Purification and characterization of the carbonic anhydrase enzyme from Black Sea trout (Salmo trutta Labrax Coruhensis) kidney and inhibition effects of some metal ions on enzyme activity

Accepted Manuscript Title: Purification and Characterization of The Carbonic Anhydrase Enzyme from Black Sea Trout (Salmo trutta Labrax Coruhensis) Kidney and Inhibition Effects of Some Metal Ions on Enzyme Activity Author: Murat Kucuk ˙Ilhami Gulcin PII: DOI: Reference:

S1382-6689(16)30088-6 http://dx.doi.org/doi:10.1016/j.etap.2016.04.011 ENVTOX 2497

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Environmental Toxicology and Pharmacology

Received date: Revised date: Accepted date:

11-9-2015 20-4-2016 23-4-2016

Please cite this article as: Kucuk, Murat, Gulcin, ˙Ilhami, Purification and Characterization of The Carbonic Anhydrase Enzyme from Black Sea Trout (Salmo trutta Labrax Coruhensis) Kidney and Inhibition Effects of Some Metal Ions on Enzyme Activity.Environmental Toxicology and Pharmacology http://dx.doi.org/10.1016/j.etap.2016.04.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Purification and Characterization of The Carbonic Anhydrase Enzyme from Black Sea Trout (Salmo trutta Labrax Coruhensis) Kidney and Inhibition Effects of Some Metal Ions on Enzyme Activity

Murat Kucuk1, İlhami Gulcin1,2*

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Atatürk University, Faculty of Sciences, Department of Chemistry, Erzurum-Turkey 2

King Saud University, College of Science, Department Zoology, Riyadh-Saudi Arabia

Address for Correspondence: Prof. Dr. İlhami GÜLÇİN Atatürk University Faculty of Sciences Department of Chemistry TR-25240-Erzurum-Turkey Phone : +90 442 2314375 Fax : +90 442 2314109 E-mails: [email protected] [email protected]

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Highlights  Carbonic anhydrase enzyme was purified from Salmo trutta Labrax Coruhensis  Enzyme was characterized.  SDS-PAGE was performed for enzyme purity and subunit.  The effect of some heavy metals was determined on CA activity.

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ABSTRACT In this study, the carbonic anhydrase (CA) enzyme was purified from Black Sea trout (Salmo trutta Labrax Coruhensis) kidney with a specific activity of 603.77 EU/mg and a yield of 35.5% using Sepharose-4B-L-tyrosine- sulphanilamide affinity column chromatography. For determining the enzyme purity and subunit molecular mass, sodiumdodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed and single band was observed. The molecular mass of subunit was found approximately 29.71 kDa. The optimum temperature, activation energy (Ea), activation enthalpy (∆H) and Q10 values were obtained from Arrhenius plot. Km and Vmax values for p-nitrophenyl acetate of the purified enzyme were calculated from Lineweaver-Burk graphs. In addition, the inhibitory effects of different heavy metal ions (Fe2+, Pb2+, Co2+, Ag+ and Cu2+) on Black Sea trout kidney tissue CA enzyme activities were investigated by using esterase method under in vitro conditions. The heavy metal concentrations inhibiting 50% of enzyme activity (IC50) were obtained. Finally Ki values and inhibition types were calculated from Lineweaver-Burk graphs. Keywords: Black sea trout, Salmo trutta Labrax, Enzyme purification, Enzyme characterization, Carbonic anhydrase, Heavy metal

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1. INTRODUCTION Carbonic anhydrase (CA, E.C.: 4.2.1.1) is a member of zinc containing metalloenzyme family that catalyses the rapid and reversible hydration of carbon dioxide (CO2) and dehydration of bicarbonate (HCO3-) (Gulcin et al., 2004; Arabacı et al., 2014; Akbaba et al., 2014a; 2014b). ⇔ This enzyme plays an important role in different processes including respiration, biosynthetic processes, controlling of physiological pH such as acid-base regulation, gas balance, bone resorption and calcification (Beydemir and Gulcin, 2004; Scozzafava et al., 2015; Akıncıoğlu et al., 2015). The CA was first isolated from mammalian erythrocytes. In the later years, the enzyme was purified from human erythrocytes, fish erythrocytes, rat erythrocytes, rat saliva, cattle bones, cattle leukocytes, some bacteria and plant sources and further that they were characterized in many sources. Molecular mass of enzyme was determined about 30 kDa in mammals (Beydemir et al., 2002). CA enzymes α-, β-, γ-, -, - and -CA have been examined in six different classes (Göksu et al., 2014; Yıldırım et al., 2015; Boztaş et al., 2015). Among them, α-CA is found in cytoplasm of green plants, bacteria, algae and vertebrates. Furthermore, the enzyme is found in tissues of fish (Coban et al., 2009; Göçer et al., 2015). Zinc ion is present in the active site of all enzyme families. Each of these families has similar catalytic function. Zn+2 ions are great importance of catalysis of reactions the CA enzymes (Güney et al., 2014; Akıncıoğlu et al., 2014; Çetinkaya et al., 2014a; 2014b). The active site of CA binds the hydroxyl group (-OH) on Zn2+ ions. The hydration direction, first, a zinc ion simplifies the proton forming hydroxide ions exit from the water molecules. Second, OH- attacks CO2 and is transformed to HCO3- ions. As seen in the Figure 1, in the next step, the catalytic region release HCO3- and is restored by the binding of another water molecule (Aksu et al., 2013; Topal et al., 2014). Figure 1 here CA enzyme contains Zn2+ ion in its structure like sorbitol dehydrogenase. Zn2+ insufficiency to cause in many metabolic defects and results are reduced production of Zn2+-containing enzymes. Decreasing the amount of CA causes metabolic imbalance and diseases (Akıncıoğlu et al., 2013). Generally heavy metals can change enzymatic activities by binding the functional groups, comprising the carbonyl,

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carboxyl and sulfhydryl or by substituting of the metal associated with the enzyme. Intense exposure to metal ions can cause different toxicological effects in fish and humans indirectly. The concentration of heavy metals in fish cause decreases the habits of the fish species (Sivaperumal and Sankar, 2006). The toxicological effects of heavy metals are usually enzyme inhibition and denaturation (Ekinci and Beydemir, 2010). Generally metal inhibition of enzyme is based on heavy metal binding to the protein. Therefore, human metabolism is affected by metal toxicity. Over consumption of fish causes a variety of diseases such as cancer, diabetes, Alzheimer and Parkinson diseases (Jomova and Valko, 2011). The CA plays a critical role on the respiration and transformation mechanism of CO2 to HCO3- in living species. Also it is known that there is a close connection between CA enzyme activity and oxygen consumption (Öztürk Sarıkaya et al., 2011; Gülçin and Beydemir, 2013). Reduction in the rate of oxygen is a vital stress factor on fish. Similarly, metal inhibition on CA enzyme activity results in a reduction in the rate of oxygen consumption and increases the stress (Şentürk et al., 2011; Innocenti et al., 2010a; Beydemir et al., 2011). Enzyme inhibitions are vital for the metabolisms of all living species. Almost all drugs and most of chemicals including heavy metals display their functions on enzyme interaction mechanism (Öztürk Sarıkaya et al., 2010; Innocenti et al., 2010b). Furthermore, heavy metals are effective to be some of the strongest naturally occurring CA inhibitors. It is known that Ag+ ions inhibit both gills CA enzyme activity and ion transport in freshwater fish (Soyüt et al., 2012). In this study, we investigated the toxicological effects of some heavy metal ions, including Fe2+, Pb2+, Co2+, Ag+ and Cu2+, on the CA enzyme purified from the kidney of Black Sea trout (Salmo trutta Labrax Coruhensis) using the esterase method under in vitro conditions.

2. EXPERIMENTAL 2.1. Chemicals Pb(CH3COO)2, FeCl2, CoCl2, AgNO3 and CuSO4.5H2O, p-nitrophenyl acetate, CNBractivated-Sepharose-4B and protein assay reagent were obtained from Sigma-Aldrich Co. (GmbH, Germany). All other chemicals were analytical grade and purchased from Merck (Germany).

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2.2. Black Sea trout (Salmo trutta Labrax Coruhensis) Black Sea trout (Salmo trutta Labrax Coruhensis) were obtained from a fish farm in the Rize province in the Black Sea cost of Turkey.

