enzyme, activity, specific activity

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We can gain some understanding of enzyme behaviour through the study ... Enzymes speed up the rate of a reaction by a definite amount, proportional to.

SCIENTIFIC ANNALS OF “ALEXANDRU IOAN CUZA DIN IAŞI” UNIVERSITY Tomul I, s. Biomaterials in Biophysics, Medical Physics and Ecology 2008

LACCASE ACTIVITY DETERMINATION Alina Manole1, D. Herea2, H. Chiriac2, V. Melnig1 KEYWORDS: enzyme, activity, specific activity, turnover number. Enzymes catalyze most biochemical reactions, governing the chemical changes which we call metabolism. We can gain some understanding of enzyme behaviour through the study of rates of enzyme catalyzed reactions. In this paper, syringaldazine oxidation to the corresponding quinone, catalyzed by laccase is studied to determine laccase activity.

1.

INTRODUCTION

Enzymes speed up the rate of a reaction by a definite amount, proportional to quantity of enzyme present. Simple measurements of enzyme reactions include activity, specific activity (activity per unit mass) and turnover number, activity per mole of enzyme. Turnover number also represents the actual number of times an enzyme molecule reacts per second. To measure reaction rate, some property difference between reactant and product must be identified. Rate can be measured as disappearance of reactant or accumulation of product [1]. Quantitative description of enzyme catalysis Rate of reaction = concentration of substrate disappearing per unit time (mol L–1 sec–1) = concentration of product produced per unit time (mol L–1 sec–1) Enzyme activity = moles converted per unit time = rate × reaction volume The SI unit is the katal, 1 katal = 1 mol s-1, but this is an excessively large unit. A more practical value is 1 international unit (IU or U), defined by necessary enzyme quantity for consuming 1 μmol of substrate or producing 1 μmol of product per minute. Specific activity = moles converted per unit time per unit mass of enzyme = enzyme activity / actual mass of enzyme present SI units: katal kg-1; Practical units: μmol mg-1 min-1 or μmol μg-1min-1. Specific activity is a measure of enzyme efficiency, usually constant for a pure enzyme. An 1 2

“Al.I. Cuza” University, Faculty of Physics, Carol I Blvd., No.11, 700506, Iasi National Institute of Research and Development for Technical Physics 47 Mangeron Blvd., 700050, Iasi

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Alina Manole, D. Herea, H. Chiriac, V. Melnig

impure sample has lower specific activity because some of the mass is not actually enzyme and if the specific activity of 100% pure enzyme is known the purity of an impure sample can be calculated with the formula: test enzyme specific activity . % purity = 100% x pure enzyme specific activity Turnover number = moles of substrate converted per unit time / moles of enzyme (usually per second) = specific activity × molar mass of enzyme (with necessary unit conversions!) Multiplying mass by molar mass converts specific activity (per unit mass) into activity per mole. If n moles of substrate are catalyzed by one mole of enzyme per second, then n molecules of substrate are catalyzed by each molecule of enzyme per second. Hence turnover number represents the number of times per second that the enzyme completes a reaction cycle. Laccases (E.C. 1.10.3.2, benzenediol: oxygen oxidoreductase) are either mono or multimeric copper-containing oxidizes that catalyze the one-electron oxidation of a vast amount of phenolic substrates. Molecular oxygen serves as the terminal electron acceptor and is thus reduced to two molecules of water [2]: O2+4e-+4H+→2H2O. Three types of copper can be distinguished Type 1 copper is responsible for the blue color of the protein at an absorbance of approximately 600 nm, Type 2 copper does not confer color and Type 3 copper consists of a pair of copper atoms in a binuclear conformation that give a weak absorbance in the near UV region. The Type 1 Cu is usually coordinated to two nitrogens from two histidines and sulphur from cysteine (figure 1). It is the bond of Type 1 Cu to sulphur that is responsible for the characteristic blue color of typical laccase enzymes. The geometry is described as a distorted trigonal bipyramidal coordination with a vacant axial position where the substrate docks. The coordination is unusual as it is intermediate between the preferred coordination states for Cu (I) and Cu (II) species. A leucine residue is present but is too far away to be directly coordinated. The Cu is therefore only coordinated to three atoms. The structure showed that type 2 and type 3 coppers are close together in a trinuclear centre. The copper atoms of the T2/T3 sites are coordinated to eight histidines, which are conserved in four His-X-His motifs. The two T3 atoms are coordinated to six of the histidines (figure 1) while the T2 atom is coordinated to the remaining two. A hydroxide ligand bridges the pair of T3 atoms (figure 1 in red), resulting a strong anti- ferromagnetic coupling. The cloned sequences of various laccases also show that the 10 histidine and 1 cysteine residues are copper ligands conserved in all laccase sequences known to date except one from Aspergillus nidulans that has a methionine ligand of type 1 copper. These conserved cysteine and histidine residues serve as a pathway for the transport of electrons from the T1 Cu site where electrons are extracted from phenolic substrates to the trinuclear site that serves as the binding site of dioxygen where the electrons are required for dioxygen reduction [3].

LACCASE IMMOBILISED ON HYDROTALCITES…

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Fig. 1 A ball-and-stick model depicting the coordination of copper atoms and ligands in the active site of Cu-2 depleted Coprinus cinereus laccase. Copper atoms are in green, sulphur in yellow and oxygen in red. Laccase is regarded as the simplest enzyme that can be used to define the structure-function relations of copper containing proteins. Only laccase presents the possibility to oxidize activated metoxiphenols like syringaldazine [4]. In this paper, we study the oxidation of 4,4’-[azinobis(methanylylidene)]bis (2,6-dimethoxyphenol) (syringaldazine) to corresponding quinone, 4,4’-[azinobis (methanylylidene)]bis(2,6-dimethoxycyclohexa-2,5-diene-1-one) (Fig. 2). The increase in absorbance is followed at 530 nm and 25oC, to determine the laccase activity in international units.

Fig. 2 Catalyzed oxidation of syringaldazine by laccase to the corresponding quinone (Sanchez-Amat and Solano, 1997). 2.

MATERIALS AND METHODS

Laccase (from trametes versicolor), syringaldazine, hydroquinone, tannic acid and vanillic acid were purchased from Sigma Aldrich.. All other chemicals are of analytical grade. Absolute methanol and ethanol were purchased from Chemical Company. 0.1M Britton buffer solutions were obtained by mixing the same amount of 0.1M acetic acid, boric acid and phosphoric acid. The desired Britton buffer solutions pH was adjusted by different amounts of NaOH solution. All solutions were made using deionised water.

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Alina Manole, D. Herea, H. Chiriac, V. Melnig

Laccase activity determination using syringaldazine The specific activity of laccase was assayed spectrophotometrically by monitoring the absorbance increase from oxidation of syringaldazine at 530 nm (ε=65 mM−1 cm−1) with a Perkin-Elmer Lambda UV-VIS spectrophotometer at room temperature (light path 1 cm). The assay mixture for the test cuvette consisted in 2.20 ml Britton buffer, 0.3ml of syringaldazine 0.216 mM in absolute methanol and 0.5 ml of laccase 1mg/ml solution. The assay mixture for the blank cuvette consisted in 2.20 ml Britton buffer, 0.3ml of syringaldazine 0.216 mM in absolute methanol and 0.5 ml of deionised water. The reaction was started by the addition of syringaldazine solution and immediate mixing by inversion [5]. The absorbance of a sample is directly proportional to concentration (Beer's Law) and to sample thickness (Lambert's Law). When these two relationships are combined, we get the Beer-Lambert equation: A = εcl , where ε = extinction coefficient, a characteristic constant for a given absorbing substance; c = concentration of substrate in mol/L; l = thickness of the sample in cm (usually 1.00 cm for standard sample cuvettes). The effect of pH on laccase activity and substrate specificity Four substrates, syringaldazine, hydroquinone, vanillic acid and tannic acid (1mM in ethanol 98%) were used to determine the effect of pH on laccase activity, as laccase enzymes tend to react differently to pH with different substrates [6]. The pH optima were determined over a range of pH 3 to 8. A 0.1 M Britton buffer was used for the entire pH range. The assays for the different substrates were conducted in the same manner described in the previous paragraph. The four different aromatic compounds were also chosen to study the substrate specificity of laccase. In (Table 1) are the wavelengths of maximum absorbance for the oxidized substrates. 3.

RESULTS AND DISCUSSIONS

Table 1. Wavelengths of maximal absorption of laccase-oxidisable aromatic compounds. Substrate λ (max. change, nm) Syringaldazine 530 Hydroquinone 390 Vanillic acid 390 Tannic acid 458 Once the assays have been run, the absorbance values versus time are then plotted and a regression analysis performed on the data. The slope of the graph is the crude velocity in ∆absorbance/second. Then that crude velocity is converted to the change in concentration per minute, and then converted again into the change in number of moles/mL/minute. Often the turn over number is then computed from the knowledge of the velocity and the concentration of the enzyme that was used in the reaction.

LACCASE IMMOBILISED ON HYDROTALCITES… 2,0

pH=3 pH=4 pH=5 pH=6 pH=7 pH=8

1,8 1,6

Absorbance (a.u.)

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1,4 1,2 1,0 0,8 0,6 0,4 0,2 0,0

0

1

2

3

4

5

6

7

8

9

10

Time (min)

Absorbance (u.a.)

