps important in the subtle culinary di¡erences between the two plants. Although the breakdown of sinigrin by myrosinase shown in Fig. 1 is thought to be a hydrolysis followed by a (non-enzymatically catalyzed) Lossen rearrangement [14,15], there have been suggestions that thiocyanates and nitriles are also produced [16]. Sulfur K-edge X-ray absorption spectroscopy can be used to identify the chemical forms of sulfur species in situ [17^19] and we have therefore addressed this possibility using X-ray absorption spectroscopy at the sulfur K-edge. We ¢nd no evidence for the generation of thiocyanates, but do see an essentially quantitative conversion of sinigrin to isothiocyanates and sulfate when cells are damaged. Furthermore, we have found evidence of the chemical precursor (IV) to the iso-
0304-4165 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 0 1 ) 0 0 1 6 1 - 1
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Fig. 1. Schematic of the degradation of L-D-S-glucosides (I) by myrosinase (L-thioglucoside glucohydrolase) and related biochemistry. The initial hydrolysis of (I) gives rise to glucose (II) and (III), which will be in equilibrium with (IV). (IV) is then thought to undergo a Lossen rearrangement to produce the isothiocyanate (V) and sulfate.
thiocyanate, which possesses the unusual thio ( s C = S) motif. 2. Materials and methods Horseradish (A. lapthifolia) and wasabi (W. japonica) were purchased fresh at local markets, and the former was also harvested at Stony Brook Apiary, NJ. The parts of the plant typically used for culinary purposes and available commercially are technically the root of horseradish and the stem of wasabi, but we will follow common parlance and refer to both as roots in this paper. Thin sections were prepared and mounted under 10 Wm thick polypropylene ¢lm to retard desiccation during exposure to the X-ray beam. Toluene extracts were prepared with an electrical food `juicer', the juice delivered directly into toluene to trap volatile organic compounds. No signi¢cant di¡erences were seen between freshly harvested (2 days between harvest and spectroscopy) and commercially obtained horseradish roots. Model compounds were purchased from Aldrich. Sulfur K-edge X-ray absorption spectra were collected on beamline 6-2 at the Stanford Synchrotron Radiation Laboratory using a Si(111) double crystal monochromator and a downstream Ni-coated harmonic rejection mirror. Energy resolution was optimized by decreasing the vertical aperture upstream from the monochromator until no further sharpening of features of the near-edge spectrum of a sodium thiosulfate (Na2 S2 O3 W5H2 O) standard was detected. The optimized energy resolution was quantitatively determined to be 0.51 eV by measuring the width of the 2471.4 eV 1sCZ*(3b1 ) transition of gaseous SO2 (using pseudo-Voigt peak deconvolution), which corresponds to a transition to a single orbital, rather than to a band of orbitals which can be the case with solid standards [20]. The incident X-ray intensity was monitored using a helium-¢lled ion chamber [17], and the X-ray absorption spectra were monitored in £uorescence using a Stern^Heald^ Lytle detector. The energy scale was calibrated with refer-
ence to the lowest energy peak of a sodium thiosulfate standard, assumed to be 2469.2 eV [21], and was reproducible to less than 0.1 eV. All spectra were recorded at room temperature, and samples had less than 10 min of exposure to the X-ray beam. More prolonged exposure to the X-ray beam (approx. 20 min) resulted in no signi¢cant
Fig. 2. Sulfur K-edge X-ray absorption spectra of compounds likely to be present in the roots of horseradish and wasabi. Allyl isothiocyanate (a), ethyl thiocyanate (b), cysteine (c) (a representative thiol) and methionine (d) (a representative thioether), sinigrin (e), and sulfate (f). Allyl isothiocyanate and ethyl thiocyanate were in toluene solution, and all other compounds were in aqueous solutions at neutral pH. The spectra are normalized to the sulfur K-edge jump, which is equivalent to total sulfur concentration (note that sinigrin (e) contains two di¡erent sulfurs). The spectrum of sulfate is very sharp, so it is displayed reduced by a factor of 3.
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changes in the spectra, indicating that X-ray photoreduction of the sample is probably insigni¢cant. Data were collected using the program XAS-Collect [22], and data analysis was carried out using the EXAFSPAK suite of programs (http://ssrl.slac.stanford.edu/exafspak.html). Quantitative speciation of the chemical forms of sulfur present in biological samples was achieved by least-squares ¢tting of sums of model compound spectra [23] using the EXAFSPAK program DATFIT. 3. Results Fig. 2 shows the sulfur K-edge X-ray absorption spectra of a range of relevant model compounds. All can be clearly distinguished under ideal conditions, even cysteine and methionine, as previously discussed [17]. The spectra of ethyl isothiocyanate and ethyl thiocyanate are clearly distinct, but the spectra of ethyl and allyl isothiocyanates were essentially indistinguishable. This is not surprising since the immediate environment of the sulfur atom is so similar in the two compounds, and likewise we do not expect to be able to distinguish allyl or ethyl isothiocyanate from larger alkylated forms (4-pentenyl- and 6-methylsul¢nylhexyl- in wasabi, L-phenylethyl- in horseradish [12]), although signi¢cant amounts of the methylsul¢nylsubstituted forms [12] would be expected to give rise to features attributable to thioether-like species. Fig. 3 shows a comparison of the spectra of sections of horseradish (a) and wasabi (b) both before and after bruis-
Fig. 3. Sulfur K-edge X-ray absorption spectra of horseradish and wasabi. Horseradish (a) and wasabi (b) are compared before and after bruising by rubbing with a polypropylene spatula (solid and dashed lines, respectively). Also shown are toluene extracts of horseradish (c) and wasabi (d) and a toluene solution of allyl isothiocyanate (e).
Fig. 4. Results of curve-¢tting analyses of sulfur K-edge X-ray absorption spectra of a section of horseradish root. Spectra are shown before (upper) and after (lower) disruption of the cells by bruising. The data are shown as ¢lled circles, the best ¢t as a solid line, and some of the model compounds of Fig. 2, scaled according to their ¢tted contributions to the horseradish spectra, as broken lines (sini., sinigrin; RNCS, allyl isothiocyanate ; RSR, methionine ; SO23 4 , sulfate). The residual (observed minus calculated) spectrum is shown beneath. The asterisk indicates a minor component present only in the bruised data set that was not modeled by the curve-¢tting analysis.
ing by rubbing with a polypropylene coated spatula. Sinigrin and sulfate are the major sulfur-containing species in both plants, accounting for approx. 50% of the total sulfur, and more than 70% of the organic sulfur. The di¡erences after bruising can be attributed to the loss of sinigrin and the generation of isothiocyanates and sulfate. Fig. 3 also shows a comparison of the spectra of the toluene extracts of horseradish and wasabi with that of a solution of allyl isothiocyanate in toluene. All three spectra (Fig. 3c^e) are essentially identical, indicating that isothiocyanates are indeed the major toluene-soluble compounds released when horseradish is ground. Etoh et al. [12] reported that 78% of the isothiocyanates in horseradish root and 82.5% of the isothiocyanates in wasabi root were allyl isothiocyanates, and our data are fully consistent with this. They also reported that about 11% of the isothiocyanates in wasabi root were methylsul¢nylalkyl derivatives, but curve-¢tting analyses (not illustrated) indicate essentially 100% isothiocyanate, and would not tolerate addition of a thioether (methionine) component. Fig. 4 shows a curve-¢tting analysis of the horseradish tissue sections. The ¢ts are very good, suggesting that the chosen model compounds are indeed appropriate for the bulk of the sulfur present. Similar quality ¢ts were obtained for wasabi (not illustrated). Table 1 shows the dis-
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Table 1 E¡ects of bruising on the distribution of sulfur-containing species in horseradish and wasabi root Horseradish Sinigrin Sulfate Isothiocyanate Thioether
Wasabi
Intact
Bruised
v% per S
Intact
Bruised
v% per S
53 (4) 31 (1) 13 (8) 3 (12)
15 47 35 2
338 16 22 31
45 29 13 14
7 49 31 12
338 20 18 32
(5) (1) (6) (7)
(2) (1) (3) (3)
(2) (1) (3) (5)
The table shows the curve-¢tting results for spectra of tissue samples both before and after bruising. The values given are in terms of percentage of total sulfur and the values in parentheses are 95% con¢dence limits, which were estimated from the diagonal elements of the covariance matrix.
tribution of sulfur-containing species before and after the bruising for the two plant materials, and it is clear that the disappearance of sinigrin is accompanied by an essentially equimolar appearance of sulfate and isothiocyanates. There is no evidence for the generation of thiocyanates, and the generation of nitriles [16] is unlikely to be occurring on a signi¢cant scale since this would probably result in the generation of additional sulfate. The concentrations of sinigrin and its breakdown products are so high in these samples that we were unable to distinguish between cysteine and methionine in the remainder [16]. As shown in Fig. 2, the di¡erences between the spectra of these two species are small. 4. Discussion Sulfur plays many essential roles in biochemistry, but tools for studying this element in vivo are not yet widely available. Previous work addressing the sulfur biochemistry of horseradish and wasabi has, of necessity, relied on extraction with organic solvents and gas chromatography. While this has provided much useful information, it cannot be used to probe intact tissues or without concerns that the extraction may cause chemical changes. Sulfur K-edge X-ray absorption spectroscopy provides a useful tool for determining the chemical forms of sulfur in intact biological samples. We have previously used it to determine the cellular levels of thiols, thioethers, organic disul¢des and other forms in erythrocytes and plasma [17]. Here we employ the technique to follow the metabolic conversion of sinigrin to its products in two cruciferous plants. Sinigrin and its congeners are clearly the most abundant sulfur-containing species in the roots of both horseradish and wasabi, accounting for approx. 50% of the total sulfur, and 70% of the organic sulfur in our measurements. The presence of organic isothiocyanates in the cut sections, before deliberate attempts to disrupt the cellular structure by bruising, probably re£ects some damage during sample preparation. If this is correct, and all the organic isothiocyanate comes from sinigrin in initially undamaged cells, then sinigrin accounted for approx. 95% of the organic sulfur in horseradish root, and more than 85% of the organic sulfur in wasabi root! Sinigrin contains two sulfur atoms, and their local
