Evaluation of the Ethylene Effect on Well Defined Copper Enzymes

1)

Megawati Santoso1), Harold M. Goff2) Department of Chemistry, Faculty of Mathematics and Natural Sciences Institut Teknologi Bandung, Bandung 2) Department of Chemistry, University of Iowa, Iowa e-mail: megawati@chem.itb.ac.id Received August 2005, accepted for publication December 2005

Abstract An indirect approach in studying ethylene binding sites of copper enzymes was carried out since ethylene pleitropic effect in plants and the general affinity of Cu(I) complexes for ethylene suggest a possibility of ethylene binding to various copper enzymes. For this purpose, superoxide dismutase (SOD) and tyrosinase were evaluated using two different methods. H-NMR spectra of three different conditions of SOD (native SOD; reduced SOD; and reduced SOD in the presence of ethylene) were acquired at 25 C. Activity of mushroom tyrosinase was monitored in the absence of ethylene by evaluating the formation of dopachrome at 475 nm. Three other assay systems were constructed the same way as the control, except one of the reagents (enzyme, DL-DOPA, or sodium phosphate buffer) was each saturated with ethylene prior to mixing all of reagents. Both systems behaved unsatisfactorily for the purpose of evaluation. The coordinated ethylene proton-NMR signal was obscured by the broad HOD signal, due to the partial oxidation of Cu(I) SOD by trace oxygen. Nonetheless, the tyrosinase assay results demonstrated that displacement of oxygen in the assay system by ethylene did not account for the loss of activity. The observed ethylene effect on tyrosinase activity, however, was minimal, since ethylene could produce threshold physiological response at concentration of 6.5 x 10-9 M. Keywords: Ethylene; Copper Enzymes; Superoxide dismutase; Tyrosinase Abstrak Penelitian tentang interaksi molekul etilen pada enzim yang mengandung ion tembaga (Cu) dilakukan berbasis pada penemuan berbagai efek molekul etilen pada tumbuhan dan pada hipotesis kemudahan enzim Cu(I) dalam mengikat molekul etilen. Dalam penelitian ini dilakukan uji terhadap dua jenis enzim Cu, yaitu superoksida dismutase (SOD) dan tirosinase dalam berinteraksi dengan molekul etilen. Uji terhadap SOD dilakukan dengan menggunakan NMR. Spektrum H-NMR SOD pada kondisi alamiah, SOD tereduksi, dan SOD tereduksi yang telah dijenuhkan oleh etilen diukur pada suhu 25 C. Uji terhadap tirosinase dilakukan dengan mengukur aktivitas enzim pada kondisi alamiahnya dan pada kondisi di mana salah satu reagen (enzim, DL-DOPA, atau dapar natrium fosfat) dijenuhkan oleh gas etilen, melalui pengukuran absorban pembentukan molekul dopakrom pada 475 nm dengan menggunakan spektrofotometer UV. Hasil penelitian menunjukkan kedua sistem tidak cukup memadai untuk digunakan sebagai bahan uji. Signal etilen yang terkoordinasi oleh Cu(I) bertumpang tindih dengan pita lebar HOD karena oksidasi parsial Cu(I) SOD oleh oksigen. Uji terhadap tirosinase menunjukkan penggantian oksigen oleh etilen. Namun kuantitas oksigen yang digantikan belum secara optimal berkontribusi pada hilangnya aktivitas enzim karena molekul etilen dapat memberikan efek fisiologi pada tanaman pada konsentrasi 6,5 x 10-9 M. Kata Kunci: Etilen, Copper enzyme, Superoksida dismutase, Tirosinase 1. Introduction The studies on ethylene physiological effects have been carried out intensively due to the significant role of ethylene in post harvest technology (Abeles et al., 1992; Roberts and Tucker, 1985; Fuch and Chalutz, 1984; Abeles, 1973). Studies in this area showed that the effects of ethylene on plants could be categorized into three major groups: phytogerontological or aging effects, developmental effects, and enzymatic or metabolic effects. Although existence of an ethylene receptor in plants is a reasonable assumption, isolation of such a receptor is difficult. Translocation of ethylene in non-significant amounts in many parts of the plant 25 and the high volatility of ethylene are primary problems in isolating the ethylene receptor. Two groups, Hall et al. and Sisler et al., however, succeeded in detecting the ethylene binding protein in Phaseolus vulgaris cv Canadian Wonder (Jerie et al., 1979; Bengochea et al., 1980; Bengochea et al., 1980; Hall et al., 1982) and mung bean, respectively (Sister, 1980; Sisler and Wood, 1987; Sisler, 1979). Low rate constants of ethylene association and dissociation in those bean cotyledons provide a better condition for detection and partial fractionation of the protein. An indirect approach in studying ethylenebinding sites was carried out by investigating the effect of ethylene analogs and inhibitors. Initial

