Accepted Manuscript

Spectroscopic Investigations on the Effect of N-Acetyl-L-cysteine-CappedCdTe Quantum Dots on Catalase

Haoyu Sun, Bingjun Yang, Erqian Cui, Rutao Liu

PII: S1386-1425(14)00730-6DOI: http://dx.doi.org/10.1016/j.saa.2014.04.157Reference: SAA 12121

To appear in: Spectrochimica Acta Part A: Molecular and Biomo-lecular Spectroscopy

Received Date: 18 March 2014Revised Date: 17 April 2014Accepted Date: 23 April 2014

Please cite this article as: H. Sun, B. Yang, E. Cui, R. Liu, Spectroscopic Investigations on the Effect of N-Acetyl-L-cysteine-Capped CdTe Quantum Dots on Catalase, Spectrochimica Acta Part A: Molecular and BiomolecularSpectroscopy (2014), doi: http://dx.doi.org/10.1016/j.saa.2014.04.157

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Spectroscopic Investigations on the Effect of

N-Acetyl-L-cysteine-Capped CdTe Quantum Dots on Catalase†

Haoyu Sun, Bingjun Yang, Erqian Cui, Rutao Liu*

School of Environmental Science and Engineering, Shandong University, China

-America CRC for Environment & Health, Shandong Province, 27# Shanda South

Road, Jinan 250100, P.R.China

*All correspondence should be addressed to:

Rutao Liu

School of Environmental Science and Engineering,

Shandong University, Jinan 250100 P.R.China

Phone/Fax: 86-531-88364868

Email: Trutaoliu@sdu.edu.cn (Liu RT)

Paper Summary

Characters with spaces: 32, 085

Number of figures: 9

Number of tables: 3

Abstract

Quantum dots (QDs) are recognized as some of the most promising semiconductor

nanocrystals in biomedical applications. However, the potential toxicity of QDs has

aroused wide public concern. Catalase (CAT) is a common enzyme in animal and

plant tissues. For the potential application of QDs in vivo, it is important to

investigate the interaction of QDs with CAT. In this work, the effect of

N-Acetyl-L-cysteine-Capped CdTe Quantum Dots with fluorescence emission peak at

612 nm (QDs-612) on CAT was investigated by fluorescence, synchronous

fluorescence, fluorescence lifetime, ultraviolet–visible (UV-Vis) absorption and

circular dichroism (CD) techniques. Binding of QDs-612 to CAT caused static

quenching of the fluorescence, the change of the secondary structure of CAT and the

alteration of the microenvironment of tryptophan residues. The association constants

K were determined to be K288K=7.98×105 L mol

-1 and K298K=7.21×10

5 L mol

-1. The

interaction between QDs-612 and CAT was spontaneous with 1:1 stoichiometry

approximately. The CAT activity was also inhibited for the bound QDs-612. This

work provides direct evidence about enzyme toxicity of QDs-612 to CAT in vitro and

establishes a new strategy to investigate the interaction between enzyme and QDs at a

molecular level, which is helpful for clarifying the bioactivities of QDs in vivo.

Keywords: Catalase, QDs, Spectroscopic studies, Static and dynamic quenching,

Activity inhibition.

1. Introduction

Quantum dots (QDs) are engineered semiconductor nanocrystals with the size of

2-100 nm [1]. Due to their novel photophysical properties and excellent chemical

processability, quantum dots have been extensively explored and used as imaging

agents or biomedical labeling [2, 3]. However, many studies reported that

nanoparticles could induce some toxic responses in vitro,, such as the production of

reactive oxygen species (ROS) and the subsequent release of toxic heavy metals [1, 4].

In terms of these findings, the developments and applications of nanoparticles are

worried by scientist, policy makers and the general public [5]. Both the environment

conditions and the physicochemical properties of nanoparticles (chemical composition,

size, shape, aggregation, and surface coating) effect on their potential toxicity to

human health [1, 5].

