Russian Journal of Bioorganic Chemistry, Vol. 29, No. 1, 2003, pp. 43–49. Translated from Bioorganicheskaya Khimiya, Vol. 29, No. 1, 2003, pp. 49–56. Original Russian Text Copyright © 2003 by Karaseva, Naumchik, Metelitza.

Noncompetitive Activation of the Peroxidase-Catalyzed Oxidation of o-Phenylenediamine by Melamine E. I. Karaseva, I. V. Naumchik, and D. I. Metelitza1 Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, ul. Kuprevicha 5/2, Minsk, 220141 Belarus Received October 2, 2001; in final form, March 6, 2002

Abstract—Peroxidase-catalyzed oxidation of Ó-phenylenediamine (PDA) is greatly activated with melamine (MA) in 15 mM phosphate–citrate buffer at pH 6.0–7.4 in a noncompetitive manner: kcat and Km increase in direct proportion to the MA concentration. An extent of the activation is quantitatively characterized with a coefficient α (in M–1), which essentially increases along with the rise in pH from 6.0 to 7.4. MA acts as a nucleophilic catalyst in the oxidation process: it most likely affects the peroxidase active site from the distal position of heme. MA noncompetitively inhibits the peroxidase oxidation of PDA at pH 4.3, since it completely loses its nucleophilic properties in acidic medium. A rapid, highly accurate, and simple analytical test system based on the kinetics of melamine-activated oxidation of PDA is proposed for the quantitative determination of melamine within the concentration range of 10–4–10–3 M. This test system uses the spectrophotometric determination of the PDA oxidation product at 455 nm. Key words: horseradish peroxidase, activation, inhibition; o-phenylenediamine; melamine, peroxidase activation, enzymatic assay; nucleophilic catalysis 1

INTRODUCTION

Soluble aromatic amines,2 such as PDA, DA, 3,3'tetramethylbenzidine, and 5-aminosalicylic acid, are mostly used in modern highly sensitive assays as substrates of horseradish peroxidase (HP, EC 1.11.1.7). The main areas of the HP analytical use are: enzyme immunoassay [1], immunocytochemistry [2], chemiluminescent analysis [3], biosensors [4], and enzymatic assay of microquantities of various organic compounds [5]. The sensitivity of all the methods may be improved by an activation (enhancement of efficiency) of peroxidase-catalyzed oxidation of the above-mentioned aromatic amines. Three essentially different ways are known for the activation of aromatic amine oxidation with peroxidase. Historically the first of them consists in the addition of nitrogen-containing bases (ammonia, imidazole and its derivatives, and pyridine) to the reaction mixture at pH > 6 [6, 7]. These activators act on HP as nucleophiles that change the basicity of functional groups of the enzyme and expand the pH optimum of its catalytic activity. The kinetics of peroxidase oxidation of p-phenylenediamine and DA was studied under activation with pyridine, imidazole [6, 7] and its numerous deriv1 Please

send correspondence to fax, (375)-(172) 63-7274 or email, [email protected]. 2 Abbreviations: DA, o-dianisidine; HP, horseradish peroxidase; MA, melamine (2,4,6-triamino-1,3,5-triazine); PCB, 15 mM phosphate–citrate buffer; PDA, o-phenylenediamine; and poly(MADS), poly(MA-disulfide).

