Blue Copper Peroxidase and Phthalocyanine Conjugate: Synthesis

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Blue Copper Peroksidase and Phthalocyanine Conjugate: Synthesis, Characterization and Applications Meltem Göksel Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00861 • Publication Date (Web): 08 Jan 2019 Downloaded from http://pubs.acs.org on January 9, 2019

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Bioconjugate Chemistry

1 1 2 3 4 5

Blue Copper Peroxidase and Phthalocyanine Conjugate: Synthesis, Characterization and Applications Meltem Göksela,b

6 7 8

a b

Kocaeli University, Kosekoy Vocational School, PO Box 141, Kartepe, Kocaeli, 41135, Turkey

Gebze Institute of Technology, Department of Chemistry, PO Box 141, Gebze, 41400, Kocaeli, Turkey

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Abstract. Trametes versicolor can degrade barks as a source for carbon necessity. Therefore it secretes lignin peroxidase, mangan peroxidase and laccase. The laccase enzyme was produced high yield at pH and glucose concentration 5 and 10 gL-1, respectively. In optimized medium, the enzyme activity was between 200-250 UL-1 when inducer was absent. It was seen that the activity reached 400 UL-1 when phenol used as an inducer. The molecular weight of purified laccase was found 80 kDa with SDS-PAGE and kinetic constant Km and Vmax values for ABTS were determined 3.66x10-4 μM and 1652 UL-1, respectively. Hence, due to these properties, these enzymes are widely used in industrial areas free or immolized. Laccase enzyme decolorization of 6 different dyes was carried out. A decolorization capacity of 50-99% was achieved by cultivation in 20 days using a beginning dye concentration of 20 ppm. The removal of color with active enzyme is obtained around 90%. Also laccase enzyme was conjugated amine functionalized low symmetrically phthalocyanine. This conjugate was examined both photodynamic therapy and chemosensor application. This conjugate fluorescence had a quantum yield of 0.32 (lifetime 3.59 ns) and generates efficiently singlet oxygen (quantum yield 0.4). The conjugate was successfully photodamage displayed in HeLa and HuH-7 cells in photodynamic therapy application. These results indicate that conjugate represent interesting agent with potential applications in photodynamic therapy. In addition that the chemosensor behavior of this compound to different metal ions has been studied and this conjugate display as fluorescence chemosensor for determination of Fe3+ions.

31 32 33

Keywords: Laccase, Zinc phthalocyanine, Metal Sensor, Conjugate, Photodynamic Therapy.

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Introduction.

2 3 4 5 6 7 8 9 10 11 12

White rot fungi can completely degrading lignin and various aromatic compounds. Most commonly studied basidiomycetes Trametes versicolor and Phanerochaete chrysosporium among the white rot fungi. These basidiomycetes cultered under suitable conditions produced several extracellular oxidoreductases and activated oxygen species1,2. Laccase (EC 1.10.3.2, oxygen reductase) is the best known enzyme. Laccases are monomeric glycoproteins, which haveapproximate 500 aminoacids with molecular weight around 60-85 kDa3. This enzyme was first characterized by Yoshida from Rhus vernifera tree5. Laccases catalyze the oxidation of different substrates including phenols, methoxy substituted phenols, and inorganic compounds6, 7.It contains “blue” copper sites that mononuclear cupper site (T1 centre), trinuclear cupper sites (T2/T3) comprising one-type 2 cupper and two-type 3 cuppers centre. These different cupper sites have been classified according to their optical, and EPR spectra 3.

13 14 15 16 17 18 19 20 21 22 23 24 25

The white-rot fungi use to degrade lignin and related compounds. Enzyme degradation mechanism is both non-specific and a radical-free process. These enzymes can be catalyzing of oxidation and reduction reactions, besides to produce reactive oxygen species (ROS). These fungi used nonspecific mechanisms so degrade a wide range of pollutant matterials such as lignin and its derivatives. Synthetic dyes widely used in the pharmaceutical, textile, cosmetics, and food industries. Especially azo-dyes which is at least one azo bonds (-N-N-) in aromatic rings. In addition azo-dyes also carry sulfonic acid groups so using these synthetic dyes cause carcinogenic pollutant4. Use of synthetic dyes causes pollution of wastewater, so decolorization of wastewaters is important because of toxicity and/or carcinogenicity. The classic wastewater treatment systems are not fully efficient for decolorization. White rot fungi characteristic possess that capability to degrade various compounds including dyes. White rot fungi produce extracellular ligninolytic enzymes such as lignin peroxidase, laccase and manganese peroxidase (MnP). These enzymes are managing for the degradation of pollutants8.

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Conventional cancer therapies including chemotherapy and radiotherapy have many side effects or drug resistance. Phototherapy is a class of non-invasive therapeutic techniques with many advantages such as selectivity, non-toxicity and controllability. Photodynamic therapy (PDT) is phototherapy method. PDT includes three key materials: light, photosensitizer molecule and molecular oxygen9. Under the appropriate light irradiation, the absorbed energy on the photosensitizers is transferred to molecular oxygen, and so, cytotoxic singlet oxygen (1O2) or reactive oxygen species (ROS) generates. Thus cancer cells are killed. Ideal phototherapy agents (photosensitizer) should exhibit minimal dark toxicity and are effective in cancer destruction under light exposure, via either photothermal or photodynamic mechanisms. Light penetration is one of major challenges in phototherapy. The NIR window (700-1000 nm) is minimal absorbed by biological tissues. This is ideal for phototherapy applications9.

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Chemosensors are portable and cheap10. Fluorescent sensors, are chemosensor subunit, use in the biological application such as probes, environmental sensors, molecular devices, nerve gas sensor, etc11-13. Fe3+ which involve important role in human and animal supports many biological pathways. It used as an oxygen-carrying in heme, and a cofactor in enzymatic reactions. In addition that high level of Fe3+ ions can be toxic for lipids, proteins and other cellular components via oxidation. On the other hand low level of of Fe3+ causes diabetes, anemia, liver damage, and cancer14.


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3 1 2 3 4

This study include five section: A) Laccase purification and characterization, B)Decolorization potention of laccase, (C) Preparation and characterization of new phthalocyanine-laccase bioconjugate, (D) Efficacy of chemosensor based on bioconjugate, and (E) Potention of bioconjugate for PDT applications.

5 6 7 8 9 10

Results and Discussions A. Laccase purification and characterization: Organism and culture conditions for laccase production

11 12 13

White rot fungus Trametes versicolor, the contents in Table 1 in the liquid medium, for 12 days at 130 rpm and incubated at 32oC. Laccase activity in the medium was monitored and the maximum laccase activity was found to be between 4 to 8 days (Fig 1).

14 15 16 17 18

The growth curves were made in 250 mL erlenmeyer flasks with 100 mL of liquid culture medium. Modified medium15 with 1 gL-1 of glucose and 50 mLL-1 TES were used. The liquid cultures were incubated at temperatures of 32°C in a rotatory shaker at 130 rpm. Inoculation was made with 0.5 cm x 0.5 cm of mycelium previously growth at 28°C in petri plates on malt extract agar during one week.

19 20 21

Figure1. Time course of laccase production by T. Versicolor during growth in modified medium15.

Optimization of modified medium

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Trace elements, phases of growth and reproduction of microorganisms because it has a significant effect on the available medium for growth of the fungus and enzyme production is necessary. In the literature, which made quite busy working groups Tien & Kirk in the 1984 publication stated that they made a few changes in trace element solution was used23. Examined the effect of glucose on the production of laccase, the presence of the excess production of the enzyme has an impact on the slowing down is indicated in the literature24. The validity of this condition and whether there is any interaction with trace elements to understand the fungi, the contents of Table 2 given in eight different media cultivated in different concentrations of glucose and trace elements were tested.

31 32

The maximum enzyme production at low glucose (1 gL-1) and trace elements (50 mLL-1) containing culture medium is observed at medium 4. In addition, the lowest enzyme production was



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4 1 2 3 4

observed in the medium 2 that maximum of glucose (5 gL-1) and absence of trace elements (Fig 2). In A relatively early study by Yu and Wong, parallel to our results found Trametes versicolor fungus will grow slowly at low glucose concentrations, although it is stated that the secretion of the maximum level of laccase24.

5 6 7 8 9

As a result, in the presence of trace elements than cellulose has been found to have a significant effect on enzyme production. Consequently, some metals present in trace elements may inhibit the enzyme production, increasing concentrations of trace elements, reduced enzyme production. The absence of trace elements, fungus and enzyme production vital activities are adversely affected.

10 11

Figure 2. The effects both TES and glucose concentration on the laccase production.

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Studied media which contain malt extract broth (0.2%) in KH2PO4 (50mM, pH:4) buffer were fixed. This media was diversifying via adding different glucose and TES amounts. The effect of the enzyme production glucose and TES in two day intervals in samples were determined by activity assay.

16 17 18 19 20 21 22 23 24

Effect of pH on laccase activity The effect of pH on the laccase production although it is not so much information, fungi, when grown in media with a pH 5 was considerable laccase production was reported19. In this study, effect of pH on Trametes versicolor laccase production was examined over a wide range such as 3.5-7.0. The effect of pH was expressed as the relative laccase activity measured by the ABTS assay method described above. Laccase activity in the sample taken from Trametes versicolor grown in medium at different pH values over 10 days was shown in Figure 3. Maximum activity was seen on day 4 and the maximum activity seen on pH 5, too. In addition, minimum laccase activity is seen on pH 3.5, 6.5, and 7.0.


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5

1 2

Figure 3. The effect of pH on enzyme production. Higher activity was observed at the pH 5.0.

3

Purification of the laccase enzyme from the culture supernatant

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Laccase purification from the supernatant were performed both precipitation and ion exchange chromatography.

Purification with Ammonium Sulphate precipitation As seen from the figure 4, the percentage of ammonium sulfate required for laccase precipitation is ≥ 70. As a result of the calculations made at 85% saturation of ammonium sulfate was used.

10 11 12

Figure 4. Enzyme precipitation by Ammonium Sulfate precipitation method. The higher activity enzyme was obtained at 85% Ammonium Sulfate.

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Precipitated protein was dissolved in a small amount buffer and was loaded on DEAEsepharose column. It was collected in 1.5 mL fractions and determined the enzyme activity. Fraction 2 including maximum laccase activity. In addition, fraction 1 and fraction 3 have activity, too. Fractions presence of both protein and laccase activity were observed in figure 5. Proteins bound to the column matrix, so that in the first three fractions thus obtained were concentrated.



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Figure 5. Elution of ion-exchange chromatography of Laccase. The ammonium sulphate precipitate was chromatographed on DEAE-Sepharose. Total protein was monitored at 280 nm. The fractions were assayed for enzyme activity. Fraction no. 1 had highest specific activity.

5 6 7

Concentrated protein fractions were performed SDS-PAGE analysis. The purified laccase protein appeared as a single band in SDS-PAGE analysis (Fig. 6). SDS-PAGE analysis of the fractions that are high compared to ̴90% was purified laccase enzyme.

8 9 10 11

Figure 6. SDS-PAGE of the concentrated protein fractions. Molecular mass markers are shown in the left lane. (30 µL of each fraction were loaded. Standard proteins (SP): lane1, fraction1:lane3, fraction2: lane4, fraction3: lane5).

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Protein standards molecular weight calibration curve was obtained by SDS-PAGE. The purified laccase molecular weight determination is calculated using this curve. The molecular mass of the denatured laccase was determined to be 78.6 kDa and did not have any isozyme (Fig 7). The distance of 28 mm on a graph obtained from the replacement value of the molecular weight of the purified enzyme is determined. The travel distance of proteins against the logarithm of the molecular weight protein standards plotted to determine the molecular weight of the weight of the equation want to calculate.

19 20 21 22

Figure 7. Protein standards molecular weight calibration curve. Molecular Weight Marker (M.W. 30,000– 200,000).

23


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7 1 2

Purification by ammonium sulphate precipitation steps of laccase are summarized in Table 3. Accordingly, 19.2% yield laccase enzyme was purified about 3 times.

3 4

Table 3. Laccase purification by ammonium sulphate precipitation. Total Volume

Total Protein

Tatal Activity

Spesific Activity

(mL)

(mg)

(U)

(Umg-1)

Culture supernatant

100

1.981

25674

Ammonium sulphate

4

0.545

Dialysis

6

DEAE-sepharose

2.7

Purification Step

Yield Purification (%)

(fold)

12960

100

1

8941

16405

34.8

1.3

0.256

5018

19600

19.5

1.5

0.128

4733

36974

19.2

2.9

5

Purification with ion-exchange chromatography method

6 7 8 9 10 11

The protein in the supernatant removed from the mycelium, any precipitation process without being subjected to DEAE-sepharose column and 1.5 mL fractions were collected. Determination of the activity of collected fractions and proteins presence were shown in Figure 8. In this method, because the sample loaded on the column was dilute, fraction was quite numerous. Although the number of fractions with enzyme activity is high, their protein content is low.

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As shown in figure 8(a), fractions between 5 and 22 were obtained from anion exchange column with enzymatic activity. These fractions were collected and then to obtain concentrated enzyme solution was loaded onto the anion exchange column. Figure 8(b), the second fraction obtained from anion exchange column appears to be more concentrated in the protein solution. The number of fractions containing laccase activity was six.

17 (a)

(b)

18 19 20

Figure 8. Anion exchange chromatography purification of laccase from Trametes versicolor (a) 1st step, (b) 2nd step.

21



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Concentrated protein solution, was obtained from the supernatant separated from the mycelium after the anion exchange column, had six fractions. The purified laccase fractions appeared as a single band in SDS-PAGE analysis (Fig. 9).

4 5 6

Protein standards molecular weight calibration curve was obtained by SDS-PAGE. The purified laccase molecular weight determination is calculated using this curve. The molecular mass of the denatured laccase was determined to be 82.5 kDa and did not have any isozyme (Fig 9).

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Figure 9. SDS-PAGE of the concentrated fractions without precipitation of the supernatant. Molecular mass

11 12 13 14

Figure 10, the distance of 25mm on a graph obtained from the replacement value of the molecular weight of the purified enzyme is determined. The travel distance of proteins against the logarithm of the molecular weight protein standards plotted to determine the molecular weight of the weight of the equation we want to calculate.

markers are shown in the left lane. (30 µL of each fraction were loaded. Standard proteins (Std): lane1, fraction1:lane2, fraction2: lane3, fraction3: lane4, fraction4: lane5, fraction5: lane6, fraction6: lane7).

15 16

17 18 19

Figure 10. Protein standards molecular weight calibration curve. Molecular Weight Marker (M.W. 30,000– 200,000).

20 21 22

The laccase of the supernatant is applied to the without precipitation purification process steps in Table 4 are summarized. Accordingly, the laccase enzyme is purified in 27% yield was about 6 times.

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9 1

Table 4. Purification without precipitation of laccase. Total Volume

Total Protein

Tatal Activity

Spesific Activity

(mL)

(mg)

(U)

(Umg-1)

Culture supernatant

100

1.981

25674

DEAE-Sepharose I

30

0.265

DEAE-Sepharose II

10

0.092

Purification Step

2 3

Yield

Purification

(%)

(fold)

12960

100

1

14347

54140

55.9

4.2

6942

75456

27.0

5.8

Consequently, as a result of comparative purification as seen in the table 4, of the highest purification constant was achieved without precipitation purification methods.

4 5

Characterization of purified Laccase:

6

Partial characterization of the purified laccase with DEAE-Sepharose is carried out as follows:

7 8 9 10 11

Effect of pH on purified laccase activity pH for the enzyme to be effective in the active site, so in the enzyme characterization is one of the parameters to be studied. During the experimental studies, activity assay using ABTS as a substrate and the highest activity when the pH optimum of laccase 3-4 was prepared in phosphate buffer solution (Fig. 11).

12 13

Figure 11. Effect of pH on enzyme activity.

14 15 16 17 18 19

Effect of temperature on purified laccase activity The effect of temperature on laccase activity was determined with ABTS at 30, 35, 40, 45, 50, 55, 60, 65 and 70°C. The effects of temperature were expressed as the relative laccase activity measured by the ABTS assay method described above. Enzyme activity is increasing with increasing temperature until 65oC (Fig. 12). It was found that the enzyme is denatured at 70 oC.



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10

1 2

Figure 12. Effect of Temperature on enzyme activity. Higher activity was observed at 65°C

3 4 5 6

Progres curve Substrate was turned into products by laccase enzyme maximum activity was examined the rate of change with time it was found that 60 minutes (Fig. 13).

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Figure 13. Time Course Study to determine the maximum laccase production.

Determination of kinetic datum Kinetic studies were performed by measuring the initial velocity in 1 mL glass cuvette with 1 cm path length, and the velocities of enzyme-catalyzed reactions were measured at 420 nm for ABTS. ABTS laccase activity is a substrate commonly used in the determination. In this study, we purify our ABTS laccase enzyme kinetic parameters aimed to compare themselves with literature values. Kinetic experiments performed in the ABTS concentration was varied between 0.04 and 0.83 mm. The Michaelis-Menten plot obtained from kinetic experiments was shown in fig.14. Figure 15 was observed the Lineweaver-Burk plot. As a result of the calculations Vmax = 1652 UL-1 and Km = 3.66 x 104 M, respectively (Figure 14 and figure 15).


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11

1 2

Figure 14. Laccase from Trametes versicolor Michaelis-Menten plot.

3 4

Figure 15. Trametes versicolor laccase Lineweaver-Burk plot.

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B. Decolorization efficiency of laccase Uninoculated controls (static and agitated) not showed colour change in the medium. Decolourization of the dye derivatives in the medium could be either adsorption on the fungal biomass or biodegradation. Azo dye derivatives were important synthetic colorants; they are very recalcitrant to biodegradation. In this study, these dyes were made to degrade with microorganisms. The organisms were efficient in secretion of laccase. Degrading the azo dyes are Remazol Red, Remazol Blue, Remazol Violet, Remazol Yellow, Remazol Orange and Remazol Black with high yield, %17.4, %90.2, %42.7, %89.8, %37 and %80, respectively. In presence of 20 ppm dyes all stationary cultures, a decrease in the dye absorbance was accompanied by visible sorption of the dyes to the fungal mycelia. It was observed that decolorized within 20 days with the decrease in absorbance in the visible range between 57.2 % and 89.8 % (Fig.16).



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1 2 3

Figure 16. Decolorization of dyes by T. versicolor in the modified medium15 with 1 gL-1 of glucose and 50 mLL-1 TES were used.

5

C. Preparation and characterization of new phthalocyanine-laccase bioconjugate

6 7 8 9 10

A common simple way to conjugation a molecule with protein is by amine coupling chemistry. The number of photosensitizer molecules conjugated with the protein can be controlled through the ratio of the reactants and the reaction time. The cartoon showing was shown that ZnPc conjugation to laccase via amine coupling (Scheme 1). It should be noted that because amine-ZnPc can conjugate to any surface carboxyl group.

4

R

N

N

N

R

NH Zn HN

R

O

N

N

N

N

NH2

NHS

N

N

NH Zn HN

R

Lac cas e DCC/

N

N

O

NH

N

R

R R:

HO

O O

O

O O

11 12

Scheme 1. Synthesis route for laccase conjugated zinc(II) phthalocyanine.

13 14

The newly synthesized bioconjugate (3) was characterized by general spectroscopic methods such as FT-IR, MALDI-TOF, and UV-vis as well (fig.17).

O

15


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13

1 2

Figure 17. a) FT-IR, b) MALDI-TOF, and c) UV-vis spectra of the synthesized laccase-phthalocyanine

3

bioconjugate (3).

4 5

This newly conjugate was useful different workspaces, such as metal sensing and/or cancer treatment.

6 7

D. Chemosensor eficacy of bioconjugate

8 9 10

All absorption and emission spectral studies were performed in DMSO/PBS at room temperature. Flouresence spectra of conjugate exhibit the most intense fluorescence spectrum due to containing enzyme moieties.

11 12 13 14 15

The fluorescence spectrum of laccase was affected in the presence of 5 µM, 1 μL cations such as Na+, K+, Ag+, Fe2+, Mg2+, Ba2+, Cs2+, Cu2+, Ca2+, Mn2+, Ni2+, Cd2+, Zn2+, Co2+, Pb2+, Hg2+, Fe3+and Cr3+ by all reduced their intensity. In addition, there was a little or no decrease of conjugate fluorescence intensity upon the addition of all cations. On the contrary conjugate fluorescence intensity reduced only in the presence of 5 µM cation Fe3+ (Fig. 18). Li+,

16 17

Figure 18. The fluorescence emission responses of the conjugate to different metal ions.

18 19 20

The specificity of the decrease of the fluorescence of this conjugate for Fe3+ was indicated in Figure 19. Titration of conjugate with Fe3+ resulted in a decrease in the fluorescence intensity. A



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Benesi–Hildebrand graph of the fluorescence datum were non-linear, indicating that the stoichiometry of the Fe3+ complex formed is different from 1:1. Results of the “Method of Continuous Variation” in a Job plot with a mole fraction of Fe3+ close to 0.33, accordingly indicating a preferred 1:2 stoichiometry for the complex25.

5

6 7 8

Figure 19. The flourencence emission changes of the conjugate by addition of Fe+3. Inset: Job’s plot for conjugate with Fe3+.

9 10 11 12 13

E. Investigation of bioconjugate for PDT applications Photophysical and photochemical properties: Ground state UV-vis spectra

14 15 16 17

Phthalocyanines have two absorption bands in the ground state UV-vis. These are known as Q- and B-band and were observed in the visible region due to the π → π* transitions from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO) of the phthalocyanine ring26.

18 19 20 21 22 23 24

The electronic absorption spectra of phthalocyanines were determined by UV-vis spectroscopy. This spectrum of bioconjugate (3) showed single narrow Q band absorptions which are characteristic for metallated phthalocyanines compounds in DMSO (Fig. 20(a)). The Q band of this compound was observed at 684 nm in DMSO. The conjugation of the phthalocyanine core caused 8 nm red-shift when compared to both unsubstituted zinc(II) phthalocyanine and Amine-Pc (2). The substitution of the laccase to the mono-amine zinc(II) phthalocyanine (2) did not any significant effect to wavelength of the absorption bands (Table 5).

25 26

Table 5. Absorption, excitation and emission spectral results for unsubstituted and enzyme conjugated zinc(II) phthalocyanine with different symmetry in DMSO.


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15 Q band

Excitation

Emission

Stokes shift

Ex, (nm)

Em, (nm)

ΔStokes, (nm)

4.99

684

692

8

683

5.10

684

693

9

672

5.14

672

682

10

Compound

Solvent

Bioconjugate (3)

DMSO

684

Amine-Pc (2)

DMSO

Std-ZnPca

DMSO

1

a Data

log 

max, (nm)

from reference 29

2 3 4 5 6 7 8

Aggregation Studies The UV-vis spectrum of conjugate (3) was also obtained in different solvents for determination of the aggregation behaviour. Conjugate formed non-aggregated species and it gave single narrow Q band absorption in DMSO, toluene, THF, ethanol, DMF and chloroform. But this conjugate formed aggregates in water and they showed a broad shoulder at around 635 nm (Fig. 20(a)).

9 (a)

(b)

10 11 12

Figure 20. UV-vis absorption spectra of laccase-phthalocyanine bioconjugate (3) (a) in different solvents and (b) in water and addition of triton X-100 to water solution. Concentration= 1.00x10-5 M.

13 14 15

Triton X-100 which is a surfactant added to reduce the aggregation, so aqueous solution conjugate turned to monomeric species and they gave single narrow Q band absorption. Because formed aggregates of this compound were broken (Fig. 20(b)). .

16 17 18 19

In this study, the aggregation properties of conjugate were also investigated at different concentrations in DMSO for investigation of the effect of concentration to aggregation behaviour. The Lambert–Beer law was obeyed at concentrations ranging from 1.2x10-5 to 2x10-6 M for this conjugate. The studied conjugate did not show any aggregation in DMSO (Fig. 21).

20



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1 2 3 4 5

Figure 21. Enzyme conjugated zinc(II) phthalocyanine (3) absorbance changes in DMSO at different concentrations:12x10-6 (orange), 10x10-6 (blue), 8x10-6 (green), 6x10-6 (cyan), 4x10-6 (navyblue), 2x10-6 (purple) M. (Inset: Plot of absorbance versus concentration).

Fluorescence spectra

6 7 8 9 10 11 12

Fluorescence spectrum of enzyme conjugated derivative (3) was carried out in DMSO at rt°. The fluorescence excitation-emission spectra of the enzyme conjugated compound (3) are similar to metallo phthalocyanine complexes. The excitation spectra were analogous to absorption spectra. According to mirror images of the fluorescent spectra for compound (3) did not show any degradation in DMSO during excitation (Fig. 22). The Stokes’ shifts (ems−exc) range was 12 nm. Fluorescence emission-excitation peaks of enzyme conjugated zinc(II) phthalocyanine (3) in DMSO were listed in Table 6.

13 14 15

Figure 22. Absorption, excitation and emission spectra for enzyme conjugated derivative (3) in DMSO. Excitation wavelength=650 nm.

16 17 18 19 20 21 22 23 24

Fluorescence quantum yields and lifetimes When the photosensitizer’s orbital electron relaxes after being excited to a higher quantum state to its ground state by emitting a photon so fluorescence consists. The fluorescence process is defined by fluorescence quantum yield (F). This value is the ratio of the number of photons emitted to the number of photons absorbed18. ΦF value of the studied zinc(II) phthalocyanine-enzyme conjugate is 0.315 in DMSO (Table 6). The zinc(II) Pc compound (3) are higher than unsubstituted zinc(II) phthalocyanine compounds in DMSO. The conjugation of the laccase enzyme to the the zinc phthalocyanine increased the ΦF values. Fluorescence lifetime (F) value (in DMSO) of the studied zinc(II) phthalocyanine compound increased when they conjugated with laccase enzyme (Table 6).


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17 1 2 3

Table 6. Photophysical-photochemical results of zinc(II) phthalocyanine derivatives with different substituent in DMSO.

Compound

4 5 6 7 8 9

cData

F

ak

F

ns

s-1(x108)

Δ

d (x10-5)

Conjugate (3)

0.32

3.59

2.78

0.39

0.59

Amine-Pc (2)

0.22

2.11

1.04

0.72

1.53

Std-ZnPc

0.20b

1.22c

1.47c

0.67c

2.61d

a k is the rate constant for F bData from reference28. dData

F

fluorescence. Values calculated using kF = ΦF/F.

from reference 27. from reference 29.

Singlet oxygen quantum yields

10 11 12 13 14

Singlet oxygen is formed from a bimolecular interaction between the triplet state of a photosensitizer and ground state (triplet) molecular oxygen. Singlet oxygen quantum yield (Φ) is quantified by generated singlet oxygen amount. Singlet oxygen generation depends on quantity of molecules in the triplet state, consequently efficient energy transfer determined by amounts of between excited triplet state of photosensitizer and ground state of oxygen28.

15 16 17 18 19 20

The singlet oxygen was measured by UV–vis spectroscopy. The 1,3diphenylisobenzofuran (DPBF) is a singlet oxygen scavenger and which absorption decays were monitored at 417 nm during the light irradiation (Fig 23). For the comparision, same measurements carried out for unsubstituted ZnPc (Std-ZnPc) in DMSO. During the singlet oxygen measurements Q band intensities of studied compounds did not exhibit any changes because compounds were not degraded by light irradiation.

21

22 23 24

Figure 23. Absorbance changes during singlet oxygen quantum yield measurements. This measurement was for conjugate in DMSO at a concentration of 1.0 x10-5 M. (Inset: Plot of DPBF absorbances versus irradiation time).

25 26

The obtained  values of zinc(II) phthalocyanine derivatives were given in Table 6. The 



Page 18 of 28

18 1 2 3 4

value of given for comparison purpose in this table. The  value of the asymmetrically substituted zinc(II) phthalocyanine bearing enzyme are lower than unsubstituted zinc(II) phthalocyanine. The conjugation of the enzyme caused decreasing of the  value could be due to the addition of the bulky enzyme on the molecule.

5 6

Photodegradation studies

7 8 9 10 11 12

Photodegradation studies are related to stability of phthalocyanine molecules against the light treatment. During the photodegradation’s process when a phthalocyanine is degraded by light. Determination of phthalocyanine stability shows that how effectively will using as a photocatalyst. The stabilities of enzyme conjugated zinc(II) phthalocyanine compound (3) was determined in DMSO by monitoring the decrease in the intensity of the Q- and B- band with light irradiation (Fig.24). The photodegradation quantum yield (d) values ofcompounds in DMSO are listed in Table 6.

13 14 15 16 17

The d values are similar phthalocyanine derivatives in the literature30. Stable phthalocyanines show d values as low as 10−6 and unstable phthalocyanines show d values of the order of 10−3 in the literature31. The d value of compound (3) is lower than the d value of unsubstituted zinc(II) phthalocyanine in DMSO. The conjugation of the phthalocyanine ring with enzyme decreased the d values of zinc(II) Pc compounds and increased the stability of these complexes under light irradiation.

18 19 20 21

Figure 24. Absorbance changes during the photodegradation study of 5 in DMSO showing the decreasing of the Q and B bands at ten minutes intervals. (Inset: Plot of Q band absorbance versus time).

22

In vitro cell studies

23 24 25 26

Figure 25a and 25b show the survival of HeLa and HuH-7 cells following illumination with 1 and 2 J.cm-2 of 690 nm (±10 nm) light after uptake of phthalocyanine conjugate. Cell survival appears to be dose-dependent and there is not observed any dark toxicity.

27 28 29 30 31

In present study, photodamage on the studied cell lines was occured after PDT. In the presence of each phthalocyanine, cell survival decreased following irradiation while the number of the cells remained the same without irradiation. The concentration-dependent dark cytotoxicity was investigated in both HuH-7 and HeLa cells exposed to increased concentrations of each phthalocyanine (up to 5 M) for 24 h in the dark. The survival curves for photosensitized cells were

J.cm-2


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19 1 2 3 4 5 6

given in Fig. 25. Studied conjugate, showed the similarly photodynamic effect in both cell lines on the lowest concentration, both cell lines’ survival degree was 50% after traetment 1M drug concentration. In addition, only HuH-7 cells showed lower survival degree after photodynamic therapy on 5M drug concentration with 2 J.cm-2 irradiation. Compared to dark conditions, a significant cytotoxicity was found in both HuH-7 and HeLa cells at 1 Joule and 2 Joules, concentrations of 0.25-5 µM (Fig.25).

7 8 9 10 11 12

Photodynamic therapy with laccase conjugated zinc(II) phthalocyanine sensitizer resulted in the death of both HuH-7 and HeLa cells at different conditions (Fig. 26a-26b). Viability studies have shown that the optimum phototoxic effects such as illumination dose or concentration of phthalocyanines were tested. Hovewer following illumination with 1 J.cm-2 cell survival results same in 2 J.cm-2 irradiation. According to these results, newly prepared conjugate (3) highly effective on HuH-7 and HeLa cell lines with low (1 J.cm-2) illumination dose.

13

14 15 16 17

Figure 25. Survival of HuH-7 and HeLa cells following illumination with 1 J.cm-2 and 2 J.cm-2 of 690 nm (± 10 nm) light after 24 h with various concentration of phthalocyanine photosensitizer (3) administration. Each data represents the mean ± SD of three experiments.

18 19 20



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20 1

Conclusion

2 3 4 5 6 7 8 9 10 11

This study was reported on the isolation of a white rot fungus producing laccase as its ligninolytic enzyme. The organism was employed in the removal of six different dyes in batch cultures. The role of Lac in the decolorization process was also investigated in in vitro enzymatic experiments. Another study of phthalocyanine-laccase conjugate is fluorescence chemosensor behavior was determined for the first time. In addition potential photosensitizing characteristics of a phthalocyanine-laccase conjugate was investigated for the first time. This enzyme group increases the solubility of conjugate and the effects of absence and presence of laser irradiation were studied on a two cancer cell line in vitro. A dose dependent study was performed to identify the appropriate laser fluence and sensitizer concentration to be used in a cell death study using a breast cancer cell line (HeLa and HuH-7).

12 13 14 15 16 17 18 19 20

Laccase is an important ligninolytic enzyme and has great biotechnological potential. In this article, much attention is focused on detailed properties and aplication areas of purified laccase. The apparent Km value of the enzyme for ABTS determined from the Lineweaver-Burk plot was estimated to be 3.66 x 10-4 M, and the corresponding Vmax value was 1652 UL-1. The optimum pH of the laccase was 4.0 with ABTS as a substrate in potassium-phosphate buffer. However, this laccase showed a high relative activity that was stable over the pH range (from 3 to 4). The temperature optimum of the laccase was 45oC with ABTS as a substrate in potassium-phosphate buffer of pH 4.0, and its activity was stable over a broad temperature range. This enzyme was very stable at temperatures ranging from 0 to 65oC for a long time, but the activity completely lost 65oC.

21 22 23 24 25 26

On other hand, decolorization efficiency by T.versicolor secretions. Treatment with the fungus of the dye eliminates around 50-80%, as a result of both the adsorption and enzymatic degradation processes. This results suppoted that decolourisation of wastewaters will be performed by Trametes versicolor. A decolorization of 90% was achieved for initial Remazol blue concentrations up to 20 ppm. T. versicolor has earlier been shown to decolorize a number of dyes either completely or partially in shake flasks32.

27 28 29 30 31 32

Bioconjugate is a new fluorescent sensor for Fe3+ ions in other application area. Titration experiments show that conjugate can be used to detect changes of Fe3+ levels in media. The detailed UV-Vis and fluorescence titration experiments and careful analysis of the Job’s plot suggest the existence of both 1 : 2 (L: Fe3+) complexation stoichiometry in solution. The experimental results of this study can utility in the future design of metal-ion sensors for a variety of chemical and biological applications.

33 34 35 36 37 38 39 40 41 42 43

Another application area is cancer treatment with photodynamic therapy. In summary, for the first time laccase conjugated phthalocyanine photosensitizer was synthesized. The photophysical and photochemical properties of this conjugate correlates with unsubstituted phthalocyanine. Fluorescence quantum yields and lifetimes are higher than unsubstituted zinc(II) phthalocyanine, but the singlet oxygen and photodegradation quantum yield are lower than unsubstituted zinc(II) phthalocyanine. In this study PDT combined the laccase conjugated phthalocyanine photosensitizer displays good in vitro characteristics that make them highly suitable for continued evaluation as PDT agents, namely dark toxicity, phototoxicity at low light dose (2 mW/cm2). Viability studies have shown, that the optimum phototoxic effect tested on HuH-7 and HeLa cells was determined in the combination of light doses of 1-2 Jcm-2 and concentration of phthalocyanine up to 5 M. Efficiency of PDT is affected by a number of factors including absorption spectrum of the photosensitizer,


Page 21 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


21 1 2

wavelength of the activation light, depth of the light penetration in the biological tissue, tissue answer on singlet oxygen.

3 4

From this point of view, this work may provide a reasonable basis for development of a biotechnological process.

5 6 7 8 9 10 11 12 13 14

Materials and Methods. Organism and culture conditions The strain was maintained on 2% malt extract agar (MEA) slants at 4oC. Two mycelial agar discs (0.5 cmx 0.5 cm) were obtained from a fresh malt extract agar culture for use as the inocula. The fungal inocula were prepared in 250 mL erlenmeyer flasks, for 12 days. The fungus was cultivated in 100 mL medium (malt extract broth 2.0%, glucose 0.1%, potassium phosphate buffer 6.8%, and Trace Elements Solution) on a rotary shaker (130 rpm, 32°C). Optimized culture medium content is shown in table 1. Table 1. Medium content15. CONTENTS

AMOUNT

Malt Extract Broth

2 gL-1

Glucose

1 gL-1

Potassium Phosphate Buffer (KH2PO4)

6.8 gL-1

Trace Elements Solution (TES)

H3BO3

0.5x10-3 gL-1

CaCl2.H2O

5x10-3 gL-1

CuSO4

5x10-3 gL-1

ZnSO4.7H2O

5x10-3 gL-1

FeSO4.7H2O

5x10-3 gL-1

NaCl

8.75x10-3 gL-1

MnSO4

25x10-3 gL-1

MgSO4

150x10-3 gL-1

15 16

Laccase activity assay

17 18 19 20 21 22

The laccase activity was measured via oxidation of 2,2’-azino-bis(3-ethylbenzthiazoline)-6sulfonate (ABTS)16. 750 µL assay mixture consisting 0.167 mM ABTS, 50 mM KH2PO4 buffer (pH 4) and 5 µL sample. Laccase contained assay mixture color turns green resulting ABTS oxidation after incubated at 45oC for 30 minutes. One enzyme unit was defined as the amount of enzyme required to oxidize 1 μmol of substrate per min, under the assay conditions. The enzyme activity was determined by color intensity in the visible region of 415 to 420 nm on UV-Vis spectrophotometer.

23

Glucose and TES amounts effect on laccase production

24 25

For optimization of culture medium, both glucose and TES amounth effect on laccase production from T.versicolor was tested in different concentration. Glucose and trace elements’



Page 22 of 28

22 1 2

effect on enzyme production are determined using the conditions specified in the prepared media. It was shown in Table 2.

3 4

Table 2. The effect of different concentrations of both trace elements and glucose on laccase production in a solution prepared eight different medium. Malt Extract

Glucose

KH2PO4

Trace Element Solution

Medium 1

2 gL-1

5 gL-1

6.8 gL-1

50 mLL-1

Medium 2

2 gL-1

5 gL-1

6.8 gL-1

-

Medium 3

2 gL-1

1 gL-1

6.8 gL-1

-

Medium 4*

2 gL-1

1 gL-1

6.8 gL-1

50 mLL-1

Medium 5

2 gL-1

-

6.8 gL-1

100 mLL-1

Medium 6

2 gL-1

-

6.8 gL-1

-

Medium 7

2 gL-1

5 gL-1

6.8 gL-1

100 mLL-1

Medium 8

2 gL-1

1 gL-1

6.8 gL-1

100 mLL-1

5 6

pH effect on laccase production

7 8 9 10 11

pH effect on laccase production investigated at different pH values (3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7). Malt extract broth (0.2%) in KH2PO4 (50mM, pH:4) buffer was prepared and autoclaved then Trametes versicolor was inoculated. pH adjusted before inoculation, but the pH was not controlled during the cultivation. Activity determination, as indicated in Section 2.3., the incubation time of the day of inoculation (day 0) were made at intervals of 2 days from.

12

Protein assay using bicinchoninic acid (BCA) copper reagent

13 14 15 16

Protein content in samples was determined using the BCA17. The method takes place in two steps. In the first step, copper is reduced by protein in the sample. The reduced copper forms a complex with BCA in the second step. Discoloration occurs during the complex formation thereby protein concentration (mg/mL) is determined18.

17

Purification of laccase from culture supernatant:

18 19 20 21 22 23 24

Optimized fungus growth medium showed that maximum laccase activity following inoculation between fourth and eighth days (4 to 8 days). So, incubation was stopped on the day of maximal activity observed. Firstly, the culture fluid was used for enzyme purification after filtration through a Whatman No. 1 filter paper. The supernatant was separated by filtration of the mycelium. The supernatant obtained after filtering divided into two equal volumes for the comparison of different methods is subjected to purification procedures. The purification experiments were performed at 4oC.

25 26 27 28 29

Purification of laccase by ammonium sulphate precipitation. For ammonium sulphate [(NH4)2SO4] precipitation, saturating amount is calculated according to the literature19. Primarily in the ammonium sulfate precipitation method in which saturation of the enzyme is completely collapsed (salting out) must be found. Percentages in the range 35-85% for this precipitation are achieved by increasing the tens and the amount of activity in the filtrate is


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23 1 2 3 4 5 6 7

checked. Activity remains in the filtrate of the saturation value is used. In this study, 85% saturation at 4°C the amount of ammonium sulphate is calculated to be 44.12 g. In the culture supernatant (100 mL) for the determination of activity and protein content was precipitated by 85% ammonium sulphate saturating. After addition ammonium sulphate to culture supernatant was stirred for 1 hour and then was incubated overnight at 4oC. After then, the culture supernatant was centrifuged at 15,000xg for 30 minutes to separated proteins from supernatant. After centrifugation, shouldn't be enzyme activity in supernatant.

8 9 10 11 12 13 14 15 16 17 18 19

The residue obtained by precipitation was dissolved in a small amount of buffer solution (20 mM Na-Acetate, pH:5) to highly concentrated protein content was obtained. This mixture corresponds to the probability of having 1 with ammonium sulfate overnight at 4oC for 10 times its volume was subjected to dialysis against distilled water. In dialyzing 10 kDa cutoff cellulose membrane dialysis bag was used. After dialyzing the protein solution was loaded onto DEAESepharose anion exchange column. The protein solution was purified by loading the desired enzyme onto the column matrix due to ion-ion interaction. Hence in the protein solution obtained from the column or laccase activity has never been seen or slightly observed. Keep matrix proteins via ionic interactions are still cut off from the matrix via ionic interactions. For this column, the concentration of NaCl ranging from 0.5-2.0 M was washed with buffer solution. Washing processes received from the column was collected in fractions of 2 ml of an enzyme solution; laccase activity (420 nm), to the presence of protein (280 nm) and BCA method (562 nm) and protein content were measured.

20

Purification of laccase by ion-exchange chromatography

21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

Filtrated culture supernatant was treated with DEAE-Sepharose ion-exchange matrix. The matrix before being treated sample to come into equilibrium with the medium 3 to 5 times its volume, washed with buffer solution (20 mM Na-Asetat, pH 5). The sample (100 mL) was applied to DEAE-sepharose column which was equilibrated with the same buffer. The enzyme fractions were eluted with a linear concentration gradient of 0-1.0 M NaCl in the same buffer, at a flow rate of 5 mL/min. Fractions were analyzed for activity and protein content again after the combined fractions were loaded onto a column of DEAE-Sepharose. Gel filtration matrix sample buffer before being treated in solution overnight at 4oC for swelling of the particles was heated thoroughly. The enzyme solution was loaded onto the column and the fractions at a time in a small volume (400 L) were taken from the column to be. Proteins with laccase activity containing fractions were combined and again loaded onto a DEAE-sepharose column. Enzyme binding and washing was repeated as described above. The important point in this process, in order to obtain a more concentrated enzyme solution used amount of DEAE-Sepharose matrix is less than that used in the first. To view the operations performed by SDS-PAGE in the purification efficiency, the activity and amount of protein purification table was prepared and observed changes in the purification step. Purified laccase stored at -20°C in 0.01 M buffer was stable for at least 3 months.

37

Molecular weight determination by SDS-PAGE

38 39 40 41 42

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), in monitoring the phases of purification, which is attempted to be purified and the molecular weight of the enzyme is used to determine homogeneity. SDS-PAGE Laemmli, 1970 were made according to the method proposed by20. Relative molecular weight of the purified enzyme, using standard protein mixture was determined by SDS-PAGE.

43

Characterization of purified laccase:



Page 24 of 28

24 1 2 3 4 5 6 7 8 9 10 11

Optimized temperature Effect of temperature on the laccase activity was determined via spectrophotometrically laccase-catalyzed oxidation of ABTS at 420 nm. 5°C interval in the range 30 to 70°C, the reaction mixture was incubated for 30 minutes and activity was determined. Optimized pH The pH of the reaction mixture prepared in the range of 3.0 to 7.0 and was investigated on enzyme activity. Progress curve The laccase activity of the temporal change of the reaction mixture the activity of samples taken every ten minutes was determined. Determination of Kinetic Parameters for ABTS

12 13 14

Kinetic datum was determined at different ABTS concentrations (0.04-0.8 mm). The initial speeds were obtained via Michaelis-Menten plot. Additionally, the Km and Vmax values were determined for ABTS by Lineweaver-Burk plotting.

15

Decolorization efficiency of laccase

16 17 18 19 20 21 22 23 24

The six dyes remazol yellow (max: 420 nm), remazol orange (max: 495 nm), remazol Red (max: 520 nm), remazol blue (max: 590 nm), remazol black (max: 600 nm), remazol violet (max: 640 nm) used in this work. Fungal strain was precultured on 2% (w/v) malt extract agar for one week. The decolourization of dyes in liquid cultures was evaluated using 250 mL erlenmeyer flasks containing 100 mL of the medium that contain 20 ppm dye. Flasks were inoculated and were incubated 20 days. Decolourisation is the colour removal efficiency between the first and last day in the medium. Five days interval a 1 mL sample was taken and was centrifuged and the supernatant was used for the absorbance of the residual dye was measured. Absorbance measurements were performed using a Shimadzu 2101 UV-Vis spectrophotometer.

25

Preparation and characterization of phthalocyanine-laccase bioconjugate (3)

26 27 28 29 30 31 32 33 34 35 36 37

Asymmetrically (t-Boc) protected (1) and mono-amine functionalized (2) zinc(II) phthalocyanine21 were synthesized according to literature procedures. Mono-amine functionalized asymmetrically zinc(II) phthalocyanine (2) (10 mg, 0.0062 mmol) was dissolved in DMF (0.5 mL) and then, the purified laccase (0.025 mL) was added to a solution. For conjugation, dicyclohexylcarbodiimide (DCC) (1.8 mg; 0.007 mmol) and N-hydroxysuccinimide (NHS) (0.82 mg 0.007 mmol) were added to the mixture and stirred 48 hours at room temperature. The resulting mixture was treated with methylene chloride and water, after than the organic phase was separated by liquid-liquid extraction. Methylene chloride was removed under reduce pressure. Yield 96% (9.6 mg). FT-IR spectrum (max/cm-1): 3437 (NH), 3073 (ArCH), 2926-2854 (Aliphatic CH), 1711 (C=O), 1609 (C=C), 1486 (NH), 1220 (Aliphatic CH), 1080 (C-O-C); UV–Vis (DMSO): max nm (log ε) 359 (4.66), 619 (4.26), 683 (4.99). MS (MALDI-TOF) m/z: Calc.: 80204 (1 bound phthalocyanine); Found: 81831[M+Na]+ (2 bound phthalocyanine).

38

Metal sensor potenties of phthalocyanine-laccase bioconjugate

39 40

Addition of metal salts to the conjugate solution was caused a spectroscopic changes, were recorded using a Varian Eclipse spectrofluorometer using 1 cm path length cuvette at room


Page 25 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


25 1 2 3

temperature. The 5x10-6 M stock solution of conjugate was prepared in phosphate-buffered saline (PBS). 0.1M metal ions stock solutions were prepared in water. Flourescence spectra were taken with metal ions by addition of conjugate to be end concentration of 5x10-5 M in media.

4 5 6 7 8

Investigation of bioconjugate for Photodynamic therapy (PDT) applications: Photophysical and photochemical properties Fluorescence quantum yields (F), fluorescence lifetimes (F) and singlet oxygen quantum yields () determinations were achieved according to literature procedures21.

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

In vitro cell studies Cell Culture All tissue culture media and reagents were obtained from PAN Biotech. Both human HeLa cells and human HuH-7 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 1% antibiotics (penicillin-streptomycin) and were incubated at 37 °C in a humidified atmosphere of 5% CO2 in air. Light source The Lumacare Model LC-122 consists of two main parts: a Quartz halogen lamp light source (100 W, and a fiber-optic probe (FOP) adaptor with filters ranging from 380 to 750 nm. The fiberoptic probes of the LC-122 offers output power from 10 mWcm-2 up to 1 Wcm-2 at their output tips, depending on filter transmission wavelength. For illumination protocol we used FOP systems with activation wavelength of 680 ± 10 nm. The light power of FOP measured with a power meter that has silicon detector (Ophir). The exposure energy is controlled from the control panel by a timer. Cytotoxicity The cells seeded onto 96-well plates at 4000 cells per well and incubated 24 h to attach. Various concentrations of phthalocyanines were added to exponentially growing cells and after 24 h the cells were irradiated with laser irradiation. For dark cytotoxicity experiments, phthalocyanine was added to triplicate wells in a final concentration of phthalocyanine of 0.25, 0.5, 1, 2 and 5 μM and the cells were incubated with phthalocyanine for 24 h. The compounds were replaced with fresh medium. And then, cytotoxicity was measured using tetrazolium compound reagent (WST-1, Roche) for the quantification of cell proliferation and cell viability22. To each well, 20 μL of WST-1 compound was added and the plate was incubated for 4 h before reading. Absprbance of WST-1 tetrazolium compound can be measured at 450 nm with a microplate reader. The average of the triplicate wells for each sample was calculated. Phototoxicity studies The phototoxicity experiments include in treated with photosensitizer concentrations of 0.25, 0.5, 1, 2 and 5 μM and then incubated for 24 h. After then, the medium was removed and replaced with fresh medium. For phototoxicity, cells were exposed to the light. The total light dose was approximately 1 Jcm-2, and 2 Jcm-2. After illumination the cells were incubated for 24 h and then the plates were measured by reading optical density at 450 nm by the Universal Microplate Reader.



Page 26 of 28

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Statistical analyses Statistical significance was assessed using two-way ANOVA tests. A p value of p

Blue Copper Peroxidase and Phthalocyanine Conjugate: Synthesis

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