2.3.Purification of Carbonic Anhydrase Kidney tissues were removed from fresh trout and were washed 3 times with 0.9% NaCI isotonic saline solution. Kidney tissues were lysed by liquid nitrogen at 10.000×g for 30 minutes. Then the samples were centrifuged at medium speed (2.000×g). Precipitate and supernatant were separated from each other. Finally the supernatant sample was transferred to a buffer solution containing Tris- HCl/Na2SO4 (25 mM/0.1 M) at pH 8.7 for using in kinetic studies (Söyüt and Beydemir, 2008; Şentürk et al., 2009). The pH-adjusted homogenate was loaded to the Sepharose-4B-L tyrosine-sulphanilamide affinity column and was washed with Na2SO4 (22 mM), in Tris-HCl buffer solution (25 mM, pH 8.7). The enzyme, after binding to the column was eluted by NaCIO4/NaCH3COO (0.5 M / 0.1 M, pH 5.6) with column flow rate 20 mL/h. All procedures were performed at 4°C (Atasever et al., 2013).

2.4.Determintion of CA activity CA activity was determined by absorbance changing at 348 nm of p-nitrophenyl acetate to p-nitrophenolate over a period of 3 minutes at 25 °C using a spectrophotometer (Topal and Gülçin, 2014). The enzymatic reaction performed containing 0.4 mL of Tris-SO4 buffer solutions (0.05 M, pH 7.4), 0.36 mL pnitrophenyl acetate (3 mM), 0.22 mL water and 0.2 mL of enzyme solution in total volume of 1 mL. Enzyme solution was not added to the control sample.

2.5.Determintion of Qualitative Protein The protein elution was recorded at 280 nm. Finally, CO2-hydratase activity was determined according to the method previously described (Çoban et al., 2007; 2008).

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2.6. SDS-PAGE Study SDS-PAGE was performed after purification of the enzyme according to Laemmle’s procedure (1970), which was described previously (Gülçin et al., 2005; Beydemir et al., 2005). The running and stacking gels contained 0.1% SDS and 10 and 3% acrylamide, respectively. The electrode buffer contained Tris-glycine (0.25 M/ 2 M, pH 8.3). The buffer solution was prepared 0.65 mL Tris-HCl (1M pH 6.8), 3 mL SDS (%10), 1 mL glycerol, 1 mL bromophenol blue (0.1%), 0.5 mL β-mercaptoethanol and 3.85 mL water by mixing. A portion of sample (20 µg) was applied in 50 µL buffer solutions. The mixture was heated in a boiling water bath for 5 minutes. The purified enzyme samples were loaded into each space of stacking gel. Firstly, an electric potential of 80 V was applied until bromophenol blue reached the running gel. Then it was increased to 200 V over 3 hours. Then, gel was kept in 0.1% Coomassie Brilliant blue R-250 reagent in 50% methyl alcohol and 10% acetic acid and distained with acetic acid for 90 minutes until its surface becomes limpid.

2.7.Determintion of Quantitative Protein The protein quantity was determined spectrophotometrically at 595 nm during the purification steps. Serum albumin was used as the standard protein (Bradford, 1976) as described previously (Öztürk Sarikaya et al., 2015; Aydın et al., 2015).

2.8.Kinetic Studies 2.8.1.Optimum and Stable pHs For determination of optimum pH, CA activity was measured in 1 M Tris-SO4 buffers ranging from pH 7.0 to 9.0 and 1 M phosphate buffer ranging from pH 5.0 to 8.0. Also for determination of stable pH, the CA enzyme activity was measured in 1.0 M Tris-SO4 buffers pH 7.0 to 9.0 and 1 M phosphate buffer pH 5.0 to 8.0. The activity measurements were performed at 12 hours period during 5 days incubation using pnitrophenyl acetate as the substrate under optimum conditions.

2.8.2.Optimum ionic strength To determine the optimum ionic strength of the enzyme activities was used different concentrations of Tris-SO4 buffer (pH 9.0) ranging from 0.1 M to 1.0 M.

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2.8.3. Optimum temperature, activation enthalpy (∆H), activation energy (Ea) and Q10 The Black sea trout kidney CA enzyme activities were measured at different temperature with an increment of 10 °C ranging from 0 to 80 °C for determination the optimum temperature, activation enthalpy (∆H), activation energy (Ea) and Q10 values. Firstly to provide different temperature from 0 to 20 °C, we used an ice water bath. Second, to measure different temperatures above 20 °C a constant temperature was used. All measurements of enzyme activities was added an Arrhenius plot of the log k- 1/T to determine the optimum temperature, activation enthalpy, activation energy and Q10 values (Söyüt and Beydemir, 2012).

2.8.4. Determination of Km and Vmax values To determine kinetic studies the Km and Vmax values were used at five different concentrations of 4-nitrophenylacetate (0.15-0.75 mM) with optimum pH at 25 °C. The activities of enzymes were measured by a spectrophotometric at 348 nm. Then the Km and Vmax values were determined from a Lineweaver-Burk plot of the kinetic data. Kcat value is turnover rate of enzyme. kcat was calculated from the equation Vmax / ET that ET is total enzyme and V0 value is a specificity constant of enzyme activity and was calculated kcat /Km (Söyüt and Beydemir, 2012).

2.8.5. Inhibition Studies The inhibitory effects of iron, lead, cobalt, silver and copper metals were evaluated on CA enzyme activity purified from black sea trout kidney tissue. To determine effects of heavy metals five different substrate concentrations were used for each metal. The enzyme activities were measured for concentrations of Fe2+ ions (0.2-1.0 mM), Pb2+ ions (0.05-0.3 mM), Co2+ ions (1.0-3.0 mM), Ag+ ions (25.0-90.0 mM), Cu2+ (20-50 mM) in 1 mL cuvette. The control of enzyme activity without of inhibitor was set at %100 on chart. An enzyme activity with concentration of inhibitor was plotted for each inhibitor. Reduced 50% activity of enzyme used metal concentration (IC50) was calculated from the graphs. Ki values were calculated with determined three different inhibitor concentrations for each metal ion: 0.6, 0.8 and 1.0 mM for Fe2+, 0.10, 0.15 and 0.20 mM for Pb2+, 1.5, 2.0 and 2.5 mM for Co2+, 65,75 and 90 mM for Ag+ and 20, 25 and 30 mM for Cu2+. In all experiments, PNF was used as the substrate at the 8

five different concentrations (0.15, 0.30, 0.45, 0.60 and 0.75 mM) for each metal ion. For determination of Ki values, type of inhibition was used in Lineweaver-Burk plots (1934). This assay explained in a previous study (Oztaşkın et al. 2015).

3. RESULTS and DISCUSSION CA catalyses the interconversion of CO2 and H2O to HCO3- and H+ in metabolism. In the present study, carbonic anhydrase enzyme was purified and characterized from the black sea trout kidney tissues using Sepharose-4B-L tyrosine-sulphanilamide affinity column. The enzyme was purified with a specific activity of 603.77 EU/mg and approximately 349-fold with a yield of 35.55% (Table 1). The results of studies conducted on CA from Black Sea trout (Salmo trutta Labrax Coruhensis) kidney are shown in Figure 2. The purity and subunit molecule mass of the CA enzyme were determined using the SDS-PAGE and a single band was absorbed. The subunit molecular mass of the enzyme was calculated as approximately 29.71 kDa (Figure 3). The molecular mass of enzyme was found to be approximately 29.7 kDa. The obtained molecular mass was similar to CAs purified from many other tissues. For instance, flounder (Platichthys flesus) gills CA 29 kDa (Sender et al., 1999), zebrafish (Danio rerio) erythrocyte CA 29 kDa (Peterson et al., 1997), Antarctica icefish (Chinodraco hamatus) gills CA 28 kDa (Rizzello et al., 2007), rainbow trout (Oncorhynchus mykiss) liver CA 29.4 kDa and sea bream (Sparus aurata) gills CA 30.5 kDa (Kaya et al., 2013) were determined respectively. The kinetic study for Km, Vmax, kcat and V0 values were determined using pnitrophenyl acetate substrate. Also, optimum temperature, Ea, ∆H and Q10 results were obtained similarly to other previous study in literature (Kaya et al., 2013). Optimum pH (Figure 4) and stable pH (Figure 5) were found to be 9.0 and 8.5 respectively in 1M Tris-SO4 for p-nitrophenyl acetate substrate of CA enzyme. Also optimum temperature, ∆H, Ea and Q10 values were determined 40 °C, 1.730 kcal/mol, 2.356 kcal/mol and 1.83 (Figure 6), respectively. Also, the other kinetic parameters, such as Km, Vmax and kcat were obtained 0.434 mM, 0.547 EU/mL and 688.05 (Figure 7), respectively. Furthermore, Vo is same of enzyme activity, was found 1.585×106 mM× s-1 for the first time in this study (Table 2). The effects of heavy metals on CA enzyme from Black Sea trout kidney were determined as millimolar levels. Ki values are ranging from 0.19 mM to 74.80 mM. Specifically, heavy metals from factories and other technological requirements have 9

