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Abstract. Methylmercury as well as mercury (II) were found to be effective inhibitors of the catalytic activity of alcohol dehydrogenase from baker's yeast in the.
Microchim. Acta 140, 29±34 (2002) DOI 10.1007/s00604-001-0893-1

Enzymatic Determination of Methylmercury Traces using Alcohol Dehydrogenase from Baker's Yeast Tatyana N. Shekhovtsova and Evgenya V. Zhmaeva Chemistry Department, Lomonosov Moscow State University, Lenin Hills, Moscow, 119899 Russia Received February 9, 2001; accepted August 10, 2001; published online June 24, 2002 # Springer-Verlag 2002

Abstract. Methylmercury as well as mercury (II) were found to be effective inhibitors of the catalytic activity of alcohol dehydrogenase from baker's yeast in the reaction of ethanol oxidation by nicotinamide adenine dinucleotide. It was stated that the methylmercury inhibitory action belonged to the non-competitive type, whereas Hg(II) inhibited the enzyme according to the mixed type. The inversely proportional dependence of the indicator reaction rate on the concentration of methylmercury allowed to develop an enzymatic procedure for its determination with a detection limit of 3 nM. The possibility of methylmercury determination in presence of mercury (II) and mercury (II) determination in presence of methylmercury (concentration ratios CH3Hg ‡ :Hg(II) were 1:1 and 1:10, respectively) was shown. In the ®rst case masking reagents, DEDTC or thiourea, were used to form stable complexes with Hg(II). Key words: Enzymatic analysis; alcohol dehydrogenase from baker's yeast; inhibitors; methylmercury; mercury (II).

Mercury and methylmercury especially, belong to the main and most dangerous pollutants of the environment. Mercury vapour is actively absorbed and accumulated in brain, kidney and ova. Hg0 and CH3Hg ‡ disturb the activity of peripheral and central nervous system, that leads to a heavy damage of organism. Therefore, the problem of developing sensitive,  To whom correspondence should be addressed

selective, rapid and simple methods for mercury determination in any of its chemical forms is urgent. Enzymatic methods of analysis combine all these requirements. Alcohol dehydrogenase (ADH) from baker's yeast is one of the promising enzymes for application in chemical analysis. This enzyme belongs to the class of oxidoreductases and catalyzes the oxidation of primary aliphatic alcohols to corresponding aldehydes by nicotinamide adenine dinucleotide (NAD ‡ ). Earlier it has been stated by us that some metal ions which are potentially dangerous for human beings, and mercury (II) among them, inhibit the catalytic activity of ADH [1]. At the same time it is known that because of microorganism activities inorganic mercury compounds are transformed in water into methylmercury, which accumulates mainly in ®sh organisms. Eating such ®shes may lead to Minamata illness with lethal outcome [2]. Thus, methylmercury is characterized by the most toxic effect on human organism in comparison with other mercury species. The aim of our investigation was to work out a procedure for methylmercury determination in absence and presence of mercury (II). Experimental Reagents A solid preparation of ADH from baker's yeast (EC 1.1.1.1, Sigma, USA) was used. Solutions with enzyme concentrations of 0.5± 2.0 mM were prepared by dissolving an enzyme preparation in phosphate buffer (pH 7.6). Solid samples and solutions of the enzyme

30 were stored frozen in a refrigerator at 18  C. Solutions of NAD ‡ were prepared from a solid preparation (Wakoo, Japan) daily by dissolving accurately weighed amounts in water. Solid samples and solutions of NAD ‡ were stored at ‡ 4  C. Ethanol recti®cate was used to prepare 6 M solutions by diluting exact volumes of 96% ethanol with water. Mercury (II) solutions (1 mg=ml) were prepared by dissolving accurately weighed amounts of pure metallic mercury in 1±2 drops of very pure concentrated HNO3 and subsequent diluting with water (pH 3±4) to the required volume. Solutions with lower metal content were prepared by successive dilution of the initial solution with water acidi®ed with 1±2 drops of concentrated HNO3. Solid preparations of methylmercury iodide, thiourea, and diethyldithiocarbamate (DEDTC) were used. Solutions of thiourea and DEDTC with concentrations from 0.015 to 15 mM were prepared by dissolving exactly weighed amounts of these compounds in water. Methylmercury iodide was dissolved in 96% ethanol-recti®cate. Their solutions were stored in the dark at ‡ 4  C. Phosphate buffer solution (0.1 M, pH 7.6) was prepared by mixing 0.1 M K2HPO4 and 0.1 M NaH2PO4 solutions and subsequent addition of 0.1 M NaOH to the required pH value. Tris-HCl buffer solution (0.05 M, pH 9.0) was prepared by dissolving accurately weighed amounts of tris-(hydroxymethyl)-aminomethan (Serva) in H2O and subsequent addition of 0.1 M HCl to the required pH value. All reagents used were of analytical grade. For preparation of all aqueous solutions doubly distilled water puri®ed by water puri®cation system ``Simplicity'' (Millipore, Austria) was used. Measurement Procedure The indicator reaction rate was monitored spectrophotometrically at 340 nm, corresponding to the maximum absorbance of the reaction product NADH. A spectrophotometer Shimadzu UV-2201 (Japan) (l ˆ 0.3 cm) was used. The pH of buffer solutions was measured by potentiometer pH-121 (Russia). For dosage of solutions of the components of the enzymatic process: ADH, NAD ‡ , ethanol, mercury (II) and methylmercury micropipettes were used. All experiments were performed at room temperature (20±25  C). Note that the rate of ethanol oxidation by NAD ‡ changes by up to 2% when the temperature changes by 1  C [8, 9] . Besides, according to literature data [7], ADH solutions are stable also at temperatures  45  C. The detection limit was calculated according to the 3s-criterion for n replicate measurements and at the con®dence level (P) 95%. Procedures Determination of Methylmercury (or Mercury (II)). 1.1 ml of 0.05 M Tris-HCl buffer solution, 0.1 ml of NAD ‡ solution (4.5 mg=ml), 0.1 ml of 6 M ethanol, 0.1 ml of methylmercury or mercury (II) solutions in the concentration range of 10±250 nM or 0.25±2.5 nM, respectively, (or distilled water in the case of a blank experiment) and 0.1 ml of ADH solution (0.075 mg=ml) were placed subsequently into a glass test-tube with a ground-glass stopper. The total volume of the reaction mixture should be equal to 1.5 ml. At the moment of ADH adding and mixing the reaction solution, a stop watch was started and the absorbance of the solution was measured at 15 s intervals for 2 min at 340 nm. The indicator reaction rate was characterized by the slope of the straight initial part of kinetic curves with the coordinates: absorbance (A) versus time (s). The calibration graphs were plotted as tg ± analyte concentration. Determination of Methylmercury in Presence of Mercury (II). 1.0 ml of 0.05 M tris-HCl-buffer solution, 0.1 ml of NAD ‡

T. N. Shekhovtsova and E. V. Zhmaeva (4.5 mg=ml) solution, 0.1 ml of 6 M ethanol, 0.1 ml of a solution to be analysed with 0.1 ml of 1 mM thiourea solution or 0.1 ml of 10 mM DEDTC solution added to it previously, and 0.1 ml of ADH solution (0.075 mg=ml) were placed subsequently into a glass testtube with a ground-glass stopper. Then measurements were carried out as described above.

Results and Discussion The reaction of ethanol oxidation by NAD ‡ catalyzed by yeast alcohol dehydrogenase was used as indicator.

Optimum conditions for this reaction were stated by us earlier [1]; concentrations in the reaction mixture: ADH ± 0.005 mg=ml (30 nM), NAD ‡ ± 0.4 mg=ml (6 mM), ethanol ± 0.4 M; pH 9.0, Tris-HCl-buffer solution. Earlier [1] it was found that among the metal ions decreasing the catalytic activity of ADH: Hg(II), Cd(II), Ag(I), Zn(II), Pb(II), Cu(II)±Hg(II) was the most effective inhibitor of the enzyme. The developed procedure for mercury (II) determination possesses the best analytical characteristics in comparison with those of the procedures for Ag(I), Zn(II) and Cu(II) determination (Table 1). It permits also to determine mercury (II) rather selectively in the presence of other metal ions: only Cd(II) and Pb(II) at 100 and 1000 fold concentration excesses, respectively, interfered with the Hg(II) determination. Cd(II) and Pb(II) at their simultaneous presence did not interfere with the Hg(II) determination when its concentration was 200 times lower than their total content. Hg(II) may be also determined in presence of Ag(I), because the conditions of their determination are different (optimum pH ± 9.0 and 7.6, respectively). According to literature data organomercury compounds (OMC) as well as mercury (II) belong to the group of speci®c reactants for SH-groups of enzymes. It was pointed out that the af®nity of mercury (II) and OMC to SH-groups is much higher than to other functional groups of enzymes [3]. It was stated [4] that OMC (in particular, phenyl- and methylmercury) as well as mercury (II), inhibited the catalytic activity of urease due to strong binding with SH-groups of the enzyme. Literature data show that ADH contains 28 free SHgroups per enzyme molecule (sub unit of the enzyme)