atives [5, 8–10], 1,2,4-triazole, 1,2,3-benzotriazole, and substituted indoles [11]. The second way for the peroxidase activation is based on the cooxidation of aromatic amines in the presence of substituted phenols and polyphenols. Oxidation of such pairs as 4-aminoantipyrine–phenol [12– 14] or luminol–phenol [3, 4, 15–17] is accompanied with a great increase (sometimes by a factor of 100–200 or more) in the rate of amine oxidation. However, the use of amine–phenol pairs may result not only in the conjugated activation of amine oxidation, but in profound inhibition of the reaction, as a rule, by a competitive mechanism [18–22]. The third type of activation may be realized upon the interaction of peroxidase with synthetic polyelectrolytes, which manifests itself in the shift of pH optimum of the enzyme and in significant changes in its catalytic characteristics (the Km and kcat values). The rates of luminol and p-iodophenol oxidation in the conjugated peroxidase reaction were shown to increase in direct proportion to the concentration of poly(N-ethyl4-vinylpyridinium bromide) added to the reaction system [23]. In general, it is presumed that electrostatic field of the polymer molecule affects the HP conformation in a way favorable for the catalysis through the modification of K‡ values of ionogenic groups in the active site of the enzyme. The complexity of the enzyme activation mechanism in this case is first of all determined by the ampholytic nature of proteins that have non-uniform distribution of charged groups over the macromolecule [23].

1068-1620/03/2901-0043$25.00 © 2003 MAIK “Nauka /Interperiodica”

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v0 × 107, M s –1 10

(a)

8 6 4 2 0

1

2

3

v0–1 × 107, M–1 s 1.0

(b)

4

5 6 7 [H2O2]0, mM

0.8 0.6 0.4 0.2 0

2

4

6

8

10 12 14 [H2O2] –1 , mM –1 0

Fig. 1. The dependence of the initial rate of peroxidase oxidation of PDA (1.0 mM) on the initial ç2é2 concentration (a) in direct and (b) double reciprocal coordinates (pH 6.0).

v0 × 107, M s –1 1 20 15

2

10 5 0

3

4

5

6

7

8 pH

Fig. 2. The dependence of the initial rate of peroxidase oxidation of PDA on pH of PCB (1) in the absence and (2) in the presence of 0.6 mM MA.

A critical analysis of the data available on the peroxidase activation in the processes of oxidation of aromatic amines demonstrates that the mechanism of this phenomenon is not completely clear for the three cases

considered above. That is the cause that the investigation of the peroxidase activation still remains a topical problem of the enzymatic catalysis. The goal of this work was a kinetic study of oxidation of PDA (one of the most frequently used peroxidase substrates) under the activation with melamine (MA), which has basic properties and can be considered as a potential nucleophilic agent. The wide application of MA in various areas calls for a highly sensitive method of its determination. Until now, MA is analyzed gravimetrically as a salt of cyanuric or picric acid or spectrophotometrically in a weakly acidic medium by absorption at ~236 nm [24]. These methods do not meet the current requirements because of low sensitivity. Therefore, another goal of our investigation was a development of an enzymatic assay system for MA in the concentration range of 10–4–10–3 M. RESULTS AND DISCUSSION Kinetics of Peroxidase-Catalyzed Oxidation of PDA The initial rate of the PDA oxidation at its 2 mM concentration in the presence of hydrogen peroxide (1 mM) is directly proportional to the HP concentration in the range of 0.2–2.0 nm; the rate of peroxidase oxidation of PDA is 1.3 × 10–6 å s–1 at [HP]0 = 2.0 nm. The dependence of v0 on the initial concentration of PDA is described by the Michaelis–Menten equation. The presentation of the dependence in the Lineweaver–Burk coordinates helps calculate the kinetic parameters kcat of 1042 s–1 and Km (PDA) of 0.39 mM at 2 mM ç2é2. The dependence of v0 on the initial concentration of ç2é2 (Fig. 1a) was studied at the fixed concentration of [PDA]0 = 1mM; the dependence is described by the Michaelis–Menten equation up to the maximal value of v0. The presentation of the data as a double reciprocal plot (Fig. 1b) yielded the constants kcat of 1100 s–1 and Km (ç2é2 ) of 0.7 mM. The difference between values of kcat obtained from the dependences of v0–[PDA]0 and v0–[ç2é2 ]0 is usual for the peroxidase-catalyzed reactions and can be explained by the HP destabilization observed at high concentrations of hydrogen peroxide, which is also characteristic of many other enzymes that function in the presence of ç2é2, such as catalases, glucose oxidases, cytochromes P-450, and others [25]. The rate of peroxidase oxidation of PDA is maximal at pH 4.0–4.8 (Fig. 2, curve 1), which is close to the isoelectric point of the acidic isoform of HP (I 5.0) [26]. The temperature effect on the initial rate of the peroxidase oxidation of PDA at pH 6.0 is plotted in Fig. 3. A low activation energy (3.8 kcal/mol) reflects a high reactivity of PDA in the peroxidase oxidation. The introduction of poorly soluble poly(MADS) into aqueous medium of the reaction mixture as well as a possible presence of compounds hardly soluble in

