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Journal of Analytical Chemistry, Vol. 60, No. 3, 2005, pp. 218-233. Translated from Zhurnal Analiticheskoi Khimii, Vol. 60, No. 3, 2005, pp. 247-263. Original Russian Text Copyright ¿ 2005 by Muginova, Zhavoronkova, Shekhovtsova.

REVIEWS

Potentialities and Prospects for the Use of Alkaline Phosphatases for Determining Metal Ions
S. V. Muginova, A. M. Zhavoronkova, and T. N. Shekhovtsova
Department of Chemistry, Moscow State University, Vorob'evy gory, Moscow, 119992 Russia
Received June 1, 2004

Abstract--The published data on the nature, strength, and mechanism of metal ion effect on the catalytic activity of alkaline phosphatases and their apoenzymes of different origins are systematized and discussed. Procedures for determining metal ions are described. These procedures are based on the activating, inhibitory, and reactivating effects of metal ions on enzymes and apoenzymes. The approaches to the intentional improvement of the sensitivity and selectivity of determining metal ions are considered. Prospects are outlined for using alkaline phosphatases isolated from different sources in chemical analysis for determining metal ions in different samples.

At present, mineral substances that the human body receives from foodstuffs and water contain seven main macroelements: sodium, potassium, magnesium, calcium, phosphorus, chlorine, and sulfur. An additional 15 essential microelements, apart from macroelements, are necessary for the human body. Worthy of mention are iron, a component of hemoglobin and tissue cytochromes; cobalt, a component of cyanocobalamin (vitamin B12); copper, a component of cytochrome oxidase; zinc, a factor in the promoting effect of insulin on the permeability of cell membranes to glucose; molybdenum, a component of xanthine oxidase; manganese, an activator of some enzymatic systems; and others [1]. In the majority of cases, the above microelements enter into the composition of enzymes, hormones, and vitamins and act as catalysts of enzymatic processes [2]. A deficiency or excess of macro- and microelements results in serious disruptions of the normal function of the human body; therefore, the correct estimation of their concentrations in biological systems and the control over their concentrations in living organisms is an important task of analytical chemistry and medicine. The use of enzymes of different classes is promising for the diagnosis of a deficiency of macro- and microelements. The following enzymes have been successfully used in chemical analysis: peroxidase from horseradish roots for determining trace amounts of iron(III) [3] and toxic mercury(II) [4]; alcohol dehydrogenase (ADH) from baker's yeast and pyruvate oxidase for determining zinc [5, 6], polyphenol oxidase for determining copper(II) [7, 8] and others. In this review, we consider the possibilities of using alkaline phosphatases from different sources for determining metal ions; describe enzymatic procedures for their determination; discuss the approaches to the intentional improvement of the sensitivity and selectivity of determining metal ions; and outline the prospects

for the use of alkaline phosphatases of different origin in chemical analysis for determining metal ions in different samples, in particular, in biological samples. It should be noted that the data published over the last thirty years on the effect of metal ions on the catalytic activity of alkaline phosphatases of different origins have not been systematized before. Only the monographs by Boyer [9], Coleman and Gettins [10], Freder [11], and Spiro [12] are worth mentioning. These monographs cover the data published up to 1971 on the sources of alkaline phosphatases, the kinetics and mechanism of their action, thermal stability, and main inhibitors and activators, and the authors systematized them. Methods for determining metal ions using alkaline phosphatases were not discussed in these monographs, which makes this review valid and reasonable. General information about alkaline phosphatases. Alkaline phosphatases (EC 3.1.3.1) belong to the class of hydrolases and catalyze the alkaline hydrolysis of a great number of different phosphoric acid esters; because of this, they are nonspecific enzymes [11]. The specificity of alkaline phosphatases and their catalytic activity depend on the source from which an enzyme was isolated [9]. The kind of living tissue (mucoid tissues of intestine, placenta, lungs, kidneys, liver, etc.) and the sizes of a microorganism or an animal from which an enzyme was isolated are of significant importance, especially for the alkaline phosphatases of animal origin [9, 10]. Alkaline phosphatases are metal-dependent enzymes bearing zinc and magnesium ions in the catalytic and allosteric sites, respectively. The concentration of zinc in the molecule of alkaline phosphatase is determined by its spatial structure and varies from two to four atoms per one enzyme subunit in the case of dimer and reaches 16 atoms for tetramer.

1061-9348/05/6003-0218 ¿ 2005 Pleiades Publishing, Inc.