31

Enzymatic Determination of Methylmercury Traces using Alcohol Dehydrogenase from Baker's Yeast

Table 1. Analytical characteristics of the procedures for the determination of metal ions using ethanol oxidation by NAD ‡ catalyzed by ADH Metal ion

Applicable

Calibration

Cmin

concentration range, mg=ml

curves, equations

mg=ml

y ˆ 12.40±0.69x mˆ5 y ˆ 11.11±0.25x mˆ4 y ˆ 11.16±0.29x mˆ3 y ˆ 10.77±0.76x mˆ2

3  10

5

0.15

2

5  10

4

5

2

2  10

3

30

5

3  10

2

500

9

Hg(II)

5  10

Ag(I)

7.5  10

Zn(II)

5  10

Cu(II)

0.1±1.0

5

±5  10 4

3

4 3

±5  10

±5  10

2

RSD , nM

%

 y ± tg  102 .  x ± concentration of an inhibitor (C  10m, mg=ml).  C ± detection limit, calculated according to the 3s criterion. min  RSD ± relative standard deviation, calculated at the value of the lower limit of the concentration range.

[5]. One may suggest that the activity of the enzyme should be in¯uenced not only by mercury (II) but by OMC also, and the most toxic compound ± methylmercury, in particular. Optimization of the Indicator Reaction Conditions The dependence of the indicator reaction rate on the pH of the reaction mixture and concentrations of its components was studied for clearing up the optimum conditions of the reaction carried out in presence of methylmercury (Figs. 1, 2). The methylmercury in¯uence was studied at pH range 7.6±9.0, because at pH above 9.0 the enzyme is denaturated and at pH lower than 7.6 the indicator reaction rate is not high enough for its exact measurement. Besides it is known [6] that ADH solutions with pH 7.6 (in phosphate or tris-HClbuffer solutions) are the most stable. It was found that a maximum inhibitory effect of methylmercury on ADH activity was observed at pH 9.0 (tris-HCl-buffer solution). Concentrations of the reaction components at maximum difference between the reaction rates in absence and presence of methylmercury were selected to be optimum: ADH± 0.005 mg=ml (30 nM), NAD ‡ ± 0.4 mg=ml (6 mM), ethanol ± 0.4 M. Thus, optimum conditions of the indicator reaction in presence of methylmercury and mercury (II) [1] are equal. Under the indicated optimum conditions methylmercury was found to inhibit the ADH catalytic activity in a wide range of its concentrations (0.0001±1000 mM) (Fig. 3). It should be noted that the indicator reaction rate did not depend on the time of methylmercury preincubation with the enzyme.

Fig. 1. Dependence of the indicator reaction rate on pH of tris-HCl buffer solution in absence (1) and presence (2) of methylmercury (concentrations: ADH ± 0.2 mM, NAD ‡ ± 6 mM, ethanol ± 0.4 M, methylmercury ± 1 mM)

Fig. 2. Dependence of the difference between the indicator reaction rate in absence and presence of methylmercury (tg  102) on the concentration of ethanol (1), NAD ‡ (2), ADH (3). (trisHCl buffer solution, pH 9.0; concentrations: ADH ± 0.2 mM (1, 2), NAD ‡ ± 6 mM (1, 3), ethanol ± 0.4 M (2, 3), methylmercury ± 1 mM)

32

Fig. 3. Dependence of ethanol oxidation rate on methylmercury concentration (tris-HCl buffer solution, pH 9.0; concentrations: ADH ± 0.2 mM, NAD ‡ ± 6 mM, ethanol ± 0.4 M)

Inversely proportional dependence of the reaction rate on methylmercury concentration in the range 0.01±0.25 mM allowed to work out an enzymatic procedure for its determination with the detection limit of 3 nM, which is higher than that of mercury (II) (0.15 nM). The calibration graph equation is as follows: y ˆ (11.11  0.09)±(0.25  0.08)x, m ˆ 8; n ˆ 5; r ˆ 0.9991 (where y ˆ tg  102, x ˆ concentration of the inhibitor ± C  10m, M; n ± the number of parallel measurements; r ± coef®cient of correlation). Type of ADH Inhibition by Methylmercury and Mercury (II) The inhibitory effect of Hg(II) and methylmercury on ADH activity seems to depend on their selective