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water that accompany MA in a number of processes (e.g., in the production of melamine–formaldehyde resins and glues) prompted us to study the peroxidase oxidation of PDA in aqueous organic solvents. The effects of (1) DMF and (2) DMSO on the initial rate of peroxidase oxidation were studied at pH 4.0 (Fig. 4a) and 6.0 (Fig. 4b). Figure 4 shows that the DMSO concentrations exceeding 3–4 vol % decrease v0 , which is more pronounced at pH 4.0 (cf. curves 2 (‡) and 2 (b)). DMF inactivates HP more strongly than DMSO, especially, at pH 4.0 (see curve 1 (‡)). Thus, DMSO is more preferable organic component for the use in water–organic media, since its use remains practically unaffected the rate of the peroxidase-catalyzed oxidation of PDA at concentrations up to 5 vol %. It was previously shown in our laboratory that the organic cosolvents (DMF, isopropanol, and others) have an effect only on kcat and do not change Km values [27]. Kinetics of Peroxidase Oxidation of PDA in the Presence of MA Three amino groups impart basic properties to the MA molecule, and its nucleophilicity greatly depends on pH. We have therefore studied an influence of MA on peroxidase oxidation of PDA within the pH range of 3.0–8.0. Figure 2 (curve 2) demonstrates a dual effect of MA on the oxidation. In the presence of MA, the initial rate of MA oxidation decreases at pH < 5.5 and, on the contrary, increases at pH > 5.5 so that the shape of the HP dependence on pH is changed and the second maximum appears at pH 6.0. It follows from the dependences of v0 on the PDA concentration in the double reciprocal coordinates (Fig. 5‡) that, at pH 4.3, MA acts as a noncompetitive inhibitor (kcat and Km values demonstrate a symbate decrease along with the increase in the concentration of MA (Figs. 5b, 5c). At the same time, the rate of peroxidase oxidation of PDA in the presence of MA significantly increases on raising pH up to 7.4 (Fig. 6). This gives a clear evidence of the noncompetitive activation of the PDA oxidation. Similar dependences were observed at pH 6.0, 6.5, and 7.0 (data not shown). Thus, MA greatly activates the peroxidase oxidation of PDA in a noncompetitive manner at pH ≥ 6.0. An estimation of the activation energy of peroxidase oxidation of PDA at pH 6.0 in the presence of 0.6 mM MA (Fig. 3, curve 2) gives the value of 2.5 kcal/mol, which is 1.5 times less than the value of 3.8 kcal/mol estimated in the absence of MA. Both values are highly effective, but we would like to stress that, at the concentration 3.3 times less than that of substrate (2.0 mM), MA 1.5 times lowers the energy barrier; i.e., it behaves (in formal terms) like a catalyst of the peroxidase reaction. The dependences of kcat and Km on the MA concentration obtained for PDA oxidation at pH 6.0 and 7.4 RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

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ln(v0, M s –1) –13.2 –13.4 2 –13.6 –13.8 1

–14.0 –14.2

–14.4 3.15 3.20 3.25 3.30 3.35 3.40 3.45 3.50 T –1 × 103, K –1 Fig. 3. Arrhenius dependences of initial rate of peroxidase oxidation of PDA (pH 6.0) on temperature (1) in the absence and (2) in the presence of 0.6 mM MA.

(v /v0), % 100 90 80 70 60 50 40 30 20 100 90 80 70 60 50 0

(a)

2

1 (b)

2 1 5

10

15 20 25 30 DMSO (DMF) content, %

Fig. 4. The dependence of the initial rate of peroxidase oxidation of PDA on the organic solvent content in mixtures (1) PCB–DMF and (2) PCB–DMSO (a) at pH 4.0 and [HP] 0.5 nm and (b) at pH 6.0 and [HP] 1 nm.

are strictly linear (Fig. 7), and both parameters show a symbate variance with the increase in MA concentration. Based on these dependences, an influence of MA on the kcat and Km values in the activated oxidation process at pH ≥ 6.0 can be expressed by equations (1) and (2):

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(1)

= K m ( 1 + α [ MA ] 0 ),

(2)

MA

MA

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v0–1 × 10 –7, M –1 s 0.5 (a)

v0–1 ×10 –7, M –1 s 0.5 2

0.4 0.3

0.4 2 3 4

0.3

1

0.2

0.2

0.1

0.1

–0.1 0

0.1 0.2 0.3 0.4 0.5 0.6 0.7 [PDA]–1 × 10 –4, M–1 –2 –1 10 × k cat, s 80 (b) 70

1

–2.5 –2.0 –1.5 –1.0 –0.5 0

0.5 1.0 1.5 2.0 [PDA]–1 × 10 –4, M–1

Fig. 6. The Lineweaver–Burk plot for peroxidase oxidation of PDA at pH 7.4 (1) in the absence and in the presence of (2) 0.3, (3) 0.6, and (4) 1.0 mM MA ([HP] 3 nm, [ç2é2] 1 mM).

60 50 40 30 10 3 × K m, M 5

(c)

4 3 2 0

0.2

0.4

0.6

0.8 1.0 [åÄ], mM

Fig. 5. (a) The Lineweaver–Burk plot for peroxidase oxidation of PDA at pH 4.3 (1) in the absence and (2) in the presence of 1 mM MA and the dependences of (b) kcat and (c) Km on MA concentration.

where α (å–1 ) is a coefficient (degree) of activation that characterizes the growth of a kinetic parameter in the presence of 1 M activator. The initial rates of activated peroxidase oxidation of PDA at the constant concentration [ç2é2 ]0 can be adequately described by equation (3): k cat ( 1 + α [ MA ] 0 ) [ èï ] 0 [ H 2 O 2 ] 0 [ PDA ] 0 - . (3) v 0 = -------------------------------------------------------------------------------------------------K m ( 1 + α [ MA ] 0 ) + [ PDA ] 0 Eq. (3) is transformed into the standard Michaelis– Menten equation at the activator concentration [åÄ]0 = 0. The table lists the values of kcat and Km determined at various pH values (6.0–7.4) and MA concentrations.

The values of the coefficient α calculated from the dependences of kcat and Km on [MA] are in good agreement with each other; they grow with pH increase. The dependences of coefficients α(kcat ) and α(Km ) on pH (Fig. 8) unequivocally confirm that the activating effect of MA in peroxidase oxidation is determined by the activator nucleophilicity that increases at pH > 6. All three amino groups of MA are protonated at pH lower than 5.0 and, as a result, MA loses its nucleophilic properties and changes from an activator of the peroxidase-catalyzed PDA oxidation into an inhibitor of this reaction (see Figs. 5a, 5b). Thus, it is pH of the reaction medium that plays a crucial role in the activation of the PDA oxidation with MA. A substitution of poly(MADS) for MA (20°ë, pH 6.0, 5 vol % DMSO) confirms an important role that three amino groups of MA play in nucleophilic activation of peroxidase oxidation of PDA. Poly(MADS) also accelerates the PDA oxidation in the noncompetitive manner. However, the acceleration amounts to only 9.5% at the poly(MADS) concentration of 0.5 mM; i.e., MA in poly(MADS) almost completely loses its nucleophilic properties. This can be connected with the participation of two amino groups of the monomer in polycondensation reaction and their transformation into –NH–S–S– groups during the synthesis of poly(MADS). It is obvious that such a transformation of MA greatly decreases nucleophilic properties of its polydisulfide derivative, which affects its behavior in peroxidase oxidation of PDA. Despite numerous studies of the nucleophilic activation of peroxidase reactions [5–11], its mechanism hitherto remains obscure. The only fact is beyond question that the activation of some peroxidase reactions with nitrogen-containing organic bases is of nucleophilic nature; this is additionally confirmed by experiments in this work. It is known that the interaction of many nitrogen-containing bases with HP does not

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kcat × 10 –2, s–1 4 (b)

22

α(kcat) × 10 –3, M –1 3

α(Km) × 10 –3, M –1 3

1 2

2

3

18

2

2

14 10 K m × 10 4, M

1 K m × 10 4, M

8

1.2

6

0.8

4

0.4

0

47

1 6.0

6.5

pH

7.0

1 7.5

Fig. 8. The dependences of coefficients (α) for the MA activation of peroxidase oxidation of PDA calculated from the values of (1) kcat and (2) Km on pH (experimental conditions for each pH value are given in the table).

0.2 0.4 0.6 0.8 1.0 0 0.2 0.4 0.6 0.8 1.0 10 3 × [åÄ], å

with this channel, which has a beneficial effect on the catalysis.

Fig. 7. The dependences of the parameters kcat and Km for the peroxidase oxidation of PDA on the MA concentration (a) at pH 6.0 and (b) at pH 7.4. [HP] 3 nM, [H2O2] 1 mM.

involve the heme [7, 28, 29]. This fact is proved by the absence of spectral changes in peroxidase and its oxidized forms upon the interaction with imidazole and pyridine [7]. An NMR study of the HP complexation with imidazole derivatives also demonstrated the impossibility of direct contact of these compounds with the heme [28]. This means, that the polypeptide chain of HP has a channel for the transfer of electrons from the substrate onto the hemin or the ferrous ion of the heme group. Nucleophilic activators probably interact

The progress in studying the primary structure of peroxidases and the role of homologous sites in catalysis helps concretize the effect of nucleophilic activators on HP. It is now accepted that the peroxidase catalysis is controlled above all by the homologous sites that coordinate the heme in the distal area (large domain, residues 40–50) and in the proximal area (small domain, residues 160–170) [26]. The amino acid sequence that coordinates the distal area of the heme, Phe-His-Asp-Cys-Phe-Val, contains a functionally important His residue and corresponds to the site responsible for the acid–base catalysis in the HP molecule. It is safe to assume that nucleophilic activators interact just with this site in the hydrophobic channel of the enzyme.

Kinetic parameters of peroxidase oxidation of PDA in the presence of MA at 20°C and various pH values of PCB Mixture component concentrations, mM HP

H2O2

1 × 10–6

2

1 × 10–6

2

pH

[MA] × 103, M

kcat , s–1

6.0

0 0.3 0.6 1.0 0 0.6 1.0 0 0.6 0 0.3 0.6 1.0

1042 1299 1923 2353 645 1513 1923 333 741 115 208 275 370

PDA 0.15–1

0.1–0.8

6.5

3 × 10–6

2

0.06–0.6

7.0

3 × 10–6

1

0.06–0.6

7.4

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1.38

1.88 2.04

2.41

2003

3.85 4.76 6.70 8.47 2.00 4.55 5.71 1.50 3.33 0.42 0.76 1.00 1.30

1.30

1.76 2.03

2.37

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The interaction of the activated form of HP, compound II (ÖII), with the molecules of the reducing substrate (amine or phenol) is believed to be a rate limiting step in the peroxidase reaction. Therefore, one may suppose that just this step is accelerated by nucleophilic activators: E II + AmNH 2

A

.

In our test system, light absorption is measured at ~455 nm where many admixtures are optically transparent. A disadvantage of the system arises from the use of PDA solutions, which are unstable in air because of PDA autooxidation. However, this drawback can be easily eliminated by the use of a pelleted form of o-phenylenediamine.

E + AmNH ,

where E is peroxidase and AmNH2 is an aromatic amine. Unfortunately, further specification of the catalytic act is not possible at present due to the lack of direct experimental proofs. The following can be said without doubt: MA activates the peroxidase oxidation of PDA at pH ≥ 6 in a noncompetitive manner and behaves like a typical nucleophilic catalyst that 1.5 times reduces the energy barrier of the reaction and substantially alters its pH profile. MA loses its activation capacity at pH ≤ 5 and becomes a weak inhibitor of the peroxidase oxidation of PDA. Peroxidase Test System for Quantitative Determination of MA We used the results of the kinetic study of the MA activated peroxidase oxidation of PDA as the basis for the development of an analytical test system for the quantitative determination of MA within the concentration range of 10–4–10–3 M. The dependences of kcat and Km on the MA concentration (see Figs. 7a, 7b) can be used for calibration. It is preferable to carry out the analysis in PCB with pH 7.4, because this ensures the maximal activation coefficient equal to 2.41 × 103 M–1 (see the table). The calibration curve in coordinates v0– [MA] is advisable for the MA express analysis. This is performed at 20°ë using the simplest spectrophotometers (colorimeters) capable of measuring absorption at ~455 nm. The analysis is rapid (1–3 min if without regard for the preliminary incubation). Its sensitivity can be improved by increasing the HP concentration. The peroxidase test system for quantitative determination of MA includes the following components: PCB, pH 7.4; 0.02 M solution of hydrogen peroxide in PCB, pH 7.4; 4 mM solution of Ó-phenylenediamine in PCB, pH 7.4; 60 nM solution of HP in PCB, pH 7.4; and MA solutions of various concentrations in PCB for the calibration (see Fig. 7). In addition, the proposed peroxidase test-system enables the MA determination in the presence of admixtures poorly soluble in water, because the peroxidase oxidation of PDA can be carried out in binary water–organic mixtures PCB–DMSO or PCB–DMF (see Fig. 4). It was already mentioned in the Introduction section that the direct spectrophotometric determination of MA in a weakly acidic medium using its absorption at ~236 nm is practically used. However, admixtures accompanying MA often intensively absorb light in the UV range, which is a great drawback of this method.

EXPERIMENTAL Reagents. The acid isoform of horseradish peroxidase (HP; EC 1.11.1.7), type A, optical purity RZ 2.4 (NPO Biolar, Latvia) was used. The enzyme concentration was determined spectrophotometrically using the molar absorption coefficient in the maximum of the Soret band (403 nm) equal to 102 000 M–1 cm–1 [31]. Diluted Perhydrol was used as an oxidant; its ç2é2 concentration was determined spectrophotometrically using the molar absorption coefficient ε230 72.1 å–1 cm–1 [32]. Ó-Phenylenediamine (PDA) of analytical grade (Kharkov Pharmacochemical factory, Ukraine) was used as a reducing reagent after its purification by a vacuum sublimation. Melamine (MA) of reagent grade was purchased from Reakhim (Russia). The UV spectrum of MA had the absorption maximum at 212 nm in distilled water and at 223 nm in PCB, pH 6.0. Poly(MADS) (an averaged molecular mass ~1500 Da, ~8 monomer units) was prepared according to the previously described procedure [33]; it was kindly donated by Yu.P. Losev (Chemical Faculty, Byelarus State University, Minsk, Belarus). Organic solvents DMSO and DMF were distilled before use. Salts and bases of the grade not lower than reagent were used for the preparation of buffer solutions. Peroxidase oxidations of PDA in the presence and absence of MA were carried out in temperaturecontrolled cells of a Specol-211 spectrophotometer (Carl Zeiss, Germany). Concentrations of HP, MA, and substrates,; pH; and temperature were varied. In typical experiments (20°ë), the reaction mixture (1 ml) contained PCB of preset pH, 1 nM HP, 2 mM PDA, 2 mM ç2é2, and MA at required concentrations. In all cases, the reaction was started by adding the ç2é2 solution (before adding ç2é2 the reaction mixture was kept for 3 min at 20°ë), and the optical absorbance was recorded for 1–2 min at 455 nm, which corresponds to the absorption maximum of 2,3-diaminophenazine, the PDA oxidation product [34]. Initial rates of the PDA oxidation were determined from the initial linear section of the A455 dependences on time using the following coefficients of molar absorption of the PDA oxidation product [35]: ε, mM–1 cm–1 (pH): 19.6 (4.5), 19.0 (5.0), 17.0 (5.5), 16.4 (6.6), 16.3 (6.9), and 16.1 (8.0).

