Biological Trace Element Research

, Volume 146, Issue 3, pp 396–401

The Interactions of Glutathione-Capped CdTe Quantum Dots with Trypsin

Authors

  • Bingjun Yang
    • Shandong Key Laboratory of Water Pollution Control and Resource ReuseSchool of Environmental Science and Engineering Shandong University, China–America CRC for Environment & Health
    • Shandong Key Laboratory of Water Pollution Control and Resource ReuseSchool of Environmental Science and Engineering Shandong University, China–America CRC for Environment & Health
  • Xiaopeng Hao
    • State Key Laboratory of Crystal MaterialsShandong University
  • Yongzhong Wu
    • State Key Laboratory of Crystal MaterialsShandong University
  • Jie Du
    • State Key Laboratory of Crystal MaterialsShandong University
Article

DOI: 10.1007/s12011-011-9262-z

Cite this article as:
Yang, B., Liu, R., Hao, X. et al. Biol Trace Elem Res (2012) 146: 396. doi:10.1007/s12011-011-9262-z

Abstract

Due to their unique fluorescent properties, quantum dots present a great potential for biolabelling applications; however, the toxic interactions of quantum dots with biopolymers are little known. The toxic interactions of glutathione-capped CdTe quantum dots with trypsin were studied in this paper using synchronous fluorescence spectroscopy, fluorescence emission spectra, and UV–vis absorption spectra. The interaction between CdTe quantum dots and trypsin resulted in structure changes of trypsin and inhibited trypsin's activity. Fluorescence emission spectra revealed that the quenching mechanism of trypsin by CdTe quantum dots was a static quenching process. The binding constant and the number of binding sites at 288 and 298 K were calculated to be 1.98 × 106 L mol−1 and 1.37, and 6.43 × 104 L mol−1 and 1.09, respectively. Hydrogen bonds and van der Waals' forces played major roles in this process.

Keywords

Quantum dotsTrypsinToxic interactionMulti-spectroscopic techniquesFluorescence spectroscopy

Introduction

Quantum dots (QDs) are semiconductor nanocrystals in the size range of 1–10 nm [1]. In the past few decades, quantum dots have been extensively investigated and present a great potential for biolabelling applications due to unique fluorescent properties such as broad excitation spectra, high emission intensity, photostability, and narrow emission spectra [24]. With the extensive application of QDs, the potential adverse effects of QDs on human health and the environment cause the concerns of researchers [57]. Due to various synthetic methods, each kind of QD possesses its own unique physical and chemical properties, including size, charge, biological activity of their surface coating materials, etc., which in turn determine the potential toxicity of QDs [8]. Few studies are specifically designed for toxicological assessment [7].

Trypsin is a kind of serine protease which functions in digestion and other essential biological processes [9, 10]. As a proteolytic enzyme, trypsin cleaves peptide bonds at the carboxylic groups of arginine, lysine, and ornithine working optimally at pH 7.5–8.5 [11]. Previous studies about the interaction between protein and QDs focus on the structure changes of protein caused by QDs [8, 12]. Quantum dots FRET-based probes were used to monitor the enzymatic activity of trypsin by several research groups [13, 14]. Further studies are still necessary to investigate the function changes of the protein when it interacts with QDs from a toxicological point of view. In this work, UV–vis absorption spectra, synchronous fluorescence spectroscopy, fluorescence emission spectra, and other experimental methods are used to explore the effect of glutathione-capped CdTe quantum dots on the activity and conformation of trypsin. This report extends the method to explore the toxicity of QDs at the molecular level.

Experimental Section

Materials

Trypsin (Amresco, cat. no.0458) was dissolved in ultrapure water to form a 5 × 10−4-mol L−1 solution, then preserved at 0–4°C and diluted as required. Glutathione-capped CdTe QDs were acquired from the State Key Laboratory of Crystal Materials at Shandong University. The average particle size is about 3 nm, emission wavelength is 530 nm, and the concentration of the stock solution is 1.0 × 10−3 mol L−1 [15]. Glutathione was bought from Sinopharm Chemical Reagent Co., Ltd. N-α-Benzoyl-l-arginine ethyl ester (BAEE, purchased from Sinopharm Chemical Reagent) was biological reagent grade and dissolved in ultrapure water to form a 1.0 × 10–2-mol L–1 solution. Phosphate buffer (0.2 mol L–1, mixture of NaH2PO4 and Na2HPO4 solution) was used to control pH at 7.6 (optimum pH for enzymatic activity tests). NaH2PO4·2H2O and Na2HPO4·12H2O were of analytical reagent grade and obtained from Tianjin Kermel Chemical Reagent Co., Ltd. All solutions were prepared with ultrapure water.

Apparatus

The ultraviolet visible absorbance spectra (UV–vis) and spectrophotometric determination were recorded on a UV-2450 spectrometer (Shimadzu, Japan) in 1-cm quartz cells. Fluorescence spectra were measured on an F-4600 Spectrofluorimeter (Hitachi, Japan) equipped with a xenon lamp light source and 1.0-cm quartz cells. The pH was measured with a pHs-3C acidity meter (Shanghai Pengshun Scientific Instrument Co., Ltd.).

Methods

Trypsin Activity Assay

The method for trypsin activity determination was described elsewhere [16, 17]. The activity of trypsin was measured by spectrophotometric method using 1 mM BAEE as the substrate. Trypsin catalyzed the hydrolysis of BAEE into N-α-benzoyl-l-arginine (BA), which is much more absorptive than BAEE at 253 nm [1820]. This process was shown in Scheme 1. The BAEE and phosphate buffer were mixed in 3-mL quartz cells and used as reference solution. After spiked with trypsin, the hydrolysis of BAEE was recorded spectrophotometrically every 30 s at 253 nm for a period of 12 min by monitoring the increase of BA. The measurements were conducted at room temperature (about 25°C) and repeated three times.
https://static-content.springer.com/image/art%3A10.1007%2Fs12011-011-9262-z/MediaObjects/12011_2011_9262_Sch1_HTML.gif
Scheme 1

The hydrolysis reaction of BAEE catalyzed by trypsin

UV–Visible Absorption Spectra

PBS (1.0 mL, 0.2 mol L−1), 2 mL trypsin (5.0 × 10−5 mol L−1), and various amounts of CdTe QDs solution were added to 10-ml colorimetric tubes, then diluted with ultrapure water to the mark. After 30 min, the equilibrated solution was poured into the quartz cells, and the spectrum was recorded in the range of 190–350 nm using CdTe QDs as references.

Fluorescence Measurements

Emission spectra were performed on a F-4600 spectrofluorimeter and recorded in the wavelength range of 290–450 nm upon excitation at 280 nm with a scanning speed of 240 nm/min and scanning voltage of 650 V. Excitation and emission slit widths were set to 5 and 10 nm, respectively. The synchronous fluorescence spectra, which were measured by scanning simultaneously the excitation and emission monochromator, were obtained at λex = 250 nm, ∆λ = 15 nm, and ∆λ = 60 nm.

Results and Discussion

Effect of CdTe QDS on Trypsin Activity

Trypsin catalyzes the conversion of BAEE into BA. Performing this trypsin assay can obtain the information of activity for trypsin. As shown in Fig. 1a, the absorbance increases until the reaction reaches chemical equilibrium. In the time between 2 and 6 min after addition of trypsin, the hydrolysis of BAEE follows zero-order kinetics: the rate of the reaction is independent of the substrate concentration [21]. The absorbance is linear with time which indicates that the formation of BA proceeded at a constant rate. The slope of the straight line defines the trypsin activity. Obvious effects on the activity of trypsin caused by CdTe QDs are observed from Fig. 1b. The CdTe QDs used in this study were stabilized by glutathione, so the effect of glutathione on trypsin activity was tested. Weak inhibition is found for glutathione-containing solutions. Therefore, the decrease of the trypsin activity is mainly caused by the interactions between CdTe QDs and trypsin. We suggest that this is likely due to the conformational changes of trypsin when CdTe QDs were added.
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Fig. 1

a Trypsin activity measured in the presence of CdTe QDs or glutathione and normalized against the control experiment. b Zero-order kinetics of BAEE hydrolysis by trypsin. Conditions: a trypsin, b trypsin–glutathione (1.2 × 10−5 mol L−1), c trypsin–CdTe QDs (2 × 10−6 mol L−1); trypsin: 1.0 × 10−6 mol L−1; BAEE: 1.0 × 10−3; PBS buffer: 0.02 mol L−1, pH = 7.6; T = 298 K

UV–VIS Absorption Investigation

As a simple and efficacious method, UV–visible absorption spectroscopy technique can be used to explore the structural changes of a protein [22]. Using a mixture of CdTe QDs and PBS at the same concentration as the reference solution, the UV–vis absorption spectra of trypsin in the presence and absence of CdTe QDs are shown in Fig. 2. Trypsin has two main absorption bands. The strong absorption peak located at about 205 nm reflects the peptide bond absorption peak which is due to the transition of π → π* of C = O [23, 24], and the absorbance of trypsin around 280 nm is primarily caused by Tyr and Trp (a very small extent on the amount of Phe and disulfide bonds) [2527]. When CdTe QDs are gradually added, the intensity of the absorption peak at about 205 nm decreased and underwent an obviously red shift. A slight decline of the peak around 280 is also observed. The results suggest that the microenvironment of the two aromatic acid residues is altered, and the protein conformation is changed [28].
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Fig. 2

UV–vis spