Annals of West University of Timisoara

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Annals of West University of Timisoara. Series Chemistry 16 (2) (2007) 191 - 200. 191. USE OF CATALASE FROM SPINACH FOR TESTING AT. MOLECULAR ...
Annals of West University of Timisoara Series Chemistry 16 (2) (2007) 191 - 200

USE OF CATALASE FROM SPINACH FOR TESTING AT MOLECULAR LEVEL THE TOXICITY OF SOME IONIC LIQUIDS A na - Mari a L a cră mă , La u ra P op e ţ, Va sil e O s taf e West University of Timişoara, Faculty of Chemistry-Biology-Geography, Department of Chemistry, Pestalozzi, 16, Timişoara, 300115, ROMANIA

SUMMARY The present work tries to evaluate catalase from spinach in order to introduce it into an ecotoxicological test battery. Previous to the determination of the influence of some ionic liquids on spinach catalase activity, the optimization of assay enzyme was performed. Optimum pH for spinach leaves catalase is between 7.5-8. Spinach catalase seems to not be inhibited by its substrate till 50 mM concentration. Spinach catalase was inhibited, in different degree, by the ionic liquids studied. The inhibition is stronger if the “heads” of molecular ions can make π – π interactions with pyrrol rings from hem b. The degree of inhibition increase in the order: pyrrolidine < imidazole < pyridine. The lengths of hydrophobic “tails” have a minor influence on the degree of inhibition. The aromatic tails inhibit stronger than the aliphatic ones. Results shown that catalase (from spinach) is a good candidate for the multi-enzymatic kit for testing at molecular level the toxicity of chemical compounds.

Keywords: spinach catalase, ecotoxicological test battery, enzyme inhibition, ionic liquids.

INTRODUCTION When new chemicals are synthesized their impact on environment and human health is unknown. This is the case of ionic liquids – a group of chemicals which have gained increasing attention in recent years as alternative solvents in organic synthesis, but their toxicity and ecotoxicity was not systematically studied [1]. We therefore developed a

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strategy to overcome this drawback, proposing a multi-enzymatic battery test kit for assessing their influence at molecular level on some enzyme chosen in accordance with following criteria: to be part from a major biochemical pathway, to be found in almost all organisms, to be extracted/purified from a cheap biological source and the assays of the enzyme has to be cheap, rapid and reliable. The present work tries to evaluate catalase from spinach in order to introduce it into an ecotoxicological test battery. Catalase is a key oxidative defense enzyme that present in the great majority of aerobic species. It biological role is to removes hydrogen peroxide before it can decompose into highly reactive hydroxyl radicals. The catalytic reaction uses hydrogen peroxide as both an electron donor and an electron acceptor, as summarized in the overall reaction (1)[2]. (1) 2HO 2HO+O 2

2

2

2

The importance of the enzyme is evident in the evolution of three distinctly different enzymes with the same catalytic activity: the monofunctional catalases, the bifunctional catalase – peroxidases and the non-heme manganese – containing catalases [3]. The most widespread type is the monofunctional catalase, examples of which are found in most aerobic organisms. Monofunctional catalases are present in both empires of life, the Prokaryota and Eukaryota. In the Eukaryota, catalases are found in all major taxa, the Protista, Animalia, Fungi, Plantae [4]. Monofunctional catalases are typically homotetrameric with one heme per monomer as active site, the small monomers (55-65 kDa) containing heme b and the large monomers (80-84 kDa) containing heme d. Phylogenetic analyses have grouped the small subunit catalases into one or two main groups, clade 1 or 3, and the large subunit enzymes exclusively into clade 2 [5]. All algal/plant catalases reside in clade 1 together with a subset of small-subunit catalases from Posibacteria and Negibacteria [5]. In plants, catalase is encoded by a small gene family. This leads to multiple isoforms of the enzyme [6]. As regards the reaction kinetics, two major considerations deserve attention: • saturation of catalase by H2O2 does not occur until a substrate concentration of about 5M; • at H2O2 concentrations above 1M the enzyme is rapidly inactivated by the substrate [7]. Most vegetal catalases studied are inhibited at rather small H2O2 concentrations of 50 mM [6]. To determine the influence of some ionic liquids on catalase activity from spinach, there was used an assay modified after Aebi [8], in which the variation of the reaction