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The optimum pH range in which the catalytic activity of alkaline phosphatase attains a maximum is mainly governed by the enzyme origin. Historical information. Alkaline phosphatase was first mentioned in 1911 in publications devoted to the isolation of this enzyme from the mucous tissues of kidney and intestine and also from different tissues of many mammals [13]. In 1923, Robinson (Great Britain) showed that mammal bony cartilage was a rich source of alkaline phosphatase [14]. These data initiated intensive studies with the aim of supporting the hypothesis for the direct participation of alkaline phosphatase in the calcification of the bony tissue of animals. In 1928, studying the properties of alkaline phosphatase from pig kidney, Erdtman (Germany) first observed the enhancement of its catalytic activity in the presence of micromolar amounts of magnesium ions [9, 15]. These studies were continued in the next twenty years by another German scientist, Cloetens, who tried to throw light on the mechanism of the magnesium activating effect on the same alkaline phosphatase and the inhibitory effect on it of some other metals [16, 17]. Interest in the elucidation of the character of ion metal effect on the activity of alkaline phosphatase was dictated first of all by the necessity of cleaning and stabilizing enzymatic preparations isolated in laboratory. In addition to the investigations of the properties of alkaline phosphatases of animal origin, a great number of publications appeared in the 1930s-1940s on the isolation of alkaline phosphatases from different microorganisms, especially from bacteria, and on the study of their properties. At present, alkaline phosphatase from Escherichia coli (E. coli) is studied most extensively. Although the first report about this enzyme source is dated to 1933, its structure and physicochemical properties were investigated only in the 1960s. The work of Horiuchi [18] and Torriani [19], who studied the rate of alkaline phosphatase synthesis in the cells of E. coli as a function of orthophosphate concentration, was a prerequisite for this. In the 1960s, clinical interest was shown in alkaline phosphatases. It was found that a change in the catalytic activity of some alkaline phosphatases in blood served an important diagnostic sign of hepatitis, different diseases of bones, and heavy hereditary diseases. At that time, highly sensitive methods for determining phosphates activity were extensively developed [20-22]. Sources of alkaline phosphatases. By now, alkaline phosphatases from various sources have been isolated and characterized. These are alkaline phosphatases isolated from cells of microorganisms (bacteria, yeast, and fungi), tissues of different organs (liver, kidneys, spleen, intestine, and placenta), and bony and connective tissues (blood and synovial fluid) of many invertebrates and vertebrates (animals and human beings). At present, in addition to the commercial preparations of alkaline phosphatases used earlier (alkaline phosphatases from E. coli, intestines of calf and
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chicken, the small intestine of the Greenland seal), commercial preparations of enzymes isolated from bovine, pig, dog, and rabbit intestines; bovine and pig kidneys; cow milk and liver; and human placenta have appeared. The demand for the production of such varied preparations of alkaline phosphatases is evidently due to an increased interest of researchers in the study of different properties of these enzymes in the context of their use for solving of a wide spectrum of analytical problems. Effect of metal ions on the activity of bacterial and animal alkaline phosphatases. In recent decades, the number of publications dedicated to the study of the effect of metal ions on the catalytic activity of alkaline phosphatases of different origins has been rather large. The active interest of researchers in this theme is first of all explained by the necessity of optimizing the conditions for isolating highly active and stable preparations of bacterial and animal phosphatases. Knowledge of the character and degree of the effect of metal ions on different alkaline phosphatases allows the catalytic activity of enzymes to be intentionally varied. Information about zinc(II) and magnesium(II) ions in the catalytic and allosteric centers of these enzymes, respectively, is especially important. The published data on the effect of metal ions on alkaline phosphatases of different origins are not systematized, and they are sometimes contradictory for enzymes isolated from the same source. The use of the revealed inhibitory and activating effects of metal ions on alkaline phosphatases of different origins in chemical analysis has received insufficient attention in the literature. To obtain a comprehensive idea of the current status of the problem under consideration, we systematized the available data on the character and strength of the effect of a wide range of metal ions on the catalytic activity of alkaline phosphatases of bacterial and animal origin (Table 1). Table 1 contains no information about the action of alkali ions on the catalytic activity of alkaline phosphatases, because these ions either do not affect the rate of enzymatic hydrolysis at all [23, 31-33] or only slightly enhanced it. The activating effect of potassium, lithium, and sodium ions on alkaline phosphatases manifested itself for concentrations 0.2-2; 0.035-0.35; and 0.1-1 mg/mL, respectively [53]. In the authors' opinion, this effect may be due to the compensation for the negative charge of the substrate phosphate group by these ions and, as a consequence, to the facilitation of the accessibility of the water hydroxyl group, which favors the hydrolysis. Other researchers [54] related an increase in the catalytic activity of alkaline phosphatases in the presence of high concentrations (0.01- 0.5 M) of alkali and alkaline-earth chlorides and sulfates to an increase of the ionic strength of the reaction solution. At the same time, Mildvan [55] supposed that alkaline-earth ions activated alkaline phosphatases by forming a ternary complex involving the enzyme (E), metal
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Table 1. Data on the effect of metal ions on the catalytic activity of alkaline phosphatases of different origins under optimum conditions of indicator processes Ordinal number Source of alkaline phosphatase 2 ConcentraAvailabiliConditions of indicator reaction: Character of the tion range ty of an en- Refesubstrate, buffer solution Me(II) effect on enzyme (concentrazymatic rences (concentration; pH) (apoenzyme) tion), ÷g/mL procedure 3 4 Bacteria I 1 E. coli p-NPP, Tris-HCl (50 Ïå; 8.0) Be Cd Co Pb Cu Hg Be Bi Zn Zn Zn 1 11 6 21 6 20 0.003-0.01 3-100 0.02-2 0.065-6.5 130 131 110 127 117 48 175 80 275 2.4-24 4 200 48.6 -a [23] 5 6 7 8

1

I R R I R

2 3

Prevotella intermedia Hyperthermophilic Termotoga neapolitana

p-NPP, Tris-HCl (1 M; 8.0) p-NPP, veronal (20 mM; 8.0) p-NPP, Tris-HCl (50 mM; 8.4) p-NPP, Tris-HCl (0.2 mM; 9.9)

+b + - - - -

[24] [23] [25] [26]

4 5

Yeast Saccharop-NPP, Tris-HCl myces cerevisiae (20 mM; 8.2) Neurospora crassa p-NPP, AMPOL (50 mM; 9.4-9.8)

Zn Mn Ni Cu Mg Sr Ca Ba Fungi Mg Ca

A A I A R A

- -

[27] [28]

Mg Animals p-NPP, carbonate-bicarbonate Mg (50 mM; 9.7) Ca Mn Co Phenyl phosphate, carbonate-bi- Zn carbonate (40 mM; 10.3) Pb Cd Cu Zn Cu

-

6

Pearl oyster Pinctaga fucata

7

Red sea bream

0.005-0.05 0.008-0.08 0.27-8.8 0.59-8.2 0.65-13 0.2-2 0.56-5.6 0.6-12.7 0.1 0.5 0.02 0.1

-

[29]

I

-

A I A I
Vol. 60

- -

[30]

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POTENTIALITIES AND PROSPECTS FOR THE USE OF ALKALINE PHOSPHATASES Table 1. (Contd.) 1 8 2 Shore crab (internals) 3 p-NPP, carbonate-bicarbonate (50 mM; 10.0) Mg Ca Ba Mn Co Ni Cd Zn Cu Hg Pb Ag(I) Bi(III) Zn Mg Co Zn Cd Pb Mg Ca Ba Al(III) Mg Mg Zn Cd Co Mg Mg Zn Zn Zn (in the presence of 30 ÷g/mL Mg(II)) Ca (in the presence of 0.0002 ÷g/mL Zn(II)) Ca(II) Mg
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4

5 12-120 20-200 69-690 1-10 1-10 1-10 6-60 0.3-13 1-10 10-100 10-100 5-50 10-100 0.000065-0.065 0.0024-24 0.589-58.9 0.065-6.5 0.001-100 1-10 0.001-100 0.07-0.33 0.001-0.1 1-100 0.001-1 0.001-0.01 0.001-1 0.001-0.01 1-10 0.001-1 0.0006-0.006 72 6.5-65 11.2-112 5.9 2.4-24 80 0.075-0.75 0.009-0.09 0.001-0.009 0.1-0.6 0.8-4 24-120

6 A

7 -

8 [31-33]

I

-

9

Rat (bony tissue) p-NPP, AMPOL (50 mM; 9.4)

A

-

[34-38]

10

Chicken (intestine)

p-NPP, Tris-HCl (50 mM; 9.8)

I I I I A I

- [39] +c + + + + +

11

Calf (intestine)

p-NPP, glycine-NaOH (50 mM; 9.3) Phosphorylsalicylic acid, glycine-NaOH (33 mM; 9.3)

A I A R R I I A A I R

+ - + - -

[40] [41] [42]

Phosphorylsalicylic acid, borate (33 mM; 9.3) 2-Naphthyl phosphate, Tris-HCl (50 mM; 9.8)

+ +

[43] [44]

p-NPP, Tris-HCl (50 mM; 9.8) p-NPP, veronal-ethanolamine (50 mM; 9.6)
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R R

+ - [45]


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MUGINOVA et al.