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studies by Burg & Burg suggested basic molecular requirements for ethylene biological activity (Burg and Burg, 1967). They found that only terminal unsaturated ethylene analogs bound to ethylene binding sites and double bond containing molecules showed more activity than that of the triple bond analogs. The activity decreased with longer hydrocarbon skeleton, larger molecular size, and lower electron density in the unsaturated site. Biological activity was not observed with a charged terminal carbon atom. The fact that only unsaturated molecules with the "restrictions" described above exhibit ethylene-like effects suggests that a metal containing receptor might be present. This prevailing hypothesis was further supported by the inhibition of ethylene action in the presence of silver nitrate ions and metal chelators such as EDTA, thiourea and diethyldithiocarbamate (Beyer Jr., 1976; Beyer Jr. 1978; Beyer, 1979; Cook and Van Staden, 1987; Thompson et al., 1983). A trans effect mechanism was proposed for the binding of ethylene and its analog to the metal containing receptor site (Sisler, 1977). Copper(I) constitutes the most logical candidate among soft" metal ions which could fit the binding criteria of various alkenes to the receptor site. The essential role of copper enzymes in many biological processes (Sigel, 1981) and low toxicity of the metal ion are other important considerations. Based on those assumption, these experiments were designed to evaluate the copper(I) hypothesis to a greater extent by studying the effect of ethylene on well-defined copper enzymes. The rationale is that the ethylene pleitoropic effect in plants and the general affinity of copper(I) complexes for ethylene suggest a possibility of ethylene binding to various copper enzymes. Two important requirements are a) copper enzymes need to be in the +1 oxidation state or cycle through the +1 oxidation state, and b) copper enzymes should have an open coordination site for ethylene binding. The second requirement precludes the use of blue copper electron transport proteins as candidates. For this purpose, commercially available superoxide dismutase (SOD) is a good candidate, since it was thought that the "vacant" olefinic spectral region of this protein would provide an opportunity to detect any coordinated ethylene signal (figure 1) (Cass et al., 1977; Cass et al., 1977).

Figure 1. Superoxide dismutase active site. Tyrosinase is another logical choice for ethylene binding studies due to its relatively simple bioassay and its monooxygenating activities of mono and diphenols (figure 2).

Figure 2. Monooxygenation reactions of (a) monophenol and (b) diphenol catalyzed by tyrosinase. Evaluation of possible ethylene binding at the tyrosinase active site was carried out by monitoring the inhibition of tyrosinase activity by ethylene as a substrate competitor. The assay is based on the tyrosinase activity to oxidize DL-DOPA {3(3,4 dihydroxyphenyl)-DL-alanine)} yielding dopachrome {2,3-dihydroindole-5,6-quinone-2carboxylic acid} in the presence of the oxygen (figure 3) (Behbahani et al, 1993). Although more sensitive and more accurate methods of tyrosinase assay are available (Winder and Harris, 1991; Li and Nappi, 1991; Rodriguez-Lpoez et al., 1991; Pomerantz, 1976), this method was chosen because of its simplicity and instrumentation accessibility.

Santoso and Goff, Evaluation of the Ethylene Effect on Well Defined Copper Enzymes 27