various effects on humans, animals and other living organisms, because they bind to membrane transport ligands and can alter their catalytic functions (Rainbow and Dallinger, 1993). In this study, we measured the in vitro inhibition effects of Fe2+, Pb2+, Co2+, Ag+ and Cu2+ ions on black sea trout kidney. The half maximal inhibitory concentration (IC50) is a measurement of the effectiveness of an inhibitor in inhibiting a specific biochemical function. Also, this value represents the heavy metal concentration required for obtaining 50% of a maximum effect in vivo (Topal and Gülçin, 2015). In this study, IC50 values were calculated as 0.78, 0.13, 2.07, 19.17 and 76.67 mM for Fe2+, Pb2+, Co2+, Ag+ and Cu2+, respectively (Table 3). On the other hand, the inhibition constants (Ki) were calculates as 0.12, 0.19, 2.04, 21.43 and 74.80 mM, respectively. These values and theirs inhibition types were calculated from Lineweaver-Burk graphs (Table 3).

4. CONCLUSIONS CA enzyme was firstly purified from Black Sea trout (Salmo trutta Labrax Coruhensis) kidney. Its molecular mass was determined using SDS-PAGE method. To determine kinetic parameters p-nitrophenyl acetate was used as substrate. The optimum temperature, Ea, ∆H and Q10 were obtained from Arrhenius plot. Km, Vmax, kcat and V0 kinetic values were measured and IC50, Ki and type of inhibitions were determined from Lineweaver-Burk graphs. Heavy metals had adverse effects on CA enzyme was purified from Black Sea trout (Salmo trutta Labrax Coruhensis) kidney. However, the most hazardous metal was found as Pb2+ with Ki value of 0.185 mM. These heavy metals can cause different toxicological effects in fish.

Conflicts of Interest The authors declare no conflict of interest.

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flounders

Platichthys

flesus

purification,characterization, and immunohisto chemical localization. J. Histochem. Cytochem. 47, 43-50. Şentürk, M., Gülçin, İ., Beydemir, Ş., Küfrevioğlu Ö.İ., Supuran C.T., 2011. In vitro inhibition of human carbonic anhydrase I and II isozymes with natural phenolic compounds. Chem. Biol. Drug Des. 77: 494-499. Şentürk, M., Gülçin, İ., Daştan A., Küfrevioğlu, Ö.İ., Supuran C.T., 2009. Carbonic anhydrase inhibitors. Inhibition of human erythrocyte isozymes I and II with a series of antioxidant phenols. Bioorg. Med. Chem. 17, 3207-3211. Sivaperumal, T.V., Sankar, P.G., 2006. Heavy metal concentrations in fish, shellfish and fish products from internal markets of India vis-a-vis international standards. Food Chem. 102, 612-620. Soyut, H., Beydemir, S., 2008. Purification and some kinetic properties of carbonic anhydrase from rainbow trout (Oncorhynchus mykiss) liver and metal inhibition. Protein Peptide Lett. 15, 528–535. Soyut, H., Beydemir, S., 2012. The impact of heavy metals on the activity of carbonic anhydrase from Rainbow trout (Oncorhynchus mykiss) kidney. Toxicol. Ind. Health 28, 296–305. Soyut, H., Beydemir, S., Ceyhun, B.S., Erdogan, O., Kaya, E.D., 2012. Changes in carbonic anhydrase activity and gene expression of Hsp70 in rainbow trout (Oncorhynchus mykiss) muscle after exposure to some metals. Turk. J. Vet. Anim. Sci. 36: 499-508. Supuran CT., Scozzafava A., 2001. Carbonic anhydrase inhibitors. Curr Med Chem. 1, 61-97. Topal, M., Gülçin, İ., 2014. Rosmarinic acid: a potent carbonic anhydrase isoenzymes inhibitor. Turk. J. Chem. 38: 894-902. Yıldırım, A., Atmaca, U., Keskin, A., Topal, M., Çelik, M., Gülçin, İ., Supuran, C.T., 2015. N-Acylsulfonamides strongly inhibit human carbonic anhydrase isoenzymes I and II. Bioorg. Med. Chem. 23, 2598-2605.