4 Zn(II) Mg Ca Sr Zn Mg Mn Mg Mn Ca Cr(III) Zn Fe(III) Sn(IV) Zn Zn Zn (in the presence of Mg(II)) Zn Co Ni Cd Mn Cu Mg Zn Co Zn Pb Hg

5 0.01-0.05 2.4-240 0.4-40 0.09-90 0.65-6.5 240 550 24 55 40-400 52-520 65-650 56-560 119-1190 1-10 0.01-0.1 0.01-0.1

6 R A

7 - -

8 [46] [47]

12

Bovine (synovial fluid)

3-[Spiro-4-methboxy-1,2-dioxetane-3,2'tricyclo[3,3,1,1]chlorodecane-4-yl]-phenyl phosphate (SPP), Tris-HCl (50 mM; 9.0) p-NPP, AMPOL (0.1 M; 9.6)

13

Seal (small intestine)

p-NPP, diethanolamine-HCl (1 M; 9.8)

I A I

- -

[48]

p-NPP, Tris-HCl (50 mM; 9.8) p-NPP, borate-NaOH (5 mM; 9.8) p-NPP, Tris-HCl (50 mM; 9.8) Human Na,-Glycerol-2-phosphate, veronal (20 mM; 9.9)

I I R

+ + +

[40] [49] [50]

14

Blood (leucocytes)

15

Placenta

p-NPP, Tris-HCl (0.5 M; 8.0) p-NPP, glycine (30 ÷M; 10.5)

0.065-0.65 0.059-5.9 0.0587 0.112 0.055-0.55 0.064-0.64 1.2 6.5 29 0.065-0.65 >155 50 >200

R

-

[51]

R

-

[52]

I

-

Notes: I and A are the inhibitory and activating effects on the enzyme, respectively; R is the reactivating effect on apoenzyme. a -: the effect on enzyme was revealed, but no procedure was developed. b +: an enzymatic procedure for determining metal was developed. c +: the effect on enzyme was revealed, the possibility in principle of developing a procedure for metal determination was stated, but it was not developed. p-NPP is p-nitrophenyl phosphate. Buffer solutions: Tris-HCl is Tris(hydroxymethyl)aminomethane hydrochloride; Veronal is sodium 5,5-diethylbarbiturate; and AMPOL is 2-amino-2-methylpropanol-1.

ion (M), and ligand (L). The enzyme substrate (S) can act as a ligand. The structure of this complex changes (E-M-L, E-L-M, or M-E-L) depending on the participant E, M, or L acting as a bridge. The complex with a ligand as a bridge between an enzyme and a metal ion

is schematically depicted in the figure (bridging ligand). In this complex, the metal ion is bound only with the ligand and is involved in catalysis without the direct interaction with the enzyme. In this case, the role of the alkaline-earth ion consists in the activation of the
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x+

substrate phosphorus atom at which the attack occurred. Additionally, metal ions can take part in enzymatic catalysis either as the only binding link between the enzyme and the ligand (Fig. 1, IIA), or as a participant in the formation of a more complex bridge (Fig. 1, IIB). Due to the unique coordination properties of the metal ion, it plays an important role in the interaction between the protein and the ligand in the bridge complexes presented in Fig. 1, II. Finally, metal ions can interact with the enzyme at a site different from that to which the ligand is bound, which results in a change in the properties of the enzyme catalytic center (or the center of ligand binding). In such complexes (Fig. 1, III), enzymes play the role of a bridge between the metal and the ligand. The mechanisms of the M-E-L complex formation are poorly studied as opposed to the mechanisms of complex formation with bridging metal ions (E-M-L) or with bridging ligands (E-L-M), although we cannot explain many of the effects of metal ions only by the two above-mentioned mechanisms. Unfortunately, it is not entirely clear which mechanism of those presented in the figure dominates under certain conditions of the indicator process and for which metal ion (alkaline-earth or transition). It is possible that the interaction between the metal ion and alkaline phosphatase occurs via several mechanisms simultaneously. The data presented in Table 1 demonstrate that magnesium(II), along with other alkaline-earth ions, activates alkaline phosphatases of plant and animal origin, and the activating effect is enhanced with an increase in the concentration of a metal ion. It is interesting to note that a significant activation of alkaline phosphatases isolated from pearl oyster and rat bony tissue was observed upon the addition of magnesium in rather small concentrations (2-50 ng/mL). Such behavior of these alkaline phosphatases may be due to the fact that the tissues from which they were isolated originally contained large amounts of calcium(II) by virtue of their biological function. Evidently, this ion coordinated to allosteric centers of the considered alkaline phosphatases is easily displaced from them by magnesium(II) introduced from the outside because of its greater affinity for the enzyme protein. Calcium ions are also capable of displacing magnesium ions from the active centers of alkaline phosphatases, although the concentrations of calcium(II) required for this are almost twice as great as those of magnesium(II) (Table 1, no. 6). Note that alkaline-earth ions can not only activate alkaline phosphatases but also stabilize their structure and endow them with high catalytic activity. Alkalineearth ions react with some amino acid residues of the enzyme protein molecule, thus preventing its inactivation, for example thermal inactivation [56, 57]. To stabilize alkaline phosphatases, magnesium(II) is predominantly used. This is why most alkaline phosphatases are kept in 2-2.5 M ammonium sulfate solutions containing 0.5-5 mM magnesium(II) sulfate or chloride solutions. Evidently because of the lower stability of
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L(S) I E +L E M II(A) E +Mx+ E Mx+ +Mx+ II(B) E +L E +L
x+

L + Mx+ E L M +L E
x+

Mx+ L E

L +Mx+ E Mx+

III +Mx+ E +L

E

+L

Mx+ E

L

L E

+Mx+

Fig. 1. Schemes of the interaction of metal ion and ligand (substrate) with enzyme [55].