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ACKNOWLEDGMENTS We are grateful to Yu.P. Losev (Chemical Faculty, Byelarus State University, Minsk) for donation of poly(melamine disulfide). REFERENCES 1. Enzyme-Immunoassays, Maggio, E.T., Ed., Boca-Raton: CRC, 1983. 2. Polac, J.M. and van Noorden, S., An Introduction to Immunocytochemistry: Current Techniques and Problems, Oxford: Oxford Univ., 1984. Translated under the title Vvedenie v immunotsitokhimiyu: sovremennye metody i problemy, Moscow: Mir, 1987. 3. Gavrilova, E.M., Itogi Nauki Tekhn., Ser.: Biotekhnologiya, vol. 3, 1987, pp. 6–55. 4. Rubtsova, M.Yu., Kovba, G.V., and Egorov, A.M., Biosensors and Bioelectronics, 1998, vol. 13, pp. 75–85. 5. Ugarova, N.N., Kinetic Regularities of the Peroxidase and Luciferase Catalysis and the Problems of Use of These Enzymes for the Determination of Microquantities of Substances, Doctoral (Chem.) Dissertation, Moscow: Moscow State Univ., 1982. 6. Fridovich, I., J. Biol. Chem., 1963, vol. 233, pp. 3921– 3928. 7. Claiborne, A. and Fridovich, I., Biochemistry, 1979, vol. 18, pp. 2327–2335. 8. Lebedeva, O.V., Ugarova, N.N., and Berezin, I.V., Biokhimiya (Moscow), 1977, vol. 42, pp. 1372–1379. 9. Ugarova, N.N., Lebedeva, O.V., Kurilina, T.A., and Berezin, I.V., Biokhimiya (Moscow), 1977, vol. 42, pp. 1577–1584. 10. Lebedeva, O.V., Dombrovskii, V.A., Ugarova, N.N., and Berezin, I.V., Biokhimiya (Moscow), 1978, vol. 43, pp. 1024–1033. 11. Dolmanova, I.F., Shekhovtsova, T.N., and Kutcheryaeva, V.V., Talanta, 1987, vol. 34, pp. 201–205. 12. Litvinchuk, A.V., Savenkova, M.I., and Metelitsa, D.I., Kinet. Katal., 1991, vol. 32, pp. 535–540. 13. Metelitza, D.I., Litvinchuk, A.V., and Savenkova, M.I., J. Mol. Catal., 1991, vol. 67, pp. 401–411. 14. Metelitsa, D.I., Litvinchuk, A.V., and Savenkova, M.I., Izv. Akad. Nauk Bel. SSR, Ser. Khim. Nauk., 1991, no. 2, pp. 75–82. 15. Vlasenko, S.B., Arefyev, A.A., Klimov, A.D., Kim, B.B., Gorovits, E.L., Osipov, A.P., Gavrilova, E.M., and Egorov, A.M., J. Biolum. Chemilum., 1989, vol. 4, pp. 164–176. 16. Metelitsa, D.I., Litvinchuk, A.V., and Savenkova, M.I., Biokhimiya (Moscow), 1992, vol. 57, pp. 103–113.

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No. 1

2003