Catalase (CAT, H2O2: H2O2 oxidoreductase, EC 1.11.1.6) is one of the most

common enzymes in organism which can catalyzes the dismutation of H2O2 to one

molecule of H2O and a half molecule of O2 [6-8]. As a kind of ROS, H2O2 can exert

toxic effects by activation of transition metal ions to Fenton chemistry, generating

hydroxyl radicals and damaging DNA [9, 10]. Since CAT can prevent the damage of

reactive oxygen species in tissues by scavenging H2O2, it plays a significant role in

the detoxification of oxidative stress [11, 12]. The catalytic activity of CAT in human

tissues will change by intake of any contaminants [13]. However, it is far from fully

understanding the inactivation and reactivation of CAT in vivo and in vitro [14].

Nanoparticles can enter the body through the lung and digestive system [15]. The

toxicity of these nanoparticles can be explored by investigating the interactions

between nanoparticles and proteins [16]. Therefore, the alterations of structure and

activity of CAT can provide the basic explanations for the bioactivities of QDs

[16-18]. With advantages of simplicity, rapidity and high sensitivity, spectroscopic

methods are powerful tools to explore the interaction mechanism between protein and

nanoparticles[7, 19].

In this study, the conformational change of CAT induced by

N-Acetyl-L-cysteine-Capped CdTe Quantum Dots with fluorescence emission peak at

612 nm (QDs-612) was investigated in vitro under physiological conditions by

various spectroscopic techniques, including fluorescence, synchronous fluorescence,

fluorescence lifetime, ultraviolet–visible (UV-Vis) absorption and circular dichroism

(CD). Since QDs-612 have strong absorption at the excitation and emission

wavelengths of CAT, the inner filter effect cannot be ignored in the fluorescence

studies [20-22]. In addition, the alteration of CAT activity was also determined by

spectrophotometer method. This study provides the basic data of the binding

mechanisms of QDs-612 with CAT, which help us deepen our understanding on

QDs-612 toxicity in vitro.

2. Materials and methods

2.1. Materials and regents

Catalase (CAT, from bovine liver) was purchased from Sigma Chemical Company,

and was freshly dissolved in ultra pure water to form a 4×10-6

mol L-1

solution for

each experiment. N-Acetyl-L-cysteine (NAC, Sigma) was stored in refrigerator at 4

oC. 0.2 mol L-1 phosphate buffer solution (PB, mixture of NaH2PO4·2H2O and

Na2HPO4·12H2O) was used to control pH at 7.4. NaH2PO4·2H2O, Na2HPO4·12H2O

and Na2TeO3 were obtained from Sinopharm Chemical Regent Beijing Co., Ltd.

CdCl2·2.5H2O and NaBH4 were bought from Tianjin Kermel Chemical Regent Co.,

Ltd. Ethanol and NaOH were produced by Tianjin Damao Chemical Regent Co., Ltd.

All water used throughout the study was ultra pure with resistivity of 18.25 Ω cm.

2.2. Apparatus and methods

2.2.1. The synthesis and purification of CdTe QDs

NAC-capped CdTe QDs were synthesized by one-pod method with slight

modifications, described by Wang Ling et al [23]. Briefly, 6 mL Cd-solution (0.1 mol

L-1

), 90 mL ultra pure water and 0.17g NAC were added to a three-necked flask in

succession. Under vigorous stirring, NaOH (1 mol L-1

) was added to tune the pH of

the mixed solution to 10. Then 0.1 g NaBH4 and 1.5 mL Te-solution (0.02 mol L

-1)

were added to the three-necked flask. After five minutes, the reaction mixture was

heated to 100 oC in an oil bath and refluxed. NAC-capped CdTe QDs with different

emission spectra were obtained by varying the reaction time (Fig. 1(A)). The QDs

with fluorescence emission peak at 612 nm (QDs-612) were used in the interaction

study. For purification, the ratio of QDs to ethanol was 1:2 in volume to centrifuge at

8,000 rpm.

2.2.2. Fluorescence measurements

Fluorescence quenching spectra of CAT were measured in the presence of

increasing concentrations of QDs from 290 nm to 450nm on an F-4600 fluorescence

spectrophotometer (Hitachi, Japan) with the excitation wavelength set to 280 nm [24].

Experiments were carried out at 288 K (15 oC) and 298 K (25 oC). The synchronous

fluorescence spectra were obtained at λex=250 nm, ∆λ=15 nm and ∆λ=60 nm at 288

K, which were measured by scanning the excitation and emission monochromator

simultaneously. The scanning speed (1200 nm/min) and scanning voltage (700 V)

were the same in these measurements. For each fluorescence measurement, the

protein concentration was 4.0×10-7

mol L-1

and the maximum concentration of QDs

was controlled to 5×10-7 mol L-1.

2.2.3. Fluorescence lifetime measurements

Time-resolved measurements were carried out on a FLS920 combined fluorescence

lifetime and steady state spectrometer (Edinburgh, England) at 288 K. While the

excitation wavelength was 280 nm, the emission wavelength was 330 nm. The

concentrations of protein and QDs were the same as the fluorescence measurements.

2.2.4. UV-visible absorption measurements

UV-vis absorption spectra were taken on a spectrophotometer (Shimadzu UV2450,

Tokyo, Japan) at 288 K equipped with 1.0 cm quartz cells. Reference solutions were

the same systems without protein. Wavelengths were set from 190 nm to 450 nm, and

slit width was 1 nm with a scanning speed of 1200 nm/min. The concentrations of

protein and QDs were the same as the fluorescence measurements.

2.2.5. CD measurements

A Jasco-810 spectropolarimeter (Jasco, Japan) was applied to record CD spectra at

288 K, over the wavelength range of 190-260 nm and with a scanning speed of 200

nm/min. In view of the possible interference of buffer solution, the buffer baseline

was subtracted from the observed CD spectra. While the concentration of protein in

the systems was controlled to 4.0×10-7 mol L-1, QDs were 2.0×10-6 mol L-1 and

4.0×10-7 mol L-1.

2.2.6. CAT activity determination

The activity of CAT was determined by spectrophotometer method at 288 K. CAT

catalyzed the decomposition of hydrogen peroxide into water and oxygen and led to

decrease in the absorbance of hydrogen peroxide at 240 nm. The inhibition rate of

CAT activity was calculated by the following equation:

Inhibition %100/ 21 ×∆∆= AArate (1)

where 1A∆ and 2A∆ were the reduction of the absorption value at 240 nm in 2 min

after the addition of CAT with or without QDs, respectively.

3. Results and discussion

3.1. Characterization of NAC-capped CdTe QDs

Fig. 1(B) depicts the UV-vis absorption spectra of NAC-capped CdTe QDs with six

sizes. Photograph of emission colors of these QDs under a UV lamp is showed in Fig.

1(C). The particle diameters of QDs are determined from the first absorption

maximum of the UV-vis absorption spectra according to the following equation [25]:

84.1940064.1107147.1108127.9 2337 −+×−×= −− λλλ ）（）（）（D (2)

where D (nm) is the size of a given CdTe QDs, and λ (nm) is the wavelength of the

first excitonic absorption peak of the corresponding sample. The particle sizes of the

NAC-capped CdTe QDs are calculated to be 2.42, 3.11, 3.34, 3.47, 3.57 and 3.62 nm.

Fig. 2 shows the HRTEM images of NAC-capped CdTe QDs (QDs-612). It is easy to

judge the edge of QDs-612 from these HRTEM images. According to statistics from

one hundred QDs, the average size of QDs-612 is 3.45 nm, which is similar to the

result from above equation.

Fig. 3 shows the FT-IR spectra of pure NAC and NAC-capped CdTe QDs. The IR

absorption bands of pure NAC around 1717 cm-1 (sν COO-) and 1400 cm-1 (mν COO-)

indicate the -COO- group, which are absent on the surface of NAC-capped CdTe QDs.