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Table 1. Summary of purification procedure of CA enzyme from Black Sea trout (Salmo trutta Labrax Coruhensis) kidney Purification step Homogenate Sepharose-4B-Ltyrosinesulfanilamide affinity chromatography

Activity (EU/mL)

Total volume (mL)

Protein (mg/mL)

Total protein (mg)

Total activity (EU/mL)

Specific activity (EU/mg)

Yield (%)

Purification factor

60

22.5

34.55

777.4

1350

1.73

100

1.00

160

3.0

0.265

0.795

480

603.77

35.55

349.00

15

Table 2. Kinetic parameters of CA enzyme from Black Sea trout (Salmo trutta Labrax Coruhensis) kidney using by p-nitrophenylacetate as substrate

Kinetic parameters

Values

Optimum pH (1.0 M Tris-SO4 buffer)

9.0

Optimum ionic strength (M, Tris-SO4 buffer)

1.0

o

Optimum temperature ( C)

40.0

Stable pH (1.0 M Phosphate buffer)

8.5

Ea (kcal/mol)

2.356

∆H (kcal/mol)

1.730

Q10

1.830

KM (mM)

0.434

Vmax (EU/mL)

0.547

-1

kcat (s )

688.05

V0 (mM×s-1)

1.585×106

Molecular mass (kDa)

29.71

16

Table 3. IC50 and Ki values and inhibition types of some heavy metal carbonic anhydrase obtained from Black Sea trout (Salmo trutta Labrax Coruhensis) kidney r2

Metal ions

IC50 (mM)

Fe2+

0.78

0.9813

0.7031 ± 0.200

Competitive

Pb2+

0.13

0.9837

0.1848 ± 0.072

Uncompetitive

Co2+

2.07

0.9612

1.6791 ± 0.760

Competitive

Cu2+

76.67

0.9353

48.812 ± 4.413

Competitive

Ag+

19.17

0.9353

18.547 ± 1.727

Uncompetitive

17

Ki (mM)

Inhibition type

FIGURE LEGENDS Figure 1. Catalytic and inhibition mechanism of carbonic anhydrase Figure 2. The results of studies conducted on CA from Black Sea trout (Salmo trutta Labrax Coruhensis) kidney Figure 3. Standard Rf–Log Mw graph molecular weight of CA from Black Sea trout (Salmo trutta Labrax Coruhensis) kidney using the SDS-PAGE results Figure 4. Determination of optimum pH for from Black Sea trout (Salmo trutta Labrax Coruhensis) kidney in 1 M phosphate and 1 M Tris-SO4 buffers Figure 5. Determination of stable pH graph of from Black Sea trout (Salmo trutta Labrax Coruhensis) kidney for five days Figure 6. The effect of temperature on CA enzyme activity from Black Sea trout (Salmo trutta Labrax Coruhensis) kidney Figure 7. Determination of Lineweaver-Burk graph for CA enzyme from Black Sea trout (Salmo trutta Labrax Coruhensis) kidney at five different concentrations of p-nitrophenylacetate substrate

18

InhInh-

OH2

Zn2+

Zn2+ His94

His119

His94

His119 His96

His96

H 2O 6

5

InhH H

H H+

O Zn2+ His94

His119

H O Zn2+

1

His94

His96

His119 His96

HCO3-

CO2 2

4 H 2O O H

O

O

O

His94

Zn2+

His119 His94

His96

Figure 1

19

C O

3 Zn2+

O

H

C

His119 His96

Enzyme Purification -Sepharose-4B-L-tyrosine- sulphanilamide affinity chromatography -SDS-PAGE electrophoresis determination -Subunit molecular mass calculation

Enzyme Kinetics - Optimum pH - Optimum temperature - Optimum ionic strenght - Activation energy (Ea) - Activation enthalpy - Q 10 values - Km values - V max values

Enzyme Inhibition -Inhibition effect ofheavy metals -Half maximal inhibitory concentration (IC 50) -Inhibition constant (Ki)

Figure 2

2.5

Log Mw

2.0 1.5 1.0 0.5 0.0

0.2

0.4 Rf

Figure 3

20

0.6

0.8

1.0

0.6 Phospate buffer (1M) Tris-SO4 (1 M)

0.4 0.3 0.2 0.1 0 5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

9.0

9.5

pH

Figure 4

0.4 0.3 Activity (EU/mL)

Activity (EU/mL)

0.5

0.2

pH=5.0 pH=5,5 pH=6.0 pH=6,5 pH=7.0 pH=7,5 pH=8.0

0.1 0.0 1.0

2.0

3.0 Time (Day)

Figure 5

21

4.0

5.0

Figure 6

8.0

1/V (EU/mL)-1

6.0

4.0

2.0

-3.0

0.0 -1.0

1.0

3.0

1/[S] (mM)-1 Figure 7

22

5.0

7.0