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Analytical and Bioanalytical Chemistry DOI 10.1007/s00216-004-2968-4

Special Issue Paper

Using enzymes isolated from diverse sources to determine metal ion cofactors
Tatyana N. Shekhovtsova () · Svetlana V. Muginova T. N. Shekhovtsova · S. V. Muginova Chemistry Department, Lomonosov Moscow State University, Leninskie Gory, 119992 Moscow, Russia T. N. Shekhovtsova Phone: +7-095-9393346 Fax: +7-095-9394675 E-mail: shekhov@analyt.chem.msu.ru Received: 29 September 2004 / Revised: 15 November 2004 / Accepted: 17 November 2004

Abstract Oxidoreductases and hydrolases isolated from different sources (horseradish and peanut peroxidases, alcohol dehydrogenases from baker's yeast and horse liver, and alkaline phosphatases from Escherichia coli, chicken and seal intestine) were used to determine their metal ion cofactors: Fe(III), Zn(II) and Mg(II), respectively. Studying the effects of the metal ion cofactors on the catalytic activity of the enzymes of different origin showed that the extent of their inhibition, activation, or reactivation of their apoenzymes depended on the structure and accessibility of the enzyme active site, which varies among the biocatalysts isolated from different sources. The developed procedures are based on the inhibiting (Zn(II)) or activating (Mg(II)) effects of the metal ions on the catalytic activity of the enzymes, or on reactivating effects (Fe(III) and Zn(II)) on the apoenzymes. The procedures are characterized by high sensitivity and selectivity; the detection limits of Fe(III) using horseradish peroxidase, Zn(II) using alcohol dehydrogenase from baker's yeast, alkaline phosphatase from seal intestine and its apoenzyme, and Mg(II) using alkaline phosphatase from chicken intestine equal 10 ng L-1, 20 ng L-1, 3 g L-1, 8 g L-1 and 0.2 g L-1, respectively.

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Keywords Enzymatic methods · Enzymes isolated from diverse sources · Determination of metal ion cofactors · Inhibition and activation of enzymes · Reactivation of apoenzymes

Presented at the 8th Symposium "Kinetics in Analytical Chemistry`', Rome, Italy, 5­8 July 2004.

Introduction
It is known that enzymatic methods of analysis are highly sensitive and selective due to the extremely high catalytic activity and specificity of biocatalysts. At the same time, these methods are simple, rapid and relatively cheap. There is growing interest in developing enzymatic methods of determination of inorganic and organic compounds. Primarily, such methods are based on the inhibiting or activating effects of analytes on enzymes of different classes. Numerous highly sensitive and rather selective methods for the determination of heavy metal ions (such as Hg(II), Pb(II), Cd(II), Bi(III)), nitrogen-, sulfur-, phosphorus-containing organic compounds, phenols, amino and fatty acids, based on their inhibiting effect on various oxidases (peroxidases, alcohol dehydrogenases), and hydrolases (alkaline and acid phosphatases), have been carried out in our laboratory [1­4]. These methods were applied successfully to the analysis of different environmental, biological, and food samples [5­7]. It should be mentioned that the inhibition, and especially the activation of enzymes, although sometimes fairly selective, are rarely specific. It is desirable, therefore, to investigate means of improving the selectivity and sensitivity of enzyme systems for trace analysis. Numerous enzymes contain a metal ion in their active site as a component (cofactor) essential for their activity. It is known [8], that, distinct from the interaction of a metal effector with an enzyme, the binding of a metal cofactor in the biocatalyst molecule is more specific and strong. The removal of the metal cofactor from the active site of a metal-dependent enzyme results in the full or partial loss of its catalytic activity because of the formation of an apoenzyme, the protein part of the biocatalyst molecule. The addition of external cofactor metal ions to the apoenzyme reactivates it, recovering the catalytic activity of the enzyme and providing high selectivity and sensitivity in the determination of these metal ions. Our recent research has been directed at investigating the inhibiting effects of different inorganic and organic compounds on the catalytic activities of a number of oxidases and hydrolases. The results of the investigations indicated that one of the newest and most promising techniques for improving the sensitivity and selectivity of enzymatic methods for determining biologically active

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compounds, and therefore enhancing their potential application in solving various problems of analytical chemistry, is the use of preparations of the same native enzymes isolated from diverse sources. This approach has been successfully accomplished with a number of oxidases: enzymes that catalyze redox processes [9­13]. Thus, using native plant peroxidases isolated from a horseradish root, peanut, the xylotrophic fungus Phellinus igniarius, and an alfalfa Medicago sativa cell culture, we have developed highly sensitive and selective procedures for determining their inhibitors and substrates (a number of metal ions and phenolic compounds) with different analytical characteristics [9­11]. Studies on the effects of heavy metal ions, methyl mercury, and other organic compounds on the activity of alcohol dehydrogenases from horse liver and baker's yeast made it possible not only to find their different sensitivities towards the action of the specified effectors, but also to develop procedures for determining inhibitors of alcohol dehydrogenase from horse liver--fatty acids--that did not affect the catalytic activity of another alcohol dehydrogenase [12, 13]. In this paper we present data from studyies on the inhibiting, activating, and reactivating effects of metal ion cofactors on the catalytic activity of horseradish and peanut peroxidases, alcohol dehydrogenases from baker's yeast and horse liver, and alkaline phosphatases from Escherichia Coli, chicken intestine and seal slim intestine, and their apoenzymes. The importance of such investigations is evident: a comparison of the previously published and recently obtained data on the character and severity of the effects of the ion cofactors on enzymes of different origin can help reveal differences in the mechanisms of action of these enzymes and, probably, in the structures of the active sites of the enzymes isolated from diverse sources. The differences found may be specifically used for the development of highly sensitive and selective procedures for the enzymatic determination of biologically active metal ions.

Experimental
Reagents
Solid preparations of plant peroxidases (EC 1.11.1.7) were used. Horseradish peroxidase was purchased from Sigma, USA (44 kDa, specific activity equals to 850 U mg-1, RZ=2.2) and cationic peanut peroxidase isolated from the cultural medium of Arachis Hypogea cells (40 kDa, 100 U mg-1, RZ=2.0) [14] was kindly provided by Prof. R.B. van Huystee (University of Western Ontario, Canada). Aqueous enzyme solutions were obtained by dissolving the enzyme preparations in

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0.15 mol L-1 borate (pH 7.0) and 0.05 mol L-1 TRIS-HCl (pH 7.5) buffer solutions, respectively. Solid preparations and solutions of the enzymes were stored in a refrigerator at +4 °C. Solutions of o-dianisidine and salicylic acid (analytical grade reagents from Reakhim, Moscow, Russia) were prepared daily by dissolving accurately weighed amounts in ethanol and water, respectively. Hydrogen peroxide was purchased from Merck, Germany. Alcohol dehydrogenases (EC 1.1.1.1.) from baker's yeast (500 U mg-1, Sigma) and horse liver (0.51 U mg-1, Fluka AG, Switzerland) were used. Solid preparations of the enzymes were stored in a refrigerator at -18 °C. Aqueous solutions of these enzymes were prepared daily by dissolving a portion of the enzyme preparation in a phosphate buffer solution (pH 7.6). Aqueous nicotinamide adenine dinucleotide (Sigma) solutions were prepared daily. Ethanol solutions were prepared using 96% rectified ethanol (Reakhim). Preparations of alkaline phosphatases (EC 3.1.3.1) isolated from three sources were used. Preparations of alkaline phosphatase from E.coli (33.8 U mg-1, Sigma) and slim intestine of a Greenland seal Phoca Groenlandica (13 U mg-1, Biolar, Latvia) were used as their homogeneous suspensions in 2.5 mol L-1 (NH4)2SO4 solution. Less concentrated solutions of the enzymes were prepared immediately before study by successive dilutions of the stock solutions with TRIS-HCl buffer solution (pH 9.8). Alkaline phosphatase from chicken intestine was a lyophilized powder from Sigma (0.4 U mg-1). The solutions of this enzyme were prepared daily by dissolving an accurately-weighed amount of the preparation in TRIS-HCl buffer solution (pH 9.8). Disodium p-nitrophenyl phosphate hexahydrate (Sigma) was used. Potassium biphthalate, KOH, H3BO3, Na2B4O7, potassium dihydrophosphate and hydrophosphate (Reakhim), and TRIS (Serva, Germany) were used to prepare buffer solutions. Stock solutions of Fe(III), Zn(II), and Pb(II) were prepared by dissolving accurately weighed samples of spectrochemically pure metals in high-purity grade HCl and HNO3, respectively. Stock solutions of Co(II), Cu(II), Ni(II), Cd(II) were prepared by dissolving accurately-weighed samples of their high-purity grade chlorides in water acidified to pH 2.5 with high-purity HCl. Solutions of lower concentrations of all metal ions were prepared daily by the successive dilution of the stock solutions with acidified water. The working solution of MgCl2 was prepared from its standard solution (Sigma). The solutions of KCl and CsCl were prepared from their salts of high-purity grade (Reakhim). Solutions of 1,10-phenanthroline (Sigma) and sodium ethylenediaminetetraacetate (EDTA) (Aldrich, USA) were prepared by dissolving their accurately-weighed samples in water.

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Deionized water purified with a Simplicity Proto system (Millipore) was used in the preparation of all aqueous solutions. The rates of all of the indicator reactions were monitored spectrophotometrically by measuring the increase in the absorbance of the reaction mixture at wavelengths of 440, 315, 400 nm (l=1 cm, reference solution was water) which correspond to the maximum absorption of the products of the processes catalyzed by peroxidases, alcohol dehydrogenases, and alkaline phosphatases, respectively. The initial rate of the indicator reactions (V0, mol L-1 min-1) was calculated as V0=c/t=A/tl=tan/ l, where c is the increase in the product concentration at the reaction time (t), A is the light absorption, l is the cell length, is the molar absorbance coefficient of the reaction product, and tan is the slope of the absorbance versus time kinetic curve.

Apparatus
Solution absorbance was measured using a UV-2201 Shimadzu spectrophotometer (Japan). The pH of aqueous solutions was measured with an accuracy of ±0.005 using a potentiometer in the pH-meter mode (Econics-Expert-001, Russia).

Results and discussion
Determination of Fe(III) using peroxidases
Peroxidases are hemoproteins that contain Fe(III) as a cofactor in their active site and catalyze the oxidation of organic and inorganic compounds with hydrogen peroxide [15]. X-ray structural analyses of plant peroxidases [16, 17] have revealed that key conservative catalytic amino acid residues in the active sites of peroxidases from diverse sources are arranged in similar ways. However, the accessibility of the enzyme active site, the conformation of the surrounding peptide chains, and the number of glycans is specific to a particular enzyme. This results in different profiles of peroxidase substrate specificity, different stabilities, and different sensitivities towards the actions of effector compounds. Earlier we demonstrated that peroxidases isolated from diverse sources have different sensitivities to the inhibiting effects of organic compounds, and phenols in particular [9]. Recently we found that inorganic and organic compounds known to form stable complexes with Fe (III) inhibit the catalytic activity of these enzymes in the reactions of aryldiamines (o-dianisidine, 3,3',5,5'-tetramethylbenzidine, and o-phenylenediamine) oxidation with hydrogen peroxide;

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however, the degrees of inhibition, the applicable concentration ranges of the inhibitors, and their preincubation times with the enzymes vary (Table 1). It is clear that there is a correlation between the lower limits of determination of inorganic and organic compounds and stability constants of their complexes with Fe(III). At the same time it is obvious that the accessibility of the ion cofactor in peanut peroxidase is much worse. This may be explained by the structural peculiarities of both peroxidases: in particular, there are 308 and 294 amino acid residues, and 8 and 3 glycans in horseradish and peanut peroxidases, respectively [16, 17]. [Table 1 will appear here. See end of document.] Thus, it is not worth using peanut peroxidase to determine the indicated acids. On the other hand, the application of horseradish peroxidase is promising not only for a highly sensitive determination of the acids, but also for the sensitive and selective determination of 0.02­0.1 g L-1 Fe(III), based on the reactivation of the enzyme inhibited beforehand with salicylic acid for 30 min. Optimum conditions for carrying out the indicator reaction of o-dianisidine oxidation with hydrogen peroxide for the Fe(III) determination are as follows: 0.1 mol L-1 phthalate buffer, pH 5.0; concentrations of the enzyme, o-dianisidine, hydrogen peroxide, and salicylic acid are 0.6 nmol L-1, 0.05 mmol L-1, 0.1 mmol L-1, and 20 nmol L-1, respectively. The limit of detection (cmin) equals 10 ng L-1. The calibration equation for the determination of Fe(III) has the form y=5.05 + 0.34x, where y=10-2tan and x=cFe(III), in ng L-1. It should be emphasized that Fe(III) is very strongly bound to the peroxidase heme [15], so it is very difficult to remove it from the active site under the conditions of the experiment: incubation time 1 h, the ligand concentration 0.1 mmol L-1. That is why we can't say that a true apo-peroxidase is obtained in the presence of the inhibiting ligand (L); in this case it is possible to propose the following scheme for the process (Fe(III)* is an external ion added to the system from outside): Scheme 1 The proposed procedure for Fe(III) determination is highly selective: Fe(II), Co(II), Cu(II), Ni(II) do not interfere with the determination of 0.02 g L-1 of Fe(III) even at 105 -fold excesses, and Cr(III) and Mn(II) only decrease the inhibiting behavior of Fe(III) significantly at 103-fold excesses. (1) (2)

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Determination of Zn(II) using alcohol dehydrogenases
Alcohol dehydrogenases (ADH) contain Zn(II) in their active sites and catalyze the oxidation of aliphatic alcohols with nicotinamide adenine dinucleotide (NAD+) at pH 9.0 [18]. Numerous effectors of alcohol dehydrogenases from baker's yeast (ADH I) and horse liver (ADH II) were studied using the indicator reaction of ethanol oxidation with NAD. It has been found that these enzymes are significantly inhibited by different classes of organic compounds (amino and fatty acids, respectively) and have different sensitivities towards the inhibiting action of nitrogen containing heterocyclic compounds, Hg(II), and organomercury compounds [12, 13]. Literature and our experimental data show that these two alcohol dehydrogenases have different active forms (the first one is the tetramer, and the second is the dimer), different numbers of SH-groups per subunit (28 and 14, respectively) [18], and are inhibited to different extents by a number of metal ions in their different concentrations (see Table 2). The degree of inhibition (I, %) was calculated from the equation: I,%=(V0 - VI)/V0â100%, where V0 and VI are the rates of the enzymatic reaction in the absence and in the presence of an inhibitor, respectively. [Table 2 will appear here. See end of document.] At the same time it was found that apoforms of both alcohol dehydrogenases may be obtained using the same organic ligands (1,10-phenanthroline, 1,2,3-benzotriazol, for instance), but under different conditions. 1,10-Phenanthroline was found to be the most promising ligand for inhibiting alcohol dehydrogenases from both sources and from preparing their apoenzymes. It was shown that preliminary incubation of ADH I and ADH II with 1,10-phenanthroline in molar concentration ratios of 1:1000 and 1:500, over 15 and 30 min, results in inhibitions of 36 and 90%, respectively. The subsequent introduction of external Zn(II) and other metal ions (Co(II), Ni(II), Cu(II))--which have ionic radii and stability constants of their complexes with 1,10-phenanthroline that are close to those of Zn(II)--without the removal of the ligand excess, provides different degrees of reactivation of previously inhibited ADH (Table 3). The degree of reactivation (A,%) was calculated from the equation: A,%=(VMe- VL)/(V0- VL)â100%, where V
0

and VL are the initial rates of the enzymatic reaction in the absence and in the presence of the ligand respectively; VMe is the rate of the reaction in the presence of the ligand and metal ion. [Table 3 will appear here. See end of document.] The data presented in Table 3 show that rather similar degrees of reactivation of both of the inhibited ADH are achieved at dramatically different metal ion concentrations. There is a partial correlation between the degrees of reactivation by Zn(II), Co(II) and Cu(II), stability constants of

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their complexes with 1,10-phenanthroline, and their degrees of inhibition on the enzymes. It should be noted that Co(II) and Ni(II) are the only metal ions among those studied which do not inhibit the native enzymes at all. Incubation of the inhibited ADH I with Zn(II), Co(II) and Cu(II) for 15 min enhances the degrees of reactivation to 218, 150, and 110%, respectively. Incubation of the inhibited ADH II with Zn(II), Co(II), Cu(II), and Ni(II) for five minutes results in degrees of its reactivation of 94, 155, 39, and 38%, respectively. We are now investigating the reasons for these different effects of metal ions on the catalytic activity of native and inhibited alcohol dehydrogenases of different origin, and this will be the theme of a future publication. Here we will only note that the obtained data show that, despite the presence of parts of polypeptide chains characterized by a high degree of correspondence located close to active sites in alcohol dehydrogenase molecules isolated from diverse sources [20], the accessibilities of their active sites are different. Thus, the results of our research show that selective reactivation of ADH inhibited by 1,10-phenanthroline ("pseudo-apoenzyme") obtained without dialysis (which allows us to eliminate an excess of the ligand) with Zn(II) is impossible. Consequently, to carry out a selective and sensitive procedure for Zn(II) determination, it was necessary to obtain a true apoenzyme without the ligand excess. When the dialysis is carried out for 24 h under dynamic conditions, full discrimination of the catalytic activity of ADH from baker's yeast (I=100%) with 1,10-phenanthroline in TRIS-HCl buffer is achieved at a molar concentration ratio of 1:100. At the same time, a 48 h dialysis of ADH from horse liver with 1,10-phenanthroline at a molar concentration ratio of 1:500 results in only 47% enzyme inhibition. Thus, we did not succeed in obtaining the true apo-ADH from horse liver, but it did seem to be possible to use ADH from baker's yeast for Zn(II) determination. Studying the effects of Zn(II), Co(II), Cu(II), and Ni(II) on the obtained apo-ADH I showed that it was only reactivated selectively with Zn(II). The proposed scheme of the reactivating process differs from that for the inhibited peroxidase reactivation. The reason for this is weaker Zn-binding in the catalytic site of ADH in comparison with iron-binding in peroxidase heme, so Zn(II) removed more easily from the native enzyme with complexing agents. The scheme of the process may be presented as follows: (1) (2)

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Scheme 2 The proposed procedure, based on the reactivating effect of Zn(II) on apo-ADH I, allows us to determine Zn(II) in the concentration range 0.05­0.5 g L-1; (RSD) relative standard deviation at 0.05 g L-1 is 3% (n=5, P=0.95); cmin equals 20 ng L-1. Optimum conditions for carrying out the procedure for Zn(II) determination are as follows: 0.05 mol L-1 TRIS-HCl buffer solution, pH 9.0; concentrations of ADH I, NAD, ethanol, and 1,10-phenanthroline are 0.2 mol L-1, 6 mmol L-1, 0.4 mol L-1, and 20 mol L-1, respectively. The calibration equation for the determination of zinc has the form y=45.1+5.7x, where y=V, mol L-1 min-1 and x=c
Zn(II)

, g L-1. Co(II) and Ni(II) do
3

not influence Zn(II) determination at 105-fold excesses, and Cu(II) does not influence it at 10 -fold excess.