At the same time, the absorption band of -COOM (1604 cm-1 and 1388 cm-1) appears

at the FT-IR spectra of NAC-capped CdTe QDs. Furthermore, the absorption band of

-S-H group (2547 cm-1) disappears at the FT-IR spectra of NAC-capped CdTe QDs,

which is obvious on the surface of pure NAC. All these distinctions indicate that

NAC-capped CdTe QDs are synthesised by formation of covalent bonds between

carboxyls, thiols and the surface of CdTe [26].

The concentration of QDs with fluorescence emission peak at 612 nm (particle size,

3.47nm) is determined by using Lambert-Beer’s law [25]:

CLA ε= (3)

12.2)(10043 D=ε (4)

where A is the absorbance at the peak position of the first exciton absorption peak for

a given CdTe QDs sample. C, L and ε stand for the molar concentration (mol L-1

) of

the CdTe QDs of the same sample, the path length (cm) of the radiation beam used for

recording the absorption spectrum and the extinction coefficient per mole of CdTe

QDs (L mol-1 cm-1) at the first excitonic absorption peak, respectively. The

concentration of QDs-612 is 1×10-6 mol L-1 approximately in this study.

3.2. Fluorescence spectra and quenching analysis

Fluorescence quenching spectra of CAT at various concentrations of QDs-612 are

shown in Fig. 4, which is already eliminated the inner filter effect by the following

equation [27]:

2/)( 2110AA

obscorFF

+= (5)

where Fcor and Fobs are the corrected fluorescence intensity and observed value,

respectively. A1 (set at 280 nm) and A2 (set at 330 nm) represent the absorbance of

samples at the excitation and emission wavelengths, respectively. As shown in Fig. 4,

the fluorescence intensity of CAT quenches gradually with the adding of QDs-612

with a strong fluorescence emission peak at 334 nm. Small red shifts (288 K, 4.4 nm;

298 K, 4.8 nm) are also observed from the emission wavelengths, which may indicate

the increased polarity of the environment surrounding the fluorescence chromophore

of CAT [28].

Quenching mechanisms include static and dynamic quenching, which can be

distinguished by whether quenchers interact with fluorescent materials to form

complexes [27]. The well-known Stern-Volmer equation can be used to confirm the

quenching mechanism [29]:

[ ] [ ]QQKF

FSV 0q

0 k11 τ+=+= (6)

where F0 and F are the fluorescence intensities of CAT in the absence and presence of

QDs-612. KSV, kq, 0τ and [ ]Q stand for the Stern-Volmer quenching constant, the

bimolecular quenching rate constant, the average lifetime of the fluorescence quantum,

and the molar concentration of QDs-612, respectively. The Stern-Volmer plots for

different temperatures are shown in Fig. 5(A), which are calculated from three parallel

tests. The slopes of these plots are different values of KSV (Table 1), which decreases

with increasing temperature. For biopolymer, 0τ is 10-8 s [30], so kq values (288 K,

5.52×1013 M-1 s-1; 298 K, 4.51×1013 M-1 s-1) are obtained and greater than kq of

biopolymer (2.0×1010 M-1 s-1) [31]. The results indicate that the type of the quenching

is static quenching in this system.

The fluorescence lifetimes of QDs-CAT are also tested to check the mechanism of

quenching further, the values of which are listed in Table 2. Because monoexponential

decay is inadequate, we use two exponentials (Fig. S1) to describe the data using the

following equation [32]:

2211av aa τττ += (7)

where avτ , 1τ and 2τ are the average lifetime of the protein and the lifetime of the

species, respectively. The amplitudes a1 and a2 correspond to the fluorescence

quantum yield weighted proportion of excited fluorophores (a1+a2=1). It is obvious

that the average values of CAT lifetimes in the absence and presence of QDs-612

change scarcely. Since fluorescence lifetimes will change to a large extent in dynamic

quenching, it is static of the fluorescence quenching in the system of QDs-CAT, which

is consistent with the result from Stern-Volmer equation [31].