Fig. 3 Absorbance values for syringaldazine oxidation catalyzed by laccase, at different pH values. 1,5 1,4 1,3 1,2 1,1 1,0 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1

pH=5 pH=6

1:1

1:2

Dilution

Fig. 4 Absorbance values for syringaldazine oxidation catalyzed by laccase (initial concentration 1:1 and dilution 1:2), at pH=5 and pH=6. 2,0

tg α = 0,4 ΔA/min

1,8

Absorbance (a.u.)

1,6 1,4 1,2 1,0 0,8 0,6 0,4 0,2 0

1

2

3

4

5

6

7

8

9

10

Time (min)

Fig. 5 Initial rate of syringaldazine oxidation reaction catalyzed by laccase, at pH = 5.

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Alina Manole, D. Herea, H. Chiriac, V. Melnig

Initial rate, v0 = 0.4 ΔA/min. To determine the concentration variation in time the Beer law is used. The cuvette dimension is 1 cm. A = εcl, then ΔA/min = εcl/min by rearranging is obtained ΔA/(minεl) = c/min. Is known that ε=65 mM−1cm−1 then concentration variation/minut = 0.4/ 65 mM/min = 0.6 10-2 mM/min = 0.6 10-2 μmoli/mL/min. The optimum pH values, for which the enzyme activity is maxim, are characteristic for each enzyme. In this pH optimum domain the proton acceptor and proton donor groups of the active centre are in ionized state necessary for the enzyme to be active. Outside this domain the binding of substrate is not possible and if the pH value exceeds o certain limit value the enzyme can be irreversible denaturized. This pH optimum depends on the environment composition, temperature and enzyme stability in acid and alkaline environment. The pH stability domain doesn’t necessary coincide with reaction rate optimum domain [6]. 0,115

Absorbance (a.u.)

0,110 0,105 0,100 0,095 0,090 0,085 0,080 3

4

5

6

7

8

pH

Fig. 6 Absorbance values for tannic acid oxidation catalyzed by laccase, function of pH (after 8 minute from the reaction start). 0,45 0,40

Absorbance (a.u.)

0,35 0,30 0,25 0,20 0,15 0,10 0,05

3

4

5

6

7

8

pH

Fig. 7 Absorbance values for vanillic acid oxidation catalyzed by laccase, function of pH (after 8 minute from the reaction start).

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Absorbance (a.u.)

0,30 0,25 0,20 0,15 0,10 0,05

3

4

5

6

7

8

pH

Fig. 8 Absorbance values for hydroquinone oxidation catalyzed by laccase, function of pH (after 8 minute from the reaction start). 2,0

Absorbance (a.u.)

1,5

1,0

0,5

0,0 3

4

5

6

7

8

pH

Fig. 9 Absorbance values for syringaldazine oxidation catalyzed by laccase, function of pH (after 8 minute from the reaction start). 0,120 0,118

Absorbance (u.a.)

0,116

hidroquinone vanillic acid tannic acid

0,114 0,112 0,110 0,108 0,106 0,104 0,102 0,100 0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

4,0

4,5

5,0

Time (min)

Fig. 10 Absorbance values for the three different aromatic compounds oxidation catalyzed by laccase, at pH=5.

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Alina Manole, D. Herea, H. Chiriac, V. Melnig

From the measurements done it can be observed that the optimum pH for syringaldazine oxidation and tannic acid respectively, by laccase, is 5 and, for hydroquinone and vanillic acid oxidation is 4. 4.

CONCLUSIONS

From the enzymatic assay of laccase, in case of using syringaldazine as substrate, according to the procedure indicated by the producer, resulted that laccase has an activity of 1,8 10-2 μmols/min. Also, by using the continuous spectrophotometric rate determination method the optimum pH determined for this oxidation reaction, catalyzed by laccase, at 25oC, is 5. In the case of using tannic acid as substrate the optimum pH is also 5, and for vanillic acid and hydroquinone is 4. From the measurements regarding laccase substrate specificity for the 4 different phenolic compounds (syringaldazine, hydroquinone, vanillic acid and tannic acid) can be observed that the enzyme is catalyzing the oxidation of all this compounds. Also, it can be observed that the activity of laccase presented in the case of syringaldazine oxidation is superior to that presented for the other 3 phenols.

REFERENCES 1. 2. 3. 4. 5. 6.

D.L. Nelson, M.M. Cox, Lehninger: Principles of Biochemistry, 2004. A. Ghindilis, Direct electron transfer catalysed by enzymes: application for biosensor development, Biochemical Society Transactions, Vol. 28, part 2, p. 84-89, 2000. Marièlle Bar, Kinetics and physico-chemical properties of white-rot fungal laccases, 2001 C.F. Thurston, The structure and function of fungal laccases, Microbiology, 140, p. 19-26, 1994. J.P. Ride, Physiological Plant Pathology, Vol. 16, p. 187-196, 1980. F. Xu, Effects of redox potential and hydroxide inhibition on the pH activity, profile of fungal laccases, The Journal of Biological Chemistry, 272 (10), p. 924-928, 1997.