Figure 3. Catalyzed oxidation of DL-DOPA to dopachrome by tyrosinase. 2. Methods 2.1 Evaluation of the Ethylene Effect on SOD Proton NMR spectra of bovine erythrocyte SOD were acquired at 25 C on a Bruker AMX-600 FT NMR irradiating at 600.14 NMz. Three different samples of bovine erythrocyte SOD were prepared: a) native SOD in the Cu(II) oxidation state, b) reduced SOD in the Cu(I) oxidation state, and c) reduced SOD in the presence of ethylene. Each sample consisted of 0.3 ml of 2 mM protein solution in 0.02 M deuterated sodium phosphate buffer, pD 7.0. Bovine erythrocyte SOD was purchased from Sigma Chemical Co., and used without further purification. Deuteration of protein exchangeable protons was carried out prior to proton NMR measurement, by stirring the enzyme in 0.02M deuterated phosphate buffer in a 3.0 ml Amicon stirred cell at 4 C. The procedure was repeated five times to promote optimum deuterium exchange. Change of pD due to the isotope effects was not corrected. After acquiring the proton NMR of this solution, 0.4 mg solid sodium dithionite (Na2S2O4, Aldrich) was added into the protein solution under N2 atmosphere. The proton NMR spectrum of this was then recorded at room temperature to determine whether the reduction of Cu(II) to Cu(I) occurred. The air on the top of the solution in the NMR tube was slowly flushed with ethylene. The ethylene gas was then gently mixed with the protein solution. The procedure was repeated six times. At room temperature approximately 1.8 moles of ethylene is dissolved in 0.3 ml of aqueous protein solution. The proton NMR spectrum of the ethylene saturated protein solution was acquired under the same conditions as the previous samples. 2.2 Evaluation of the Ethylene Effect on Mushroom Tyrosinase Mushroom tyrosinase was purchased from Sigma Chemical Co. The control assay, where activity of tyrosinase was monitored in the absence

of ethylene, consisted of a) two enzyme units (0.2 mL of a solution of 1 mg tyrosinase in 100 mL of 0.1M sodium phosphate buffer, pH 6.0), b) 0.5 mL of 5 mM DL-DOPA solution in 0.1 M sodium phosphate buffer, pH 6.0, and c) 2 mL of 0.1 M sodium phosphate buffer, pH 6.0. All reagents were placed in a 3 mL cuvette equipped with a magnetic stirring bar. The solution was stirred for four minutes prior to the measurement to optimize O2 solubility. The formation of dopachrome was monitored at 475 nm (= 3.7 x 103 M cm), with the rate constant calculated from the first five minutes of reaction (Behbahani et al., 1993). Three other assay systems were constructed the same way as the control, except one of the reagents (enzyme, DL-DOPA, or sodium phosphate buffer) was each saturated with ethylene prior to mixing all of reagents. Ethylene was not bubbled directly into the reaction mixture to prevent total removal of O2 from the solution. The amount of ethylene dissolved in the enzyme solution, DLDOPA, and sodium phosphate buffer at 25 C is approximately 1.2, 3.0, and 12 moles, respectively. Hence, three assay systems with three different ethylene concentrations (0.5, 1.1, and 5 mM) were prepared. The assay was run in triplicate to evaluate reproducibility of results. 3. Result and Discussion The proton NMR spectrum of native SOD (figure 4) showed two groups of signals. The upfield region (chemical shift -1 to 4 ppm) consisted of aliphatic methyl, methylene, and methine protons, whereas the low field signals (chemical shift 6 to 10 ppm) were due to aromatic and unexchanged protons. Signals in this low field region were derived from four phenylalanine, one tyrosine, and eight histidine residues. Intensities corresponded to approximately one proton per signal (Cass et al., 1977; Cass et al., 1977). This information was useful for estimation of the coordinated ethylene signal intensity. Assuming that one ethylene was bound to the SOD active site, the intensity the four coordinated ethylene protons was expected to be four times higher compared to the intensity of each proton signal in the low field region. The downfield spectral region of reduced SOD, from 6 to 10 ppm (figure 5) shows a rather distinct appearance compared to that of native SOD. The signals line widths were narrower and multiplet splittings were observed. The loss of paramagnetic properties due to reduction of Cu(II) to Cu(I) accounted for the spectral changes. The proton NMR spectrum of reduced SOD in the presence of ethylene showed the free ethylene proton signal at 5.42 ppm (Signal 1 in figure 6). No coordinated ethylene proton signal was observed. The coordinated ethylene proton signal (expected to be shifted 0.5 ppm upfield from the free ethylene proton signal) might be obscured by the broad HOD

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signal. The line broadening of the HOD signal was presumably due to the partial oxydation of Cu(I) SOD by trace oxygen which could have been introduced during addition of ethylene to the enzyme solution. Attempts to reduce this water signal intensity by deuterium exchange and by water suppression techniques were not successful. A rapid

exchange process between free and coordinated ethylene molecules could not be ruled out as another cause for the unseen coordinated ethylene proton signal. Therefore, evaluation of ethylene binding in SOD was not conclusive.