For the static quenching, the binding constant (Ka) and the number of binding sites

(n) can be obtained by the following formula [33]:

( ) [ ] ( )[ ]( )[ ]000 //1loglog/log FPFFQnKnFFF TTa −−−=− (8)

where F0 and F are the fluorescence intensities of CAT in the absence and presence of

QDs-612. [QT] and [PT] refer to the total concentration of the QDs and the protein,

respectively. Ka is the binding constant and n is the number of binding sites. The log

(F0-F)/F against log [1/([QT]-(F0-F)[PT]/F0)] plots of QDs-CAT system at different

temperatures are shown in Fig. 5(B). The values of Ka and n are listed in Table 1. The

number of binding sites is approximately 1, indicating that three is one binding site in

QDs-CAT system during their interaction. The values of Ka are of the order of 105,

which indicate that three is a strong interaction between CAT and QDs-612.

The thermodynamic parameters can be calculated to judge the binding force using

the following equations [34]:

−

∆=

21

0

1

2 11ln

TTR

H

K

K

a

a (9)

aKRTG ln0 −=∆ (10)

( ) TGHS /000 ∆−∆=∆ (11)

where Ka1 and Ka2 are the binding constants at T1 (288 K) and T2 (298 K), respectively,

while R is the gas constant of value 8.314 J mol-1

K-1

. 0H∆ , 0G∆ and 0S∆ refer

to enthalpy change, free energy and entropy change, respectively, the values of which

are shown in Table 1. The negative values sign for 0G∆ mean that the binding

process is spontaneous. It is obvious that the value of 0H∆ is negative and the 0S∆

value is positive, which indicate that the predominant force in the interaction between

CAT and QDs-612 is hydrophobic interaction [35, 36].

3.3. Synchronous fluorescence and analysis of the interaction mechanism

Synchronous fluorescence spectrum is obtained by scanning at the excitation (λex)

and emission (λem) wavelengths at the same time. When the fixed intervals

( λ∆ =λem-λex) is 15 nm, a spectrum characteristic of tyrosine residues is obtained.

When λ∆ is fixed at 60 nm, the synchronous fluorescence of CAT is characteristic

of a tryptophan residue [37]. Because the shifts of emission maximum are related to

the changes of polarity of these amino acid residues, synchronous fluorescence

spectroscopy technique can be used to analyze the change of molecular

microenvironment [38].

The Synchronous fluorescence spectra of CAT in the absence and presence of

NAC-capped CdTe QDs are shown in Fig. 6. It can be seen in this figure that the

maximum peaks of tryptophan residues are red-shift whereas the maximum emission

of tyrosine residues keep unchanged, which indicate that tryptophan residues expose

to a hydrophilic environment and the binding site of QDs-612 should be near to the

tryptophan residues.

3.4. UV-vis absorption spectra and analysis of the conformational changes of protein

UV-vis absorption spectroscopy can be used to explore the structural changes of

protein. While the strong absorption peak at 208 nm reflects the framework

conformation of CAT [39], the absorption band maximum at 405 nm is the

characteristic absorbance of heme in CAT [7, 40].

The absorbance of CAT at 208 nm decreases with a red-shift of maximum peak

position in Fig. 7(A) with gradual addition of QDs-612 to CAT solution, which

indicate that the binding of QDs-612 to CAT leads to the loosening of the protein

skeleton and decreasing in hydrophobicity of microenvironment of aromatic acid

residues [41]. As shown in Fig. 7(B), a slight increase of the absorption intensity at

405 nm is observed by adding QDs-612. The change of the absorption band maximum

about heme of CAT should be induced by the alteration of the secondary structure of

CAT.

3.5. Circular dichroism spectra and analysis of protein structure changes

To detect sensitive secondary structure changes of CAT, circular dichroism (CD)

measurement was performed. Fig. 8 shows the CD spectra of CAT in the absence and

presence of QDs-612. Two main negative bands at 209 nm and 221 nm are observed,

which are characteristic of secondary structure of CAT [42]. The content of four types

of secondary structure were analyzed by using CDPro software package. The results

are listed in Table 3. The α-helix of CAT decreases and β-sheet has the opposite

tendency, which indicate that the interaction between CAT and QDs-612 induces

unfolding of secondary structure of CAT [43]. The results prove that the QDs-612

bind to the amino acid residues of the skeleton polypeptide chain and destroy the

hydrogen bonding in the protein, which will affect CAT function in vivo.

3.6. Effect of NAC-capped CdTe QDs on CAT activity

The impacts of NAC-capped CdTe QDs on CAT activity were performed in vitro at

physiological pH 7.4. It can be seen from Fig. 9 that the activity of CAT decreases

with adding QDs-612. When the concentration of QDs-612 is 5×10-1 mol L-1

(maximum), the activity of CAT decreases to 70.8% of the initial level. As mentioned

previously in Fig. 7 (B), the change in CAT activity is similar with the variation of

absorption intensity at 405 nm about heme, which indicates that the change of

substituents on the porphyrin rings of hemes leads to the inhibition of CAT activity.

Furthermore, the influence of QDs-612 on CAT activity keeps stably when the

concentration of QDs-612 comes from 3×10-1 mol L-1 to 5×10-1 mol L-1 because

hemes (the active center of CAT) are buried inside the structurally highly conserved

CAT deep and QDs-612 cannot bind to them directly.

4. Conclusions

In this paper, we investigated the interaction between NAC-capped CdTe QDs

(QDs-612) and CAT by using spectroscopic techniques under physiological

conditions. It is found that binding of QDs-612 to CAT is spontaneous with about 1:1

stoichiometry. The predominant force of the binding is hydrophobic interaction. The

results of Synchronous fluorescence, UV-vis absorption spectroscopy and circular

dichroism indicate that the microenvironment and secondary structure of CAT

changes and tryptophan residues expose to a hydrophilic environment. The CAT

activity is inhibited after adding QDs-612. This study provides valuable information

of the toxicity of NAC-capped CdTe QDs to CAT, which is useful for better

understanding the impact of CdTe QDs to human health.

†Acknowledgements

This work is supported by NSFC (21277081), the Cultivation Fund of the Key

Scientific and Technical Innovation Project, Research Fund for the Doctoral Program

of Higher Education, Ministry of Education of China (708058, 20130131110016),

Independent innovation program of Jinan (201202083) and Independent innovation

foundation of Shandong University natural science projects (2012DX002) are also

acknowledged.

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Figure Legends

Fig. 1 (A) Normalized fluorescence spectra (λex=280nm) of NAC-capped CdTe QDs

refluxed for different times of 10 min, 20 min, 30 min, 40 min, 50 min, 60 min. (B)

Corresponding UV-vis spectra of NAC-CdTe QDs. (C) Photograph of emission colors

under a UV lamp.

Fig. 2 HRTEM images of NAC-capped CdTe QDs.

Fig. 3 FT-IR spectra of pure NAC and NAC-capped CdTe QDs.

Fig. 4 Effect of NAC-capped CdTe QDs on CAT fluorescence (corrected) at the

following conditions. CAT: 4×10-7mol L-1; NAC-capped CdTe QDs/(10-7 mol L-1): (a)

0, (b) 1, (c) 2, (d) 3, (e) 4, (f) 5; pH: 7.4; T: 288 K and 298 K.

Fig. 5 (A) Stern-Volmer plots for the quenching of CAT by NAC-capped CdTe QDs at

288 K and 298 K (n=3); (B) The log (F0-F)/F against log [1/([QT]-(F0-F)[PT]/F0)]

plots of QDs-CAT system at 288 K and 298 K (n=3).