Determination of Zn(II) using alkaline phosphatases
Alkaline phosphatases are hydrolases that catalyze the hydrolysis of various phosphoric acid esters. The specificity of the action of phosphatases and their catalytic activity depend on the source of the enzyme [21]. Alkaline phosphatases are known to be metal-dependent enzymes containing two metal ions: zinc and magnesium at their catalytic and allosteric sites, respectively. The zinc content of the enzyme molecule is governed by the spatial structure of the biocatalyst, and varies from two to three atoms per enzyme subunit for the dimer to 16 atoms for the tetramer. We studied three alkaline phosphatases (APh) isolated from different sources (Eschericia coli (E. coli) (I), chicken intestine (II) and slim intestine of Greenland seal (III)) in order to develop procedures for determining zinc. The choice of bacterial phosphatase and two phosphatases of animal origin for our studies is due to the fact that the first enzyme is the only phosphatase whose active site structure is established exactly. The two other phosphatases are poorly studied; their structures and properties can be judged by comparing the action of the same effectors on their catalytic activity with the actions of the effectors on the activity of the previously studied enzyme. In addition, it was interesting to compare the properties of alkaline phosphatases from E. coli and chicken intestine, which are dimers, with those of the phosphatases from seal intestine, which is a tetramer (Table 4). The hydrolysis of p-nitrophenyl phosphate (p-NPP)--giving rise to a colored product, a p-nitrophenolate ion--at pH 9.8 was used as the indicator reaction for monitoring the catalytic activity of alkaline phosphatases. [Table 4 will appear here. See end of document.] The effects of zinc ions on the catalytic activities of the three enzymes were studied over a wide range of their concentrations (from 0.05 to 50 mg L-1) under the selected optimum conditions of 9


the indicator reaction (Table 4). It follows from Fig. 1 that zinc inhibits the catalytic activity of all alkaline phosphatases to a variable extent. The same degree of inhibition--25%--was observed for the alkaline phosphatases I, II, and III at zinc concentrations of 5.0, 0.5, and 0.25 mg L-1, respectively.

Fig. 1 Degrees of inhibition (I,%) of alkaline phosphatases from E.coli (1), chicken intestine (2), and seal intestine (3) as functions of zinc concentration (experiment performed under optimum conditions for the indicator reaction)

To compare the properties and to investigate the analytical possibilities of alkaline phosphatases of different origin, the effects of zinc and other metal ions, primarily the alkaline earth metals magnesium and calcium, were studied. The choice of these metal ions was prompted by published data on the catalytic activity of enzymes that transfer phosphate, including alkaline phosphatases [22]. The authors of [23] proved that magnesium ions occur at active sites of alkaline phosphatase I. Of the alkali metals, potassium and cesium were chosen, because it is known that the former is, as a rule, present in physiological fluids and activates many enzymes, including alkaline phosphatase from E. coli; cesium together with magnesium can enhance its catalytic activity [24]. Data on the effects of cesium and potassium on the catalytic activities of APh II and III are unavailable. We also studied the effects of Co(II), Ni(II), Cu(II), and Cd(II). These ions, whose ionic radii are close to that of Zn(II), may replace zinc ions at the active site of the enzymes. It was also of interest to study the effect of Pb(II) on the catalytic activity of APh I and APh III, because the inhibiting effect of Pb(II) on APh II has been used previously [1] for developing a highly sensitive (cmin=0.1 g L-1) enzymatic method for its determination. It was shown that potassium and cesium in different concentrations (7­70 and 0.2­7 g L-1, respectively) produced rather low activating effects (A=16­35%) on APh I and II. Magnesium

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only activated APh from chicken intestine significantly (A=600%). Calcium in concentrations from 0.01 to 0.5 g L-1 had no effect on the catalytic activities of the phosphatases. The rate of the indicator reaction did not change when the enzymes were exposed to ions of specified metals for 30 min or less. Co(II), Ni(II), Cu(II), and Cd(II) in concentrations of about 1 mg L-1 had no effect on the rate of the indicator process catalyzed by the enzymes from all three sources. The addition of Pb(II) at the same concentration did not change the activities of APh I and APh III. When Co(II), Ni(II), Cu(II), and Cd(II) were introduced together into the indicator system in amounts comparable with those of Zn(II), and exposed to the enzyme for at least three minutes, the inhibiting effect of the zinc later manifested itself to only a minor extent. This effect of foreign ions may be caused by their competition with zinc for binding sites in the molecules of phosphatases. Thus, the results of our investigations indicate that the inhibiting effect of zinc ions is selective and manifests itself at rather low concentrations only in the case of alkaline phosphatase from seal intestine. It was revealed that the inhibiting effect of Zn(II) depended on the nature of a buffer solution in the indicator reaction (Fig. 2). Among a number of buffer solutions--borate, carbonate, glycine and TRIS-HCl--borate buffer seemed to be the most promising for maximum inhibition of phosphatase from seal intestine with Zn(II). The same degree of inhibition--50%--was observed in the borate, carbonate, TRIS-HCl, and glycine buffers at zinc concentrations of 0.04, 0.25, 1.5, and 2.2 mg L-1, respectively. Thus, varying the buffer nature makes it possible to enhance the sensitivity of Zn(II) determination. Application of 0.05 mol L-1 TRIS-HCl and 5 mmol L-1 borate buffer solutions allows us to determine zinc over the concentration ranges 1­10 mg L-1 and 0.01­0.1 mg L-1 with RSDs (at lower limits of determination) of 3 and 14% (n=5, P=0.95), respectively. The calibration equation for the determination of zinc in borate buffer has the form y=-1.34-4.58x, where y=V, mol L-1 min-1, x=c
Zn(II)