T. N. Shekhovtsova and E. V. Zhmaeva

interaction with SH-groups of the active site of the enzyme. So the same character of inhibitory in¯uence of mercury (II) and methylmercury on the enzyme activity may be supposed. However, as it is pointed out above, mercury (II) is a stronger inhibitor of ADH than methylmercury. The type of ADH inhibition by Hg (II) and methylmercury was studied to explain the reasons of the different extent of their inhibitory effect. It is known [8] that enzyme inhibitors may be reversible and irreversible. An inhibition type may be de®ned only for reversible inhibitors, that is, for those inhibitors which are able to form dissociating complexes with an enzyme. The method of dilution [9] was used while studying the reversibility of ADH inhibition by mercury (II) and methylmercury. According to this method, the reversible inhibition takes place in those cases when 10 times dilution of an enzyme and inhibitor mixture gives the following ratio: Enzyme activity after dilution 1=10 of enzyme activity without dilution. It was shown that ADH interaction with Hg (II) and CH3Hg ‡ as well, was reversible (the above indicated ratio was 6.6 and 7.0, respectively) [8, 9]. The presentation of the obtained data in Lineweaver-Burk and Dixon coordinates stated the mixed type of the inhibitory effect for mercury (II) and the non-competitive type for methylmercury (Figs. 4, 5). As it is known the mixed type of inhibition assumes that an inhibitor affects upon both a binding site of an enzyme with

Fig. 4. Data on studying the type of ADH inhibition by mercury (II), presented in the coordinates of Lineweaver-Burk (a) and Dixon (optimum conditions for mercury (II) determination; (a) concentrations of Hg(II), mg=ml: 1 0.5; 2 0.3; 3 0.2; 4 0.1; 5 0; (b) concentrations of ethanol, M: 1 0.05; 2 0.1; 3 0.2; 4 0.4; 5 0.6)

Enzymatic Determination of Methylmercury Traces using Alcohol Dehydrogenase from Baker's Yeast

33

Fig. 5. Data on studying the type of ADH inhibition by methylmercury, presented in the coordinates of Lineweaver-Burk (a) and Dixon (optimum conditions for methylmercury determination; (a) concentrations of methylmercury, mM: 1 ± 1.5; 2 ± 0.75; 3 ± 0.25; 4 ± 0.025; 5 ± 0; (b) concentrations of ethanol, M: 1 ± 0.2; 2 ± 0.3; 3 ± 0.4; 4 ± 0.6; 5 ± 0.8)

a substrate and a catalytic site of a biocatalyst. According to the non-competitive type of inhibition, an inhibitor is not able to join an enzyme, but it joins an enzyme-substrate complex. Thus, the different extent of the inhibitory effect of Hg (II) and CH3Hg ‡ may be explained by different types of ADH inhibition. Determination of Methylmercury in Presence of Hg(II) and of Hg(II) in Presence of Methylmercury To study a possibility of selective determination of methylmercury in presence of Hg(II) we chose thiourea and DEDTC as masking reagents, capable to form stable complexes with Hg(II). It was stated that DEDTC in a wide range of its concentrations (0.01±1000 mM) had no in¯uence on the catalytic activity of ADH. Thiourea inhibited the enzyme at

pH 9.0 only in the interval of its concentrations from 10 to 100 mM and pre-incubation time of 5 min. Thiourea and DEDTC at concentrations 1 mM and 10 mM, respectively, eliminated the inhibitory effect of mercury (II) at its concentrations of 2 ng=ml (10 nM) and 20 ng=ml (100 nM), that correspond to 4  MPC and 40  MPC, respectively ( MPC ± maximum permissible concentration of mercury (II) in natural waters equals to 0.5 ng=ml). At the same time these reagents did not interfere with the determination of methylmercury. The obtained data are presented in Table 2. Thus, using thiourea or DEDTC as masking reagents for Hg (II) allows to determine methylmercury in presence of 10-fold excess of mercury (II). Besides, it was found that methylmercury interfered with mercury (II) determination only at 1000-fold excess (Table 3). Thus, the procedure for mercury

Table 2. Results of methylmercury determination in presence of mercury (II) and masking reagents (CCH3 Hg‡ ± 10 nM (a, b); CHg(II), nM: 10 (a), 100 (b)) (P ˆ 0.95; n ˆ 5) Concentration ratio