Fig. 6 Synchronous fluorescence spectra of CAT in the absence and presence of

NAC-capped CdTe QDs (corrected). (A) ∆λ=15 nm; (B) ∆λ=60 nm. Conditions: CAT:

4×10-7mol L-1; NAC-capped CdTe QDs/(10-7 mol L-1): (a) 0, (b) 1, (c) 2, (d) 3, (e) 4,

(f) 5; pH: 7.4; T: 288 K.

Fig. 7 UV-vis absorption spectra of CAT in the absence and presence of NAC-capped

CdTe QDs. Conditions: CAT: 4×10-7

mol L-1

; NAC-capped CdTe QDs/(10-7

mol L-1

):

(a) 0, (b) 1, (c) 2, (d) 3, (e) 4, (f) 5; pH: 7.4; T: 288 K.

Fig. 8 CD spectra of CAT in the absence and presence of NAC-capped CdTe QDs.

Conditions: CAT: 4×10-7mol L-1; NAC-capped CdTe QDs/(10-7 mol L-1): (a) 0, (b) 2,

(c) 4; pH: 7.4; T: 288 K.

Fig. 9 Effect of NAC-capped CdTe QDs concentration on the activity of CAT.

Conditions: CAT: 4×10-7

mol L-1

; NAC-capped CdTe QDs/(10-7

mol L-1

): (a) 0, (b) 1,

(c) 2, (d) 3, (e) 4, (f) 5; pH: 7.4; T: 288 K.

Table 1 Binding parameters and thermodynamic parameters of the QDs-CAT system

at different temperatures according to Fig. 5 (n=3).

Table 2 Fluorescence lifetime constants of the QDs-CAT system at the following

conditions. CAT: 4×10-7

mol L-1

; NAC-capped CdTe QDs/(10-7

mol L-1

): (a) 0, (b) 1,

(c) 2, (d) 3, (e) 4, (f) 5; pH: 7.4; T: 288 K.

Table 3 Effects of NAC-capped CdTe QDs on the percentage of secondary structural

elements in CAT at 288 K in accordance with Fig. 8.

450 500 550 600 650 700

0.0

0.2

0.4

0.6

0.8

1.0

Flu

ore

scen

ce In

ten

sit

y (

a.u

.)

Wavelengh (nm)

Reflux time:10min,20min,30min,40min,50min,60min

(A)

400 450 500 550 600 650 700

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

Reflux time

10min,20min,30min,40min,50min,60min

Ab

so

rban

ce

(a.u

.)

Wavelength (nm)

(B)

Fig. 1

Fig. 2

4000 3000 2000 1000 0

10

20

30

40

50

60

70

80

90

Tra

ns

mit

tan

ce

(%

)

Wavenumbers (cm-1)

-SH-COOH

NAC

4000 3000 2000 1000 0

0

20

40

60

80

100

Tra

ns

mit

tan

ce (

%)

Wavenumbers ((((cm-1))))

NAC-capped CdTe QDs

-COOM

Fig. 3

280 320 360 400 440

0

2000

4000

6000

8000

10000

12000

Flu

ore

scen

ce In

ten

sit

y (

a.u

.)

Wavelength (nm)

a

f

(A)

288 K

280 320 360 400 440

0

2000

4000

6000

8000

10000

12000

Flu

ore

scen

ce In

ten

sit

y (

a.u

.)

Wavelength (nm)

298 Ka

f

(B)

Fig. 4

0 1 2 3 4 5

1.00

1.05

1.10

1.15

1.20

1.25

1.30

288 K

298 K

F0/F

QDs Concentration ((((10-7

mol L-1))))

(A)

6.4 6.6 6.8 7.0

-1.6

-1.4

-1.2

-1.0

-0.8

-0.6

-0.4

288 K

298 K

log

(( ((F

0-F)) ))/F

log [[[[1/(((([[[[DT]]]]-((((F0-F))))[[[[PT]]]]/F0))))]]]]

(B)

Fig. 5

240 260 280 300 320

0

400

800

1200

1600

2000

Flu

ore

sce

nc

e I

nte

nsit

y (

a.u

.)

Wavelength (nm)

a

f

(A)

240 260 280 300 320

0

2000

4000

6000

8000

10000

Flu

ore

sc

en

ce

In

ters

ity

(a

.u.)

Wavelength (nm)

a

f

(B)

Fig. 6

200 250 300 350

0.0

0.5

1.0

1.5

2.0

2.5

Ab

so

rban

ce

Wavelength (nm)

a

f

(A)

380 400 420 440

0.02

0.03

0.04

0.05

Ab

so

rban

ce

Wavelength (nm)

a

f

(B)

Fig.7

200 220 240 260

-6

-4

-2

0

2

4

6

8

CD

(m

de

g)

Wavelength (nm)

CAT

CAT+QDs (1:0.5)

CAT+QDs (1:1)

Fig. 8

0 1 2 3 4 5

0.0

0.2

0.4

0.6

0.8

1.0

Re

lati

ve

ac

tiv

ity

QDs Concentration ((((10-7

mol L-1))))

Fig. 9

Table 1

Binding parameters and thermodynamic parameters of the QDs-CAT system at

different temperatures according to Fig. 5 (n=3).

T KSV Ka n

0H∆ 0S∆

0G∆

(K) (105 M

-1) (10

5 L mol

-1) (kJ mol

-1) (J mol

-1 K

-1) (kJ mol

-1)

288 5.52±0.02 7.98 1.18 -7.24 87.85

-32.54

298 4.51±0.03 7.21 1.28 -33.42

Table 2

Fluorescence lifetime constants of the QDs-CAT system at the following conditions.

CAT: 4×10-7mol L-1; NAC-capped CdTe QDs/(10-7 mol L-1): (a) 0, (b) 1, (c) 2, (d) 3,

(e) 4, (f) 5; pH: 7.4; T: 288 K.

Number Lifetime (ns) Amplitude (%)

τAV χ2

τ1 τ2 A1 A2

a 1.66 5.15 32.47 67.53 4.02 0.992

b 1.50 5.18 33.50 66.50 3.95 0.975

c 1.79 5.46 40.58 59.42 3.97 0.916

d 1.59 5.31 36.95 63.05 3.94 1.101

e 1.80 5.60 42.76 57.24 3.98 1.054

f 1.90 5.69 45.37 54.63 3.97 1.167

Table 3

Effects of NAC-capped CdTe QDs on the percentage of secondary structural elements

in CAT at 288 K in accordance with Fig. 8.

Molar ratio of

CAT to QDs

Content(%)

α-helix β-sheet β-turn Random coil

1:0 16.5 28.9 22.8 30.9

1:0.5 15.9 29.8 23.5 29.2

1:1 15.1 30.9 24.1 28.4

Graphical abstract

N-Acetyl-L-cysteine-Capped CdTe Quantum Dots with fluorescence emission peak

at 612 nm (QDs-612) could interact with catalase (CAT) which leads to

conformational and functional changes of the enzyme. The potential toxicity of

QDs-612 to CAT was investigated by multi-spectroscopic techniques. Binding of

QDs-612 to CAT caused static quenching of the fluorescence, the change of the

microenvironment of tryptophan residues and the secondary structure of CAT. The

interaction between QDs-612 and CAT was spontaneous through hydrophobic force

with about 1:1 stoichiometry. Besides, the CAT activity was also inhibited for the

bound QDs-612. This work clarifies the fact that QDs-612 can not only contribute to

the conformational changes of CAT but also alter the enzyme function.

Research highlights

1. The interaction between CdTe QDs (QDs-612) and catalase (CAT) is spontaneous.

2. The predominant force of the binding is hydrophobic interaction.

3. The interaction changes the secondary structure of CAT.

4. Tryptophan residues of CAT expose to a hydrophilic environment.

5. QDs-612 could affect the function of CAT by decreasing its catalytic activity.