chemical environments are quite distinct (Fig. 1). This is re£ected in the sulfur K-edge absorption spectrum (Fig. 2). The spectral feature near 2471 eV arises from the thioether that bridges the glucose and nitrogen-containing portions of the molecule (Fig. 1, I) and is dominated by a (S)1sC(S-C)c* transition at 2471.0 eV. The features near 2480 eV are attributable to the formally hexavalent sulfur of the C = N-OSO3 3 group (see Fig. 1). PseudoVoigt deconvolution indicates that this is comprised of three major peaks, at 2477.6, 2478.5 and 2480.3 eV. In general, the R-OSO3 3 group is expected to give two major transitions, which for idealized C3v symmetry can be assigned as 1sCa1 and 1sCe. However, the symmetry at sulfur will actually be lower than C3v due to the in£uence of the nitrogen (the S-O-N angle is expected to be about 121³), and the 1sCe peak might therefore split, giving rise to the transitions at 2477.6 and 2478.5 eV. Alternatively, two di¡erent conformations might be present (in approximately equal amounts) which could give rise to two di¡erent 1sCa1 peak energies (2477.6 and 2478.5 eV). As expected, the spectra of organic thiocyanates and isothiocyanates are substantially di¡erent. The spectra of
Fig. 5. Spectroscopic identi¢cation of a species with novel sulfur coordination in bruised horseradish. A close-up of the spectrum of the bruised horseradish sample is shown in (a), clearly indicating a small peak of unknown origin at about 2468 eV. Pseudo-Voigt peak deconvolution of this spectrum yielded a peak energy of 2468.2 eV (aP). The spectrum of the thioketone, 2,2,4,4-tetramethyl-1,3-cyclobutanedithione, is compared in (b) (peak energy is 2467.5 eV).
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organic thiocyanates (R-S-C = N) resemble those of thioethers, with the major low energy feature arising from a (S)1sC(S-C)c* transition at 2471.1 eV. The organic isothiocyanates (R-N = C = S) are quite di¡erent, with lower energy features attributable to nearby (S)1sC(N = C = S)Z* and (S)1sC(N = C = S)c* transitions at 2469.2 and 2470.5 eV, respectively. As discussed above, the enzyme that hydrolyzes sinigrin to yield isothiocyanates is known as myrosinase [10,11,24]. Our work conclusively shows that no signi¢cant quantities of organic thiocyanates are produced during degradation of sinigrin upon cellular disruption, as has been suggested by Stoewesand [16]. Similarly it seems unlikely that the generation of nitriles [16] is a signi¢cant pathway, since this would be expected to release sulfate rather than isothiocyanate, and we do not see signi¢cant excess of sulfate over isothiocyanate in either plant (Fig. 5). Enzyme catalysis in vivo thus follows the scheme of Fig. 1. The spectrum of both the bruised horseradish and the bruised wasabi showed a peak at low energy (Fig. 3) that was not accounted for in the curve-¢tting analysis (see Fig. 4). Features in this region are characteristic of compounds containing a thio group, and an example of a spectrum from such a compound is shown in Fig. 5 (the thioketone, 2,2,4,4-tetramethyl-1,3-cyclobutanedithione [25]). This low energy peak at 2467.5 eV is attributable to the (S)1sC(C = S)Z* transition, associated with the Z-bonding of the C = S linkage, and the energy of this transition is expected to decrease with decreasing bond length (increasing covalency). Peak deconvolution of the low energy part of the plant spectrum indicates a peak energy of 2468.2 eV, some 0.7 eV higher than that of the thioketone. The intensity of this peak also suggests that it arises from at most 2% of the total sulfur. Organic thio compounds are very unusual in biological systems, and it seems likely that this peak arises from the thio-acid amide sulfate (IV), which is thought to be unstable, spontaneously undergoing a Lossen rearrangement to form the isothiocyanate (V) (Fig. 1). Ab initio calculations provide some support for this assignment. Hartree Fock calculations, using the STO-3G basis set, were performed upon the thioketone 2,2,4,4-tetramethyl-1,3-cyclobutanedithione and upon the thio-acid amide sulfate (IV) (Fig. 1). Geometry optimizations were ¢rst performed, and these gave C = S bond î , which is within lengths for the thioketone of 1.58 A î of the crystallographically determined value [25] 0.02 A î ) and gave an estimated C = S bond length for (1.60 A î . Examination of the orbital energies indi(IV) of 1.61 A cated that the lowest unoccupied molecular orbital Z* level of (IV) lies at 0.5 eV higher energy than that of the thioketone. We note that although these calculations were of the ground state, rather than the X-ray excited state (i.e. they did not include a 1s core hole), the relative orbital energy di¡erences are likely to be propagated to the excited states. Thus, sulfur K near-edge spectra provide strong evidence of the presence of the postulated intermediate compound (IV) in the bruised tissue samples.
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