, mg L-1; the limit of detection is 3 g L-1.

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Fig. 2 Dependence of inhibiting degree (I,%) of alkaline phosphatase from seal intestine on Zn(II) concentration in borate (1), carbonate (2), TRIS-HCl (3), and glycine (4) buffers

To obtain apo-alkaline phosphatases, EDTA was used to bind zinc ions in a stable complex compound. This ligand is frequently used for such purposes [21]. To reveal the most promising phosphatase for obtaining the apoenzyme, the degrees of inhibition of the three enzymes as functions of EDTA concentration were studied. As can be seen in Fig. 3, EDTA significantly inhibited APh II and APh III of animal origin. The inhibiting effect of EDTA on APh I was weakly pronounced, even at high concentrations (at a level of 10 mmol L-1), and its value (I10%) was almost independent of the ligand concentration. It is likely that this effect of EDTA in the case of APh I might be explained either by steric hindrance to the approach of EDTA molecules to this enzyme and their coordination to Zn(II), or by a higher stability of [Zn-apoenzyme] complexes. The conditions for preparing apo-phosphatases II and III were revealed later on.

Fig. 3 Degrees of inhibition (I,%) of alkaline phosphatases from E.coli (1), chicken intestine (2), and seal intestine (3) as functions of EDTA concentration (at optimum conditions for the indicator reaction)

It was stated that the inhibiting effect of EDTA on both of the enzymes increased with their preincubation time. The greatest degree of inhibition (I=90­95%) was reached at equal incubation times of the enzymes (E) with EDTA (L) of 15­30 min at the same ligand concentration (60 mol L-1) but at different E:L concentration ratios of 1:100 and 1:6000, respectively. These concentration ratios were chosen as optimum for obtaining apo-APh II and apo-APh III. An excess of EDTA required for obtaining apo-APh II, which is smaller by a factor of 60, suggests that zinc ions in the active site of this enzyme are more accessible to the action of EDTA, as compared with apo-APh III. The different availabilities of zinc ions can be related to the different spatial configurations of these enzymes (Table 4). The low residual catalytic activity (5­10%) of APh II

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and APh III may indicate that zinc ions were incompletely removed from the enzyme active sites during their 15 min of contact with EDTA, evidently because of their strong binding with the apoenzymes [25]. The residual activities of phosphatases at incubation times of 30 min were found to be almost independent of the time that the enzymes were exposured EDTA, which is why it was unreasonable to perform long-term dialysis for preparing apo-phosphatases. It was found that the addition of external zinc (1 mg L-1) caused partial reactivation of apoenzymes II and III. The degrees of reactivation of apo-APh II and apo-APh III were 86 and 25%, respectively. The effect of a number of metal ions--Co(II), Ni(II), Cu(II), and Cd(II), which have ion radii and stability constants of their complexes with EDTA similar to those of Zn(II)--on the activity of two apo-phosphatases was studied. It was found that all of the above metal ions at concentrations of 1 mg L-1, as well as zinc, reactivated apo-phosphatase from chicken intestine (Table 5). The apoenzyme from seal intestine was only reactivated by Zn(II). [Table 5 will appear here. See end of document.] At the same time, we found that a much higher degree of reactivation (90%) of apo-phosphatase from seal intestine could be achieved when traces of magnesium (2 mg L-1) were added to the apoenzyme simultaneously with Zn(II). It seemed probable, therefore, that reactivation of the apoenzyme from seal intestine could be developed into a sensitive and selective procedure for the determination of zinc using its reactivating effect on this apoenzyme in the presence of magnesium. The calibration equation for the determination of zinc (0.01­0.1 mg L-1) has the form y=6.15+89.92x, where y=V, mol L-1 min-1 and x=c P=0.95). The limit of detection is 8 g L-1.
Zn(II)

, mg L-1; RSD=24% at 0.01 mg L-1 (n=5,

Determination of Mg(II) using alkaline phosphatases
While studying the effect of Mg(II) on three alkaline phosphatases isolated from diverse sources, we found that the presence of this metal ion at concentrations of 2­20 g L-1 activates APh from chicken intestine (A=600% at c
Mg(II)

=20 g L-1), while the degree of reactivation of APh from E.