Components added to the indicator reaction

CH3Hg ‡ : Hg(II)

tg  102 Thiourea (1 mM)

DEDTC (10 mM)

± (blank experiment) CH3Hg+

13.2  0.2 12.2  0.2

1:1 (a)

Hg(II) Hg(II) ‡ masking reagent Hg(II) ‡ masking reagent ‡ CH3Hg ‡

11.8  0.2 12.8  0.2 12.2  0.2

12.9  0.2 12.0  0.2

1:10 (b)

Hg(II) Hg(II) ‡ masking reagent Hg(II) ‡ masking reagent ‡ CH3Hg ‡

10.5  0.2 13.0  0.2 12.0  0.2

12.8  0.2 11.9  0.2

34

Enzymatic Determination of Methylmercury Traces using Alcohol Dehydrogenase from Baker's Yeast

Table 3. Interfering effect of methylmercury on mercury (II) determination using the ethanol oxidation catalyzed by ADH (P ˆ 0.95; n ˆ 5) Inhibitor

Concentration, nM

tg  102

± Hg(II) Hg(II) ‡ CH3Hg ‡

±

13.7  0.1 12.7  0.1

0.5 0.5

5 50 100 250 500

12.7  0.2 12.5  0.2 12.5  0.2 12.3  0.3 8.0  0.2

(II) determination presented in [1] allows to determine mercury (II) rather selectively not only in presence of cadmium (II), lead (II), silver (I), but in presence of methylmercury also. The developed enzymatic procedure for the determination of methylmercury is more sensitive, selective and rapid in comparison with other more often applied methods: atomic absorption with cold vapour [10], atomic ¯uorescence [11], various types of chromatography (GC [12], GLC [13], HPLC [14]). Analytical characteristics of this procedure may be compared with those of the enzymatic procedure for the determination of organomercury compounds based on their inhibitory action on urease [15]. Conclusions The developed enzymatic procedure for the determination of methylmercury based on its inhibitory effect on alcohol dehydrogenase from baker's yeast is

sensitive (Cmin ˆ 3 nM), selective (allows to determine methylmercury in the presence of 10-fold excess of mercury (II)), simple, rapid and relatively inexpensive. Acknowledgements. This work was partially supported by grant N-00-03-32548 from the Russian Fundamental Research Foundation.

References [1] E. V. Zhmaeva, T. N. Shekhovtsova, J. Analyt. Chem. (transl. from Russian). 2000, 55(8), 782. [2] H. Sigel, A. Sigel, Concepts on Metal Ion Toxicity. Metal Ions in Biological Systems. Marcel Dekker Inc., New York Basel, 1986, Vol. 20, p. 336. [3] Yu. M. Torchinskiy, Sulphur in Proteins. Mir. Nauka., 1976. p. 43 (in Russian). [4] B. Krajewska, M. Lezko, W. Zaborska, J. Chem. Technol. Biotechnol. 1990, 48, 337. [5] P. Boyer, H. Lardy, K. Myrback, The Enzymes. Vol. 7. Academic Press, New York London, 1963, p. 25. [6] C. Branden, H. J ornvall, H. Eklunol, B. Furugren. The Enzymes, Vol. 11, Part A. Academic Press, New York, 1975, p. 103. [7] L. M. Shimon, M. Kotorman, A. Bene, B. Sayani, Prikl. Biokhim. Microbiol. (in Russian), 1991, 7(1), 86. [8] T. Keleti, Basic Enzyme Kinetics. Academia Kiado, Budapest, 1990, p. 234. [9] M. Dixon, E. Webb, Enzymes. Longman Group. Ltd., 1979, 1, 98. [10] Yu. V. Zelyukova, M. M. Novoselov, Mercury in Rivers and Bodies. Proceeding of USSR Symposium, (Novosibirsk, Russia, October 9±11, 1990), p. 63. [11] S. C. Edwards, C. L. Macleod, W. T. Corns, A. D. Williams, J. N. Lester, Intern. J. Anal. Chem. 1991, 63, 187. [12] M. K. Behlke, P. C. Uden, M. M. Schantz, S. A. Wise, Anal. Chem. 1996, 68, 3859. [13] C. J. Cappen, T. J. Joribara, LC & GC. 1986, 74, 1010. [14] S. Corrado, Proceeding of Pittsburgh Conference, New Orleans, USA, March 9±12, 1992, p. 310. [15] L. Orgen, Anal. Chim. Acta. 1981, 125, 45.