Figure 4. Proton NMR Spectrum of 2 mM native SOD in 0.02 M deuterated phosphate buffer, pD 7.0, at 25 C.

Figure 5. Proton NMR Spectrum of 2 mM reduced SOD in 0.02 M deuterated phosphate buffer, pD 7.0, at 25 C.

Santoso and Goff, Evaluation of the Ethylene Effect on Well Defined Copper Enzymes 29

Figure 6 : Proton NMR Spectrum of 2 mM reduced SOD in 0.02 M deuterated phosphate buffer, pD 7.0, in the presence of 6 mM of ethylene, at 25 C. Data for tyrosinase assays are presented in (table 1). The reproducibility of the assays is demonstrated by the results of triplicate experiments listed in table 1. The presence of ethylene in the assay solution clearly decreases the tyrosinase activity. In most cases, external application of ethylene requires at least 1l L-1 (6.5 x 10-9 M liquid phase at 25 C), although in extreme cases it was found that ethylene effect could occur in the system with concentration as low as 7 x 10-9 M. Inhibition of tyrosinase activity mostly occurred when ethylene was dissolved directly into the enzyme solution prior to mixing, although the concentration of ethylene in this assay system (0.5 mM) was the smallest compared to the other two systems. An explanation was that dissolving ethylene directly into tyrosinase prior to assay provided a better condition for ethylene to compete more effectively with O2 and other substrates for the tyrosinase active site. Ethylene might presumably reduce the Cu(II) resting enzyme prior to irreversible binding or coordinate to the enzyme active site and prevent the binding of other substrates. The irreversible binding was confirmed by reexamining the proton NMR spectra of the same enzyme system after one or two hours of measurement. Indifferent NMR spectra were obtained suggesting the stability of the system. Table 1. Effect of ethylene on the tyrosinase
Number of Experiments Sample Specific Activity (units/mg protein)* Ethylene Ethylene Ethylene Control Saturated Saturated Saturated DOPA Buffer Tyrosinase 127 121 119 69 71 63 61 59 63 61+1.33 55 53 55 54+0.9

1 2 3 Average

123+3.09 68+3.11

*)Units = mole oxidized substrate per minute The amount of O2 displaced by ethylene corresponded to the amount of ethylene dissolved in the solution. Thus 1.2, 3.0, and 12 moles of oxygen were removed from the tyrosinase, DL-DOPA, and sodium phosphate buffer solutions, respectively. Inhibition of the tyrosinase activity was mostly observed in the "ethylene saturated tyrosinase" system where the least amount of O2 was displaced. Therefore the assay results also demonstrated that displacement of oxygen in the assay system by ethylene did not account for the loss of activity.

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The observed ethylene effect on tyrosinase activity, however, was minimal, since ethylene could produce threshold physiological response at concentration of 6.5 x 10-9 M. In conclusion, both bovine erythrocytes SOD and mushroom tyrosinase were not ideal models for evaluation of ethylene binding, therefore these systems could not suit as methods for evaluating constitution of Copper(I) as the most logical candidate among soft" metal ions which could fit the binding criteria of various alkenes to the receptor site. Other well defined copper enzymes such as laccase and cerruloplasmin should be tried. With respect to ethylene binding KD values of 10-10 M, more sensitive techniques such as a radiocarbon method combined with a spin-label ESR method should probably be applied. Acknowledgement The author would like to thank Prof. Soekeni Soedigdo for her valuable input on the discussion and on the further development of this research project. References Abeles, F. B., 1973, Ethylene in Plant Biology, Academic Press, Inc., New York. Abeles, F. B., P. W. Morgan, and M. E. Salveit Jr., 1992, Ethylene in Plant Biology, 2nd (ed.), Academic Press, Inc., San-Diego California. Behbahani, I., S. A. Miller, and D. H. O'Keefe, 1993, A comparison of mushroom tyrosinase dopaquinone and dopachrome assays using diode-array spectrophotometry: dopachrome formation vs. ascorbate-linked dopaquinone reduction, Microchem. J., 47, 251-260. Bengochea, T., M. A. Acaster, J. H. Dodds, D. E. Evan, P. H. Jerie, and M. A. Hall, 1980, Studies on Ethylene Binding by Cell-free Preparations from Cotyledons of Phaseolus vulgaris. II. Effects of Structural Analogs of Ethylene and of Inhibitors, Planta, 148, 407-411. Bengochea, T., J. H. Dodds, D. E. Evan, P. H. Jerie, B. Niepel, A. R. Shaari, and M. A. Hall, 1980, Studies on Ethylene Binding by Cell-free Preparations from Cotyledons of Phaseolus vulgaris. I. Separation and characterization, Planta, 148, 397-406. Beyer Jr., E. M., 1976, A Potent Inhibitor of Ethylene Action in Plants, Plant Physiol., 58, 268-271. Beyer Jr., E. M., 1978, Method for Overcoming the Antiethylene Effects of Silver(1+) ion, Plant Physiol., 62, 616-617. Beyer Jr., E. M., 1979, Effect of Silver ion, Carbon dioxide, and Oxygen on Ethylene Action and Metabolism, Plant Physiol., 63, 169-173. Burg, S. P., and E. A. Burg, 1967, Molecular Requirements for the Biological Activity of Ethylene, Plant Physiol., 42, 144-152.