alkaline phosphatases of animal origin, higher concentrations of magnesium(II) are used for their stabilization as compared to the bacterial alkaline phosphatases. Alkaline-earth metal ions do not always act as activators and/or stabilizers of the catalytic activity of alkaline phosphatases. Thus, the inhibitory effect of magnesium(II), calcium(II), and barium(II) on alkaline phosphatase from chicken intestine (Reanal) was found for low concentrations (1-100 ng/mL). The authors of [39] assumed that these ions inhibit the alkaline phosphatase by binding with the hydroxyl group of serine or another coordinating group in the active center of the enzyme. Thus, different explanations for the interaction of alkaline-earth ions with alkaline phosphatases were proposed in the literature. However, although a great number of investigations have been carried out, regularities in the behavior of alkaline-earth ions have not been revealed and the conditions under which each of the proposed mechanisms or their combination occurs have not been found. According to the data presented in Table 1, metal ions much more often act as inhibitors than as activators of alkaline phosphatases. An inhibitory effect is
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Table 2. Radii (r) and effective charges () of some metal ions; logarithms of stability constants of their complexes with Tris (log K1) [59] and of hydroxo complexes (log n) [60] Metal ion Zn(II) Co(II) Cu(II) Cd(II) Ni(II) Pb(II) Mn(II) Be(II) Mg(II) Ca(II) Ba(II) Sr(II) Fe(III) Al(III) Bi(III)
-a,

r, Å 0.83 0.82 0.80 1.03 0.78 1.32 0.46 0.34 0.78 1.06 1.43 1.27 0.67 0.57 1.20

= z2/r 4.82 5.13 5.00 4.04 5.41 3.18 8.70 11.76 5.13 3.77 2.80 3.15 13.43 15.79 7.5

log K 1.9 1.7 4.2 1.7 2.6 2.7 -a - - - - - - - -

1

log n(n) 14.80(4) 10.50(3) 16.40(4) 8.65(4) 12.00(3) 13.95(3) 7.70(4) 18.57(4) 2.56(1) 2.55(2) 1.17(2) 0.71(1) 34.40(4) 33.00(4) 34.20(4)

Data are unavailable.

mainly exhibited by transition and heavy metal ions, such as zinc, cobalt, copper, cadmium, lead, and others. Zinc(II) is the most effective inhibitor of virtually all alkaline phosphatases without regard to the source from which they were isolated. However, it is important to note that the degree of inhibition of enzymes by zinc(II) is mainly determined by the nature of the phosphatase. For example, it was found that zinc(II) in concentrations of 0.5 ÷g/mL and higher completely suppressed the catalytic activity of alkaline phosphatase from the liver of red sea bream and only slightly inhibited alkaline phosphatase from the internals of shore crab. The scheme of inhibiting the catalytic activity of alkaline phosphatase from the internals of shore crab by zinc(II) was proposed in [31]: E + Zn2+
K1

E ž Zn2+

k+0 k-0

E*,

where E is the enzyme; Ö ž Zn2+ is the complex formed by the fast and reversible association of the enzyme and zinc(II); E* is the irreversibly inactivated enzyme; K1 is the equilibrium formation constant of the E ž Zn2+ complex; and k+0 and k-0 are the rate constants of the forward and back inactivation. First, the enzyme quickly and reversibly binds zinc(II), and then it undergoes a slow irreversible inactivation with a change in the native conformation of the protein. The rate of inactivation depends on the concentration of the Ö ž Zn2+ complex and, consequently, on

the concentration of zinc. The rate constant of this reaction becomes independent of the zinc(II) concentration when the latter becomes sufficiently large (>3 ÷g/mL). Thus, the inhibition of the catalytic activity of alkaline phosphatases from the liver of red sea bream and from the internals of shore crab is accomplished via the same mechanism of the displacement of zinc and magnesium ions in the catalytic and allosteric sites of the phosphatases by metal ions from the outside. It should also be noted that zinc(II) inhibits bacterial alkaline phosphatases in higher concentrations (130 ÷g/mL) than it inhibits phosphatases of the animal origin with a looser molecular structure. Ions of other metals whose radii and effective charges are close to those of zinc(II) are also capable of inhibiting alkaline phosphatases [58] (Table 2). The authors of [23] relate the inhibitory effect of Cd(II), Co(II), Pb(II), and Cu(II) on alkaline phosphatase from E. coli to the replacement of the zinc ion in the active center of the enzyme by these ions. The inhibitory effect of metal ions, in particular zinc(II), depends on the nature of the buffer solution in which the indicator reaction is carried out. It follows from the data presented in Table 1 that, regardless of the source from which alkaline phosphatase was isolated, the concentrations of zinc(II) and other metal ions in which they inhibited different alkaline phosphatases were significantly lower in inorganic solutions than in organic buffer solutions with components of which a fraction of zinc(II) formed complexes (Table 2). The concentration of a buffer solution is also an important factor that affects the degree of inhibition of alkaline phosphatases by metal ions. For example, for alkaline phosphatase from E. coli, the concentration of metal ions required for attaining the same degree of enzyme inhibition in going from a 0.05 to 0.1 M TrisHCl buffer solution increased by two orders of magnitude (Table 1). It should be noted that, in low concentrations, zinc(II) enhances the catalytic activity of some alkaline phosphatases. For example, the activating effect of zinc ions on the catalytic activity of alkaline phosphatases from the liver of red sea bream and from rat bony tissue manifests itself in concentrations of 0.1 ÷g/mL and 0.065 ng/mL, respectively (Table 1). This effect has not been explained, and the information about the development of a highly sensitive procedure based on this activating effect for determining zinc(II) is also unavailable. It was shown that MOH+ hydroxy complexes of bivalent metal ions or MOH2+ and M(OH) 2 hydroxy complexes of trivalent metal ions whose concentrations increased with increasing pH rather than metal ions themselves can exhibit both activating and inhibitory effects [61]. Table 2 demonstrates that all metal ions are
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capable of forming rather stable hydroxy complexes, especially in the alkaline pH region, where alkaline phosphatases are most active. It may be assumed that the larger the hydroxy complex stability constant, the weaker the tendency of the metal ion to polarization and the weaker its effect on the enzyme. However, it follows from Table 1 that easily hydrolyzed metal ions, such as Pb(II), Mn(II), Be(II), Al(III), Bi(III), and Fe(III) in the specified concentrations have a significant effect (activating or inhibitory) on some alkaline phosphatases, although the indicator reaction is performed at rather high values of pH 9.7-10.5 and the fraction of metal ions unbound into hydroxy complexes is minimal. Thus, it is likely that hydroxy complexes of metal ions rather than these ions themselves produce the inhibitory effect by blocking the binding sites of the substrate molecule. Alkaline phosphatase from E. coli is the only enzyme that is inhibited by metal ions in a weakly alkaline solution (pH 7.8-8.0) (Table 1, no. 1). In this case, the fraction of hydroxy complexes is much lower; therefore, the probability that the metal ion itself or its complex with the component of the buffer solution will manifest an inhibitory (activating) effect is much higher. The character of the effect of manganese ions on different alkaline phosphatases should be taken up separately, because this ion is often mentioned in publications as an effective activator of alkaline phosphatases. In earlier publications (dating from the 1960s), by analogy with magnesium, the activating effect of manganese was related to an increase in the ionic strength of the reaction system in its presence [54]. Later, the attempts were made at explaining the activating effect of manganese on alkaline phosphatases by its similarity to magnesium ions. The similarity between these ions is in the closeness of their ionic radii and effective charges and in their frequent occurrence in many biological systems. Because enzymatic hydrolysis is mainly carried out at rather high values of pH, it is better to speak about the interaction of manganese(II) complexes or, more probably, its hydroxy complexes with the components of a buffer solution rather than about the manganese(II) effect. Thus, ions of some transition and heavy metals inhibit alkaline phosphatases of different origins, and the degree of their inhibitory effect is determined by the nature of metal ions. Zinc(II) is the most effective inhibitor of the majority of bacterial and animal alkaline phosphatases, and its effect depends on the source from which enzyme was isolated and on the conditions under which the indicator reaction is carried out (mainly on the nature and concentration of a buffer solution). The inhibitory effect of zinc ions on alkaline phosphatase from calf intestine in the hydrolysis of 2-naphthyl phosphate served as the basis for developing a procedure for determining zinc in its concentration range from 0.075 to 0.75 ÷g/mL with the spectroJOURNAL OF ANALYTICAL CHEMISTRY Vol. 60

photometric monitoring of the rate of the enzymatic process [43]. We observed the inhibitory effect of zinc ions on alkaline phosphatase from seal intestine in the hydrolysis of p-nitrophenyl phosphate in Tris-HCl and borate buffer solutions and used it as the basis for selective enzymatic procedures for determining this element with the spectrophotometric control of the reaction rate in the concentration ranges from 1 to 10 and from 0.01 to 0.1 ÷g/mL, respectively. In borate buffer solutions, only 2.5 × 105-fold amounts of potassium and 7 × 105-fold amounts of cesium interfered with the determination of zinc at a level of its detection limit (cmin), which corresponded to its MPC in water [62]. This procedure surpassed the procedure for determining zinc by atomic-absorption spectroscopy in sensitivity (cmin = 0.5 ÷g/mL) [63] but was less sensitive than the procedures for determining zinc by stripping voltammetry(5-200 ng/mL) and X-ray fluorescence with sorption preconcentration (cmin = 4 ng/mL) [64]. Thus, it was shown that varying the nature of a buffer solution in enzymatic processes allowed the regulation of the performance characteristics of the procedures for determining metal ions acted as inhibitors. The alkaline phosphatase isolated from seal intestine and immobilized on polyurethane foam (PUF) was used for the development of a test procedure for determining zinc [65]. The enzyme was modified with a solution of naturally occurring polysaccharide, N-phthalylchitosan, to impart high stability and activity to it. To improve the accuracy of the visual control over the rate of the indicator reaction of p-nitrophenyl phosphate hydrolysis, a mixture of 4% ammonium molybdate solution in 0.3 M H2SO4 and a 0.1 M Malachite Green solution in the 4 : 1 volume ratio was added to a PUF tablet to intensify the color contrast of the product of the indicator reaction. After the addition of the dye solution, a timer was switched on and the time of the development of the bright green color of the PUF tablet was measured. The developed test procedure for determining zinc(II) is rather selective (only 5 × 105-fold amounts of magnesium(II) interfered with the determination of 0.1 ng/mL zinc(II)) and ten times more sensitive than the procedure for its determination with the use of native alkaline phosphatase from seal intestine. The difference in the inhibitory effects of zinc(II) on the native and immobilized alkaline phosphatases from the seal intestine is, evidently, due to the high adsorption capacity of the PUF on which the zinc was preconcentrated. The test procedure is comparable in the sensitivity of zinc determination with electrochemical, fluorometric, and atomic-absorption procedures [63]. The authors of [66] developed highly selective and sensitive visual test procedures for determining lead(II) by its inhibitory effect on the catalytic activity of alkaline phosphatase from chicken intestine that was immobilized on silica gel, microcrystalline cellulose, and
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PUF with cmin = 1.0; 0.5; and 0.05 ng/mL, respectively. The test procedure that employed the enzyme immobilized on PUF was more sensitive than the known procedures for determining lead and is comparable only with the procedure for its determination by atomic absorption spectroscopy [67]. Ions of alkaline-earth metals and magnesium activate virtually all alkaline phosphatases, especially mammalian phosphatases, but in rather high (~20- 200 ÷g/mL) concentrations. The significant activating effect of magnesium(II) on the catalytic activity of phosphatase from chicken intestine found by the authors of this review manifested itself after a 5-min incubation of magnesium ions with the enzyme and formed the basis for the highly sensitive and rather selective enzymatic procedure for determining these ions in the concentration range from 0.6 to 6 ng/mL. Only 105-fold amounts of barium and calcium ions and 103- and 102-fold amounts of cadmium and zinc, respectively, interfered with the determination of magnesium(II) at a level of its determination limit [40]. The proposed enzymatic procedure for determining magnesium(II) by its activating effect on alkaline phosphatase from chicken intestine is more sensitive than the procedures for its determination by atomic absorption and atomic emission spectrometry [68] and by the fluorometric procedure based on the formation of a magnesium complex of 8-hydroxyquinoline-5-sulfo acid (cmin = 12 ng/mL) [69]. Of the enzymatic procedures, the only procedure was found for determining magnesium(II) with such a low detection limit. This procedure is based on the magnesium-activated bioluminescence conversion of luciferin into hydroxyluciferin in the presence of ATP catalyzed by the firefly luciferase immobilized on Br-CN-sepharose (cmin = 0.1 ng/mL) [70]. Effect of metal ions on the apoenzymes of bacterial and animal alkaline phosphatases. Metal ions acting as cofactors and built in the enzyme active center are bound into the molecule of a biocatalyst more specifically and firmly than metal ions acting as effectors (inhibitors or activators) [71, 72]. The removal of cofactor metal ions from the active center of a metaldependent enzyme results in the complete or partial loss of its catalytic activity because of the formation of the apoenzyme, the protein part of the biocatalyst molecule. When small amounts of cofactor metal ions are added to the apoenzyme from the outside, the apoenzyme is selectively reactivated, that is, the catalytic activity of the enzyme is restored. The use of the reactivating action of cofactor metal ions for analytical purposes ensures the high sensitivity and selectivity of their determination [44]. To obtain apoenzymes in general, and those of alkaline phosphatase in particular, cofactor metal ions (in the case of alkaline phosphatases, ions of zinc and/or magnesium) are bound with different ligands into a stable complex compound.