coli is much lower (A=20% at cMg(II)=0.2 g L-1), and magnesium has no influence on the catalytic activity of APh from seal intestine. Preincubation of magnesium at a concentration of 2 g L-1 with APh from chicken intestine for five minutes enhances the activation degree by ~50%. Further incubation of the mentioned mixture does not change the activation degree. The activation of alkaline phosphatase with magnesium might be explained, for instance, by the presence of so-called "liophylic" part in the enzyme molecule, the interaction of which with magnesium ions may provide better binding of 13


the substrate; the activation degree therefore depends liophylic content of the enzyme molecule [26]. Thus, only APh from chicken intestine was found to be suitable for the development of a sensitive procedure for Mg(II) determination at concentrations of 0.6­6.0 g L-1. The calibration equation for the determination of magnesium has the form y=3.30+0.53x, where y=V, mol L-1 min-1, x=c
Mg(II),

g L-1; RSD=4% at 0.6 g L-1 (n=5, P=0.95); the limit of detection is 0.2 g L-1.

Studying the influence of a number of metal ions (Ca(II), Ba(II), Mn(II), Al(III) Zn(II), Cu(II), Ni(II), Cd(II), Pb(II), Fe(III), Na, K, Cs) on the determination of 0.6 g L-1 of magnesium showed that only Ca(II), Ba(II), Zn(II), and Cd(II) interfere with the determination at 105-fold, 105-fold, 103-fold and 102-fold excesses, respectively. The interfering effects of these ions may be explained by the partial substitution of magnesium ions into the allosteric site of phosphatase with a subsequent decrease in the enzyme catalytic activity. Thus, the proposed procedure for magnesium determination is not only sensitive, but also selective. The developed procedures for zinc and magnesium ion determination were successfully applied to the analysis of biological fluids (Table 6). The results of the analysis carried out by the enzymatic procedures were confirmed by the data from alternative methods: atomic absorption (AAS) and spectrophotometric (SPh) techniques. [Table 6 will appear here. See end of document.]

Conclusions
A novel approach to determining different enzyme inhibitors--organic compounds (phenols, fatty acids), some heavy metal ions, and anions--proposed by the authors of this paper in an earlier work, has been successfully developed and used for the first time in order to determine enzyme cofactor metal ions (Fe(III), Zn(II), Mg(II)). This approach is based on the application of metalloproteins (various oxidoreductases and hydrolases) isolated from diverse sources. The data from our investigations demonstrate that enzymes of different origins, and their apo-forms, exhibit different sensitivities and selectivities towards the action of the cofactor and other metal ions. This application of such enzymes holds much promise for the selective and sensitive determination of metal ion cofactors using their different effects--inhibition or activation of enzymes, and reactivation of apoenzymes. The developed approach can potentially extend the range of metalloproteins used in chemical analysis and the samples that can be analyzed.

14


Acknowledgements This work was supported by the Russian Foundation for Basic research, project N 04-03-33116.

References
1. 2. 3. 4. 5. 6. 7. 8. 9. Dolmanova IF, Shekhovtsova TN, Kutcheryaeva VV (1987) Talanta 34:201­205 Shekhovtsova TN, Muginova SV, Migunova UM, Dolmanova IF (1996) Quim Anal 15:312­20 Bagirova NA, Shekhovtsova TN, Shopova EA, van Huystee RB (1998) Mendeleev Comm 4:155­157 Zhmaeva EV, Shekhovtsova TN (2000) J Anal Chem (Translated from Russian) 55:782­790 Shekhovtsova TN, Muginova SV, Dolmanova IF (1998) Int J Environ An Ch 69:191­ 205 Shekhovtsova TN (2000) Egypt J Anal Chem 9:11­14 Shekhovtsova TN, Bagirova NA, Veselova IA, Muginova SV (2001) Radionuclides and heavy metals in the environment. Kluwer, Dordrecht, pp 201­208 Dixon M, Webb EC (1979) The enzymes. Academic, New York Gazaryan IG, Loginov DB, Lialulin AL, Shekhovtsova TN (1994) Anal Lett 27:2917­2930

10. Bagirova NA, Shekhovtsova TN (1999) Kinetika i Kataliz (translated from Russian) 40:241­237 11. Bagirova NA, van Huystee RB, Shekhovtsova TN, Gazarian IG (2001) Talanta 55:1151­1164 12. Shekhovtsova TN, Zhmaeva EV (2002) Microchim Acta 140:29­35 13. Bychkov PV, Shekhovtsova TN (2003) Mendeleev Comm 2:75­76 14. Sesto PA, van Huystee RB (1989) Plant Sci 61:163­165 15. Welinder KG (1992) Curr Opin Struct Biol 2:388­393 16. Schuller DJ, Ban N, van Huystee RB, McPherson A, Poulos TL (1996) Structure 4:311­321 17. Henriksen A, Welinder KG, Gajhede M (1998) J Biol Chem 273:2241­2248 18. Boyer PD, Lardy H, Myrback K (1963) The enzymes. Academic, New York, 7:25­30 19. Lur'e YY (1989) Spravochnik po analiticheskoi khiomii (Handbook on analytical chemistry). Khimia, Moscow 20. Jornvall H (1977) Eur J Biochem 72:443­452 21. Fernley HN, Boyer PD (1971) The enzymes, 3rd edn. Academic, New York, 4:417­419 22. Spiro TG (1973) Alkaline phosphates. In Eichhorn GL (ed) Inorganic biochemistry. Elsevier, Amsterdam, 1:624 23. Gettings P, Coleman JE (1984) J Biol Chem 259:4991­4996 24. Miki Y, Toshima N, Tanaka K (1998) Jpn Kokai Tokyo Koho JP 10042896 A2 25. Dong G, Zeikus JG (1997) Enzyme Microb Tech 21:335­339 26. Cloetens R (1941) Biochem Z 307:352­358

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Table 1 Characteristics of the inhibiting effects of various compounds on the catalytic activity of horseradish (I) and peanut (II) peroxidases in the oxidation of o-dianisidine with

hydrogen peroxide (n is the stability constant of a complex formed by an inhibitor (Inh) with Fe(III) ­ {FeInhn})

Inhibitor

log

n (n)

HF HCN Tartaric acid EDTA Oxalic acid Sulfa salicylic acid Salicylic acid

16.1 43.9 11.9 14.6 20.2 33.1 36.3

(5) (6) (2) (1) (3) (3) (3)

Applicable concentration range (mol L-1) I II 0.5­100 1­100 0.0008­0.01a 0 20­100 10­100 0.01­0.1 0.5­100 1­10 1­100 0.0008­0.01 100­1000 0.00002­0.0001 100­1000 inc(min) I 5 60 12 h 60 30 30

II 0 15 0 15 18 h 18 h

a

the influence was not studied

16


Table 2 Degrees of inhibition (I,%) of alcohol dehydrogenases from baker's yeast (ADH I) and horse liver (ADH II) by a number of metal ions at their indicated concentrations

Me Degree of inhibition (%) 80 47 32 25 38 ADH II Concentration of Me (mg L-1) 1.0 1.0 1.0 10 10

Hg(II) Ag(I) Zn(II) Cu(II) Pb(II)

ADH I Concentration of Me (mg L-1) 0.01 0.01 1.0 1.0 0.01

Degree of inhibition (%) 52 48 26 7 3

17


Table 3 Ionic radii and logarithms of stability constants (log3) of the 1,10-phenanthroline complexes of the studied metal ions {MeL3} [19] and the degrees of reactivation (A,%) of

ADH I and ADH II inhibited with 1,10-phenanthroline, at the indicated Me(II) concentrations
log3 17.00 20.00 20.41 17.57 ADH I cMe (II) (mg L-1) 0.1 1.0 0.01 1.0 A (%) 82 100 86 119 ADH II cMe (II) (mg L-1) 100 100 100 100

Me (II)

Ion radius (å)

Zn(II) Co(II) Cu(II) Ni(II)

0.83 0.82 0.80 0.78

A (%) 86 117 78 77

18


Table 4 Some characteristics of the studied alkaline phosphatases and the optimum conditions for the hydrolysis of p-NPP catalyzed by alkaline phosphatases from E. coli (I), chicken

intestine (II) and seal intestine (III) (0.05 mol L-1 TRIS HCl buffer solution, pH 9.8)
II 100 2.6 Dimer 450­800 0.55­0.65 260 50 Tetramer 10­11 0.55­0.80 III

I

Characteristics of the enzyme and concentrations of the reaction components Molecular weight (kDa) Specific activity (U mg-1) Spatial configuration Enzyme concentration (nmol L-1) Concentration of p-NPP (mmol L-1)

89 130 Dimer 10­14 0.40­0.70

19


Table 5 Ionic radii and logarithms of stability constants of the EDTA complexes of the studied metal ions (log {MeEDTA}) [19], and degrees of reactivation of apo-phosphatase

from chicken intestine (A, %)
log {MeEDTA} 16.50 16.21 18.80 18.62 16.59 A (%) 86 86 68 69 60

Me(II) Zn(II) Co(II) Cu(II) Ni(II) Cd(II)

Ion radius (å) 0.83 0.82 0.80 0.78 1.03

20


Table 6 The results of Zn(II) and Mg(II) determinations in biological fluids using the enzymatic (I) and alternative (II) methods
Enzyme ADH from baker's yeast APh from seal intestine APh from chicken intestine II 3.8 ± 0.3 9.29 ± 0.02 33.76

Analyzed sample

Analyte

Blood serum Urine

Zn(II) Zn(II) Mg(II)

Metal ion content (mg L-1) I 3.9 ± 0.3 9.20 ± 0.01 37.4 ± 0.4

Alternative method AAS AAS SPh

21