Cass, A. E. G., H. A. 0. Hill, B. E. Smith, J. V. Bannister, and W. H. Bannister, 1977, Investigation of the Structure of Bovine Erythrocyte Superoxide Dismutase by Proton Nuclear Magnetic Resonance Spectroscopy, Biochem., 16, 3061-3066. Cass, A. E. G., H. A. 0. Hill, B. E. Smith, J. V. Bannister, and W. H. Bannister, 1977, Carbon-2 proton exchange at histidine-41 in bovine erythrocyte superoxide dismutase, Biochem. J., 165, 587-589. Cook, E. L., and J. Van Staden, 1987, Silver Action in the Cut Carnation Flower, Plant Physiol. Biochem., 25, 485-492. Fuch, Y., and E. Chalutz, 1984, Ethylene, Martinus Nijhoff/Dr. W. Junk Publ., The Hague. Hall, M. A., A. J. Cairns, D. E. Evans, A. R. Smith, P. G. Smith, J. E. Taylor, and C. J. R. Thomas, Binding Sites for Ethylene in P.F. Waeiing (ed.), 1982, Plant Growth Substances, Academic Press, London, 375-383. Jerie, P. H., A. R. Shaari, and M. A. Hall, 1979, The Compartmentation of Ethylene in Developing Cotyledons of Phaseolus vulgaris L, Planta, 144, 503. Li, J. and A. J. Nappi, 1991, Analysis of monophenol oxidase activity using high pressure liquid chromatography with electrochemical detection, J. Liq. Chromatogr., 14, 2089-2108. Pomerantz, S. H., 1976, A sensitive new assay for the oxidation of 3,4-dihydroxy-L-phenylalanine by tyrosinase, Anal. Biochem., 75, 86-909. Roberts J. A. and G. A. Tucker, 1985, Ethylene and Plant Development, Butterworths, London. Rodriguez-Lopez, J. N., P. Serna-Rodriguez, J. Tudela, R. Varon, and F. Garcia-Canovas, 1991, A continuous spectrophotometric method for the determination of diphenolase activity of tyrosinase using 3,4-dihydroxymandelic acid, Anal. Biochem., 195:2, 369-374. Sisler, E.C., 1977, Ethylene Activity of Some Acceptor Compounds, Tob. Sci., 21, 43. Sisler, E. C., 1979, Measurement of Ethylene Binding in Plant Tissue, Plant Physiol., 64, 538. Sisler, E. C., 1980, Partial Purification of an Ethylene-Binding Component from Plant Tissue, Plant Physiol., 66, 404-406. Sisler, E. L., and C. Wood, 1987, Ethylene Binding and Evidence that Binding in vivo and in vitro is to the Physiological Receptor, NATO ASI Series. Series H., 10, 239-248. Sigel, H., (ed.), 1981, Metal ions in Biological System, vol. 13, Marcel Dekker, Inc., New York. Thompson, J. S., R. L. Harlow, and J. F. Whitney, 1983, Copper(I)-Olefin Complexes. Support for the Proposed Role of Copper in the

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