The most commonly used technique for preparing apoenzymes is the treatment of an enzyme (native or immobilized) by the solution of the corresponding chelating agent followed by the removal of its excess by dialysis or by chromatography. It follows from the data in Table 3 that the following organic compounds are most often used as chelating agents for removing zinc(II) from the active center of alkaline phosphatases: EDTA, 8-hydroxyquinoline-5sulfonic acid, and 1,10-phenanthroline. However, to obtain apoenzymes of alkaline phosphatases, some researchers used inorganic reagents along with organic reagents, for example, potassium cyanide for preparing the apoenzyme of alkaline phosphatase from pig kidney [16]. The concentrations of chelating agents used for preparing apoenzymes of alkaline phosphatases of bacterial and animal origins differed insignificantly (Table 3). According to the published data, the time of dialysis varied from 8 h to several days (Table 3). Short-term dialysis resulted in the rather fast and reversible loss of zinc ions of the catalytic center, while the removal of the remaining zinc ions from the structural enzyme center required up to 20 days [44]. As a consequence, the majority of authors, without resorting to long-term dialysis, mean by apoenzyme the enzyme from which the catalytic zinc ions are removed by the fast reversible interaction with a chelating agent and which exhibits very insignificant catalytic activity (5% as compared to the initial activity). Taking this into account, by "apoenzyme," researchers mean the enzyme depleted of the zinc ions of the catalytic center. Attention should be given to the pH at which dialysis is carried out. The value of pH for all alkaline phosphatases regardless of their origin virtually coincides with the optimum pH of the indicator reaction. This is evidently due to the fact that this value of pH favors the formation of Zn(II)-chelating agent complex, on the one hand, and does not result in the instant, irreversible destruction of the enzyme structure, which is especially important in the subsequent reactivation of apoenzyme with metal ions. Temperature is also an important parameter of dialysis. Table 3 shows that, in the majority of cases, dialysis is performed at 4œC. Commercial preparations of enzymes are usually kept at this temperature. It may be supposed that this temperature ensures the stability of alkaline phosphatases in the course of a long-term dialysis. Let us dwell separately on the removal of excess chelating agent and/or its complex formed with a cofactor metal. For these purposes, along with dialysis (at the step of washing), chromatography is used. It consists in the passage of a solution of an enzyme-inhibitor complex through a column filled, for example, with Sephadex G-25 [81-83]. This method is very efficient, but rather expensive because of the large consumption of enzyme. This explains why dialysis is most widely used
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POTENTIALITIES AND PROSPECTS FOR THE USE OF ALKALINE PHOSPHATASES Table 3. Conditions for preparation and reactivation of apoenzymes of alkaline phosphatases of different origins Conditions for obtaining apoenzyme Source of alkaline phosphatase Conditions for indicator reaction: substrate, pH of dialysis, buffer solution (conchelating agent buffer solutime (concentration) tion (concen- of dialysis; t, œC centration; pH); t, œC tration) 2 3 4 Bacterial E. coli 1,10-Phenanthro- 7.0; Tris120 h (fourfold line (0.05 M) HCl (0.01 M) change of dialyzate); 4œC p-NPP; Tris-HCl (1 M; 8.0); 25œC p-NPP; Veronal (0.02 M; 8.0); 25œC H2O 26 h; 4œC p-NPP; Tris-HCl (1 M; 8.0); 27œC At cZn(II) = 0.02 ÷g/mL: ReAa = 86% At cZn(II) = 0.065 ÷g/mL: ReA = 89% cZn(II) = 321 ÷g/mL: ReA = 100% Eactivated by Co(II) (59 ÷g/mL) Reactivated by Zn(II) (9.8 ÷g/mL) and Co(II) (44 ÷g/mL) 4-NPP; Tris-HCl (1 M; 8.0); 25œC Reactivated by Zn(II) (0.85-8.5 ÷g/mL) and by Zn(II) + Mg(II) (cMg = 2.4 ÷g/mL) Reactivated by Zn(II) (0.65 ÷g/mL) and Co(II) (59 ÷g/mL) At cZn(II) = 0.16 mg/mL, ReA = 92%; at cCu(II) = 0.59 mg/mL, ReA = 71%; at cCd(II) = 0.28 mg/mL, ReA = 35% 5 Notes

227

References

1

6

7

[23]

[73] [74-76]

8-Hydroxyquino- 7.5; Tris2-3 weeks (six- p-NPP; Tris-HCl line-5-sulfonic ac- HCl (0.01 M) fold change of (0.01 M; 8.0); 25œC id (0.05 M) dialyzate); 4œC 8-Hydroxyquinoline-5-sulfonic acid (0.01 M) 8-Hydroxyquino- 8; Tris-HCl line-5-sulfonic ac- (0.01 M) id (0.01 M) H2O 8 h; -

[77]

24 h (threefold change of dialyzate); 23œC -
b

[78]

p-NPP; Tris-HCl (0.01 M; 8.0); 25œC p-NPP; Tris-HCl (1 M; 8.0); 25œC

Incubation with NaCN (0.01 M)

-

-

[79]

EDTA (0.1 M)

8.0; TrisHCl (1 M)

20 h; 20œC

p-NPP; the degree Reactivation was not of zinc removal was performed found by atomic absorption spectroscopy Reactivated by Cd(II) (0.05 ÷g/mL)

[80]

Incubation with EDTA (0.05 M)

7.0; TrisHCl (1 M)

12 h; 25œC, p-NPP; - then the solution was passed through the column with Sephadex-25 - p-NPP; -

[81]

Incubation with EDTA (0.01 M) in Tris (0.01 M)

-

Partially reactivated by Zn(II), inc (apoE + Zn(II)) = 30 min. Excess Zn(II) was removed on a column with Sephadex-25

[82]

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228 Table 3. (Contd.) 1 2 Incubation with EDTA (0.1 M) in a Tris-aminoethane-sulfonic acid buffer solution (0.01 M) E. coli, immobi- EDTA lized on a graph- (0.05 M) ite electrode using carbodiimide 3 7.0; H2O

MUGINOVA et al.