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mixture’s extinction to 240 nm is determined in order to establish the variation of H2O2 concentration based on its molar extinction coefficient at 240 nm. MATERIALS AND METHODS Reagents and materials Potassium phosphate (KH2PO4), sodium phosphate (Na2PO4), acetate buffers 0,05M of pH=4 and pH=5, glycocol buffers 0,05M of pH=9 and pH=10 had analytical purity (Reactivul Bucureşti). Hydrogen peroxide (H2O2) was purchased from Sigma, as a stabilized 30 wt% solution in water and was stored at 40C. Ionic liquids: •

[OMPy] [Cl] (4-methyl-N-octyl pyridinium choloride),



[OMPyr] [Cl] (1-octyl-1-methyl-pyrolidinium chloride),



[BMPy] [BF4] (1-buthyl-3-methyl-pyridinium tetrafluoroborate),



Cytec 111 (trihexyl-(tetracedyl)-phosphonium tetrafluoroborate),



[BMIM] [BF4] (1-buthyl-3-methyl-imidazolium tetrafluoroborate),



[HMIM] [BF4] (1-N-hexyl-3-methyl-imidazolium tetrafluoroborate),



[OMIM] [BF4] (1-N-octyl-trimethyl-imidazolium tetrafluoroborate),



[BzMIM] [BF4] (1-benzyl-3-methyl-imidazolium tetrafluoroborate)

were supplied by Prof. Bernard Jastorff from Bremen University, Germany. Catalase [EC 1.11.1.6] was extracted from fresh spinach leaves (Spinacia oleracea). Apparatus In order to assay catalase activity there was used a Beckman DU-7 spectrophotometer and 2 mL quartz cuvettes. Catalase extraction Spinach leaves (5 g) were triturated in 50 mL phosphate buffer 0,05 M, pH=7 (at 0

4 C) through homogenization in a Warring Blender at 10000 rpm, 2 times for 10 seconds, with 2 minute pause in between. The extract was centrifuged at 1500 g for 30 minutes. The clear supernatant was diluted 10 times before the assay was performed.

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Enzyme assay A spectrophotometric assay modified after Aebi [8] was used. The method is based on the property of H2O2 to absorb UV radiation at 240 nm. One activity unit represents that quantity of the enzyme that produces the decomposition of 1 μmol H2O2 in 1 minute, in the assay conditions. The molar extinction of H2O2 at 240 nm was taken 0,0436 μmol-1cm2 [9]. The reaction mixture was realized using: •

0,1 mL diluted spinach leaves extract



1,9 mL H2O2, of different concentrations in the appropriate buffer

Enzyme activity was determined using the following formula: Where ΔE is the extinction variation at 240 nm in a Δt ΔE 1 v f period (in minutes); ε=absorption molar coefficient; A= ⋅ ⋅ ⋅ dil Δt ε v p vf=final volume of the reaction mixture; vp=volume sample, dil=dilution factor The determination of optimum pH The activity of the enzyme in buffers of different concentrations was determined in order to establish the optimum pH for catalase action. The following pH-s values were chosen: 4, 5, 6, 7, 8, 9 and 10 (acetate, phosphate, tris and bicarbonate buffers). Catalase was extracted in distilled water. Crude extract was diluted 10 times, using each buffer. Hydrogen peroxide dilutions were also realized in the buffers with the above mentioned pH values. The influence of substrate concentration on spinach catalase activity The catalase extract was realized in phosphate buffer 0,05M, pH 7, and H2O2 dilutions were made in the same buffer. A solution of 352,9 mM H2O2 was prepared, solution which was diluted to obtain the following concentrations: 50,41 mM, 25,2 mM, 12,6 mM, 6,3 mM and 3,15 mM. The determination of the influence of ionic liquids on spinach catalase activity Prior to the determination of catalase activity in presence of ionic liquids, these were incubated 10 minutes together with the spinach extract: 0,5 mL extract diluted 10 times (with an extinction at 280 nm of about 0,150) + 0,5 mL ionic liquids with a concentration of about 10 mM. Out of each of these mixtures there were taken 0,2 mL

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which were put straight in the cuvette of the spectrophotometer and 1,8 mL hydrogen peroxide 25,2 mM in phosphate buffer 0,05M, pH 7 was added. The decrease of the extinction at 240 nm was monitored over about 10 minutes. Enzyme activity in presence of the inhibitors was then calculated and it was compared with enzyme activity determined in experiments made in the absence of ionic liquids. After that, the concentration needed to inhibit 50% of catalase activity of each ionic liquid, was appreciated. RESULTS Previous to the determination of the influence of some ionic liquids on spinach catalase activity, the optimization of assay enzyme is needed. The following parameters were being considered: pH and substrate concentration. The determination of optimum pH For most catalases, literature data indicate an interval of optimum pH between 6.58.5 [7]. Optimum pH for spinach leaves catalase results to be 7.5-8. The influence of substrate concentration on spinach catalase activity As there wasn’t found any data in literature concerning spinach catalase inhibition, there was assumed that this enzyme too, could be inhibited by H2O2 concentrations of more that 50 mM. The catalase from spinach seems to not be inhibited by its substrate till 50 mM concentration (Figure 1). Spinach leaves catalase is inhibited by concentration of about 100 mM H2O2 (Figure 2).

The determination of the influence of ionic liquids on spinach catalase activity The ionic liquids used in this study were grouped into 2 kits, depending on their structure. The influence of the ionic liquids from kit 1, respectively kit 2 on spinach catalase activity is presented in Table I, respectively Table II.

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Figure 1.The variation on spinach leaves catalase

Figure 2. Comparison between the allure of the

depending on H2O2 concentrations

experimental curve (continuous line) and that of the Michaelis-Menten ecuation (dotted line)

Table I. The concentrations of the ionic liquids included in kit 1, which inhibit 50% of the initial activity of spinach catalase Ionic Liquids [BMIM][BF4] [HMIM][BF4] [OMIM][BF4] [BzMIM][BF4]

Concentration that inhibit 50% of catalase 61,68 μM 48,31 μM 60,86 μM 20,90 μM

% inhibition at 10μM ionic liquid 8,1 % 10,3 % 8,2 % 23,9 %

SD % 5,8 3,7 11,0 4,3

Table II. The concentrations of the ionic liquids included in kit 2, which inhibit 50% of the initial activity of spinach catalase Ionic Liquids Cytec 111 [OMPyr][Cl] [OMPy][Cl] [BMPy][BF4]

Concentration that inhibit 50% of catalase 12,24 μM 49,71 μM 21,14 μM 57,57 μM

% inhibition at 10μM ionic liquid 40,8 % 10,5 % 23,6 % 8,7 %

SD % 2,8 2,8 3,5 9,2

DISCUSSION In order to be able to explain the way in which tested ionic liquids influence spinach catalase activity one needs to know their structure. The structure of ionic liquids from kit 1 is presented below: These compounds have the same an imidazole ring which has a methyl group on the third position. All of the four compounds also have the same type of counter-ion –

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tetrafluoroborate. The difference between the four compounds is made by the length of the hydrophobic “tails” from the first position of the imidazole ring.

Figure 3. Ionic liquids with the same imidazole “head”

Experimental data from table I indicates (with an error of about 10%) that although the nature of the hydrophobic “tails” influences the degree of inhibition, the length of the tails has no significant effect on the degree of inhibition of spinach catalase. It is possible that the imidazole rings of the ionic liquids from kit 1 to interfere with the attachment of the substrate to the active center or with the tertiary structure of the catalase, interacting with the pyrrole rings from the catalytic center. Although we suppose that these ionic liquids interfere with the active site, this is no competitive inhibition. On the other hand, the fact that ionic liquids with aliphatic tails are less powerful inhibitors than BzMIM, might be explained through the reduced accessibility to the active center. The active site heme groups are deeply buried inside the molecular structure requiring the movement of substrate and products through long channels [10]. There are tree channels that have been implicated as potentially having a role in access to the active site. The perpendicular or main channel has long been considered to be the access channel. In small subunit enzymes the first 20 Å of the perpendicular channel starting from the surface of the protein is funnel shaped presenting little obstacle to even large molecules. The next

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15 Å of the channel extending from a conserved aspartate to the heme is constricted, restricting access to only small molecules [11].

Figure 4. Models of molecular surfaces of BMIM, OMIM, BzMIM respectively

As one can see in Figure 4, both BMIM and OMIM are asymmetric molecules and the fact that they inhibit catalase less than BzMIM does might be the result of their difficult access to the funnel’s neck of the channel which leads to the active site. The “ball” shape of BzMIM makes its access to this site easier. Another explanation could be the different nature of the hydrophobicity of BzMIM molecule and the other ionic liquids from kit 1. The number of possible π- π interactions between BzMIM and heme b is bigger than the numbers observed for the other ionic liquids from kit 1. In order to estimate the influence of the “heads” of ionic liquids on catalase activity, OMPyr and MOPy were studied, both of them having the chloride ion as counter ion (kit 2).

Figure 5. Ionic liquids with different “heads”

One can notice that pyridine is a higher inhibitor of catalase than pyrrolidine, when they are used as “heads” in ionic liquids (table II). The explanation, as in the example above, might be given by the bigger number of π- π interactions between OMPy and heme b from the catalytic center of catalase compared to OMPyr. We present below, Figure 6, the chemical structures of BMPy and MOPy. In this case, unfortunately, there are tree structural elements modified at the same time: the nature of the counter-ion, the position of the methyl “little tail” and the length of the hydrophobic tail bounded to the N atom.

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USE OF CATALASE FOR TESTING AT MOLECULAR LEVEL THE TOXICITY OF SOME IONIC LIQUIDS

Figure 6. Ionic liquids with tree structural elements modified at the same time

Although experimental data break a bit our theory according to which the degree of catalase inhibition might be related directly to the number of π- π interactions that ionic liquids might realize with the heme b from active site, yet, considering that MOPyCl has a smaller diameter than BMPyBF4, the former would enter much easier and would inhibit the enzyme more. The last compound analyzed was Cytec 111 and its structure is presented below, Figure 7:

Figure 7. Ionic liquid-Cytec 111

Its structure can hardly be compared with the structures of the other compounds, being thus difficult to explain its influence on catalase activity and to present theories in this regard. In order to be able to create a theory which should satisfactorily explain the effects of ionic liquids on catalase activity, there would be necessary to study very many related compounds, in which more features vary and the degree of modifications includes at least 45 successive modifications. This way SAR and QSAR studies could be carried out, studies which would give a base to the theory. Considering only the experimental data presented in this study alone, we can say that those ionic liquids which have imidazole, pyridine, pyrrolidine, phosphonium “heads” and aliphatic and aromatic hydrophobic “tails” inhibit, at different degree, spinach catalase activity. The method used in obtaining the extract which shows catalase activity from

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spinach leaves, the assay for the enzyme and the method for testing the effects of certain ionic liquids on this activity are fast and cheap procedures which offer a great deal of essential data referring to the toxicity of these compounds at a molecular level. CONCLUSIONS Catalase (from spinach) is a good candidate for the multi-enzymatic kit for testing at molecular level the toxicity of chemical compounds. A strategy for selecting the compounds was proposed. The ionic liquids studied inhibit, in different degree, spinach catalase. When the anion (as a counter-ion) was maintained constant (tetrafluoroborate), the nature of “heads” of cation, and also the nature of their “tails” influence the degree of inhibition of catalase. This inhibition seems to be allocated to interaction with pyrrole rings from the catalytic center. The inhibition is stronger if the “heads” of molecular ions can make π-π interactions with pyrrol rings from heme b. The degree of inhibition increase in order: pyrrolidine< imidazole< pyridine. The lengths of hydrophobic “tails” have a minor influence, but the aromatic tails inhibit stronger than aliphatic ones. REFERENCES 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

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