4 -; 25œC

5 p-NPP; TrisHCl (0.01 M; 8.0); 25œC

6

7

A column with Sephadex-25 [83] was used for obtaining and reactivating apoenzyme; reactivated by Co(II) (59 ÷g/mL)

-

48 h; -

o-Hydroxyphe- cZn(II) = 65-520 ng/mL, nyl phosphate; cmin Zn(II) = 50 ng/mL Tris-HCl (0.1 M; 8.0); 25œC

[84]

Animals Pig (kidney) KCN (0.01 M) 9.0; NaHCO3-HCl 6 days (two Na--Glycerol dialysis + four phosphate washing procedures); 4œC 12 days; 25œC Reactivated by Zn(II) (ReA = [16] 70%) and by Ca(II) and Hg(II) (ReA = 30%) without incubation Reactivation was not performed Na--Glycerol phosphate; 38œC p-NPP; -

7.8; Acetate- veronal, HCl + toluene (NH4)2C2O4 (4%) 9.1; NaHCO3 (5 mM)

-; 25œC

At c (Ca(II), Mg(II), Mn(II)) = [17] 0.1 mM, ReA = 100%

Calf (intestine)

(NH4)2SO4 (2 M)

-

24 h; 4œC

Analytical range for Zn(II) [85] is (0.5-5), (2-15), and (10- 50) ÷g/mL at an activity of alkaline phosphatase of 293, 30, and 22 sp. un/mL, respectively incZn(II) = 2 min. Analytical range for Zn(II) is 10-50 ng/mL [46]

Calf (intestine)

9.0; -

-

SPPc; -

EDTA

5.0; -

7 days; 4œC

p-NPP; -

Analytical ranges for Zn(II) [44] and Ca(II) are 6.5-65 ng/mL and 0.8-4 ÷g/mL, respectively [45]

EDTA (0.1 M)

8.5; H2O

6-16 h; 2-4œC

Phenyl phosReactivated by Mg(II) phate; veronal- (0.03 mg/mL) and Mn(II) ethanolamine (0.024 mg/mL) (0.05 M; 9.6); 38œC p-NPP; - Analytical range for Zn(II) is 0.01-0.1 ÷g/mL

Seal (small intestine)

Incubation with EDTA (60 mM) in Tris (0.05 M)

-

-

[50]

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POTENTIALITIES AND PROSPECTS FOR THE USE OF ALKALINE PHOSPHATASES Table 3. (Contd.) 1 Blood (leucocytes) 2 Incubation with EDTA 3 - 4 5 6

229

7

Placenta

EDTA (0.005 M) in acetate buffer solution (0.05 M)

5.0; -

Human 1 h; 37œC -Glycerol-2-phosphate; veronal (0.02 M; 9.9) with an additive of MgCl2 18 h; - p-NPP; glycine (30 ÷M; 10.5)

Reactivated by Zn(II) [51] (0.65 ÷g/mL), Co(II) (5.9 ÷g/mL), and Cu(II) (6.4 ÷g/mL) At cZn(II) = 65 ÷g/mL, cCa(II) = [52] 40 ÷g/mL, cCa(II) = 24 ÷g/mL, ReA = 100, 54, and 24 %, respectively; inc = 15 min

ReA is the percentage of reactivation (ReA, % = V/V0 × 100%, where V0 is the rate of indicator reaction in the presence of chelating agent and/or metals. -b Data are unavailable. -c SPP is 3-[spiro-4-methoxy-1,2-dioxetane-3,2'-tricyclo[3,3,1,1]chlorodecane-4-yl]phenyl phosphate:
-a

OO

O CH3

OO

O CH3

Cl OPO3
2-

2- HPO4 Cl

O- O- (pH 10.0).

O + Cl

OCH3 O

h

as before, although the procedure is time-consuming and multistage. Some authors [51, 80, 81] replaced dialysis with the exposure of alkaline phosphatase to chelating agent in a buffer solution for certain time under batch conditions. This technique was used, for example, in the case of alkaline phosphatase from E. coli immobilized on the graphite electrode, which was kept in a 0.05 M EDTA solution for 12 h (Table 3). An analysis of the data presented in Table 3 showed that, almost in all cases, along with zinc ions, ions of other bivalent metals (Co, Cd, Mn, Cu, Ni, and alkaline-earth metals) whose radii are close to those of Zn(II) or Mg(II) also restored the catalytic activity of alkaline phosphatase (Table 2). Metal ions taken in the same concentration (2 mM) can be arranged in the following order of decreasing efficiency of reactivating the apoenzyme of alkaline phosphatase from hyperthermophilic bacterium Termotoga neapolitana: Co > Zn > Mg > Mn > Sr > Ca > Ba > Ni > Cu. A similar dependence was observed for the most alkaline phosphatases regardless of the source of their isolation with the only difference that, in the majority of cases, zinc(II) and sometimes simultaneously zinc(II) and cobalt(II) were the first elements in this series. Such a significant reacJOURNAL OF ANALYTICAL CHEMISTRY Vol. 60

tivating effect of the cobalt ion is due to its greatest similarity to zinc ion. The data in Tables 1 and 3 indicate that the nature and concentration of a buffer solution have a pronounced effect on both the degree of inhibition of alkaline phosphatase and the degree of the reactivation of its apoenzyme by zinc(II). Thus, it was shown in [23] that, in 1 M Tris-HCl and 0.02 M veronal solutions, of the wide spectrum of test metal ions (cobalt(II), mercury(II), magnesium(II), manganese(II), nickel(II), iron(II), copper(II), and cadmium(II)), only zinc(II) significantly reactivated the apoenzyme. However, the concentration of zinc(II) required for the attainment of the same degree of reactivation of the apoenzyme in the veronal solution was approximately three times higher than that in the Tris-HCl buffer solution. When studying the effect of alkaline-earth ions on the apoenzyme of alkaline phosphatase from rat bony tissue [36], it was found that Zn(II) and Mg(II) added separately to the apoenzyme obtained with the use of EDTA did not completely restore the activity of the enzyme. The addition of a mixture of these ions in concentrations of 0.5 ÷g/mL and 0.1 mg/mL, respectively, restored 90% of the enzyme activity. Some other results were obtained when zinc(II) was removed from the active center of the same enzyme
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using 1,10-phenanthroline. It was found by atomic absorption that, after a 3-h incubation of the enzyme with 1,10-phenanthroline, only 20 and 30% of zinc and magnesium, respectively, were removed from the enzyme. When a mixture of zinc and magnesium of the same concentration as that added to the apoenzyme obtained with the use of EDTA was added to the apoenzyme prepared with 1,10-phenanthroline, it completely regained its activity. In this case, reactivation was nonselective, because magnesium(II) was replaced with Mn(II), Co(II), Ni(II), and Ca(II) [36]. The earlier investigations of the same authors showed that alkaline phosphatase from rat bony tissue is a polyfunctional enzyme with two binding sites for zinc(II) and with one binding site for magnesium(II) [36]. Manganese ions enhanced the activity of the enzyme in the presence of magnesium and zinc ions; however, they cannot replace zinc(II) and magnesium(II) simultaneously. Cobalt(II) can also play the catalytic role of zinc(II) or magnesium(II), activating the enzyme in the presence of magnesium(II) or zinc(II), respectively. Moreover, in contrast to manganese(II), cobalt(II) can replace magnesium(II) and zinc(II) simultaneously; however, the Cosubstituted enzyme thus obtained was very unstable [36]. It was shown [37] that a 0.1 mM calcium(II) solution did not reactivate the apophosphatase from rat bony tissue isolated by ion chromatography. An increase in the concentration of calcium ions in the presence of a magnesium solution with a concentration of 0.24 ÷g/mL resulted in the significant inhibition of the catalytic activity of the enzyme that was restored after the addition of 0.03-1 mM Mg(II) rather than in the anticipated enhancement of the degree of reactivation. The activity of the alkaline phosphatase decreased by a factor of 2.4. As the concentration of zinc ions was increased and the concentration of calcium ions remained constant, the following two effects were observed: zinc(II) in a concentration of 0.1 ÷M almost doubled the catalytic activity of the enzyme (from 130 to 240 un/mg); in higher concentrations, it acted as a strong inhibitor. Thus, the conclusions may be drawn about the strong synergistic effect of the above-mentioned ions on the apoenzyme of alkaline phosphatase from rat bony tissue and about the absence of its selective reactivation for this reason. The experiments on the simultaneous reactivation of the apoenzyme of alkaline phosphatase from rat bony tissue by two metals, Zn(II) and Mg(II), are not unique. Thus, Thownshend with co-workers attained a tenfold increase in the sensitivity of zinc determination based on its reactivating effect on the apoenzyme of alkaline phosphatase from calf intestine by introducing Mg(II) and Ca(II) along with zinc in the reaction mixture [44]. Having revealed the reactivating effect of these ions on the apoenzyme in the absence of zinc(II), the authors of [44] used its value as a reference point for estimating

the effect of zinc(II) in the presence of magnesium(II). This technique allowed them to detect a statistically significant increase of the phosphatase catalytic activity when they introduced ten times lower concentrations of zinc(II), as compared to the blank experiment carried out in the absence of zinc(II) but in the presence of 9.6 ÷g/mL magnesium(II). The technique mentioned was used by the authors of this review for the reactivation of apophosphatase from seal intestine by zinc(II) to which magnesium or calcium ions were added. The experiments were carried out for the concentrations of these ions (2 and 4 ÷g/mL, respectively) at which their reactivating effect on this apophosphatase was pronounced and well reproducible. The revealed direct proportionality of the degree of the reactivating effect of the cofactor ion in the presence of magnesium(II) to the zinc(II) concentration allowed us to develop an enzymatic procedure of Zn determination in the concentration range from 0.01 to 0.1 ÷g/mL. Studies of the effect of cobalt, nickel, copper, and cadmium ions on the degree of the reactivating action of zinc(II) showed that only zinc(II) reactivated apophosphatase from seal intestine under the specified conditions. Therefore, the developed procedure for determining zinc(II) exhibits both high sensitivity and high selectivity. The sensitivity of this procedure is comparable with that of the procedure based on the inhibitory effect of zinc(II) on the same enzyme in a borate buffer solution, and it is more sensitive than the procedures for determining zinc(II) by atomic-absorption spectroscopy (cmin = 0.5 ÷g/mL [63]) and amperometry with the use of the reactivating effect of Zn on the alkaline phosphatase from E. coli immobilized on a graphite electrode (cmin = 0.065 ÷g/mL) [84]. The developed procedure is less sensitive than the procedures for determining zinc(II) by stripping voltammetry (5-200 ng/mL [64]) and X-ray fluorescence with sorption preconcentration (cmin = 4 ng/mL [63]) and the procedure for determining zinc(II) by its reactivating effect on the apoenzyme of the ADH from baker's yeast (0.05-0.5 ng/mL [86]). Thus, it follows from the published data on the effect of metal ions on the catalytic activity of alkaline phosphatases of different origins that the character of the effect (inhibitory, activating, and reactivating) and the degree of the effect (I, %; A, %, ReA, %, respectively) of metal ions on the enzyme or apoenzyme of alkaline phosphatase depend on the nature of the metal ion, the source of enzyme, and the conditions of the indicator reaction (the nature of the buffer solution, its concentration, and pH) (Tables 1-3). Varying the nature of the buffer solution in which an enzymatic reaction is carried out should be one of the obligatory experimental steps in the development of enzymatic procedures for determining effectors of alkaline phosphatases and in general metalloenzymes. This approach can be used, for example, for the preparation of apoVol. 60 No. 3 2005

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phosphatases of different origins by dialysis. It is advisable to perform dialysis in buffer solutions that loosen the enzyme structure and thus favor the more efficient interaction of the chelating agent with the biocatalyst and the reactivation of apoenzymes with metal ions. From the analytical viewpoint, zinc and magnesium ions exhibit the most significant and interesting effects on the catalytic activity of bacterial and animal alkaline phosphatases (Table 1). The enzymatic procedures for determining cofactor ions that have been developed on the basis of the revealed effects are few in number. At the same time, a few of the procedures proposed are highly sensitive and selective. In this connection, it seems advisable and highly promising to optimize the previously developed enzymatic procedures for determining metal ions in various samples and to develop new enzymatic procedures, including test procedures for determining small amounts of magnesium and zinc ions by their effects on alkaline phosphatases isolated from different sources and by the reactivation of their apoenzymes. Proceeding from our findings of the activating effect of magnesium ions on the alkaline phosphatase from chicken intestine, we started investigations aimed at developing test procedures for determining these ions with the visual detection of the rate of the indicator reaction of p-nitrophenyl phosphate hydrolysis. To attain this and to enhance the stability of the enzyme to external factors (the change in pH of a medium, temperature, etc.), it is necessary to develop procedures for its immobilization on different supports (microcrystalline cellulose, papers, and silica gels of different types, polyurethane foam, etc.). As an indicator substance in the determination of magnesium, evidently not only the p-nitrophenolate ion, which has been used up to now in the specified indicator reaction, but also another product of hydrolysis, the phosphate ion, can be used. The rate of the indicator process occurring on the support with the immobilized enzyme can be controlled by the time it takes for the blue color of the reaction product to develop. The product was obtained after the addition of a sulfuric acid solution of ammonium molybdate and the reduction of the resulting heteropoly acid to Molybdenum Blue using a reducing agent. It might be expected that the performance of enzymatic process on a support would enhance the sensitivity of determining magnesium(II) by virtue of its sorption preconcentration. We hope that the high sensitivity and selectivity of the above procedures for determining metal ions with the use of alkaline phosphatases isolated from different sources; the approaches to the intentional alteration of performance characteristics of enzymatic procedures for determining metal ions that were elucidated when systematizing the published data; and the wide assortment of commercial preparations of alkaline phosphatases of different origins will give a new impetus to
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active investigations in the field of enzymatic methods of analysis and in chemical analysis as a whole. ACKNOWLEDGMENTS This work was supported by the Russian Foundation for Basic Research, project no. 04-03-33116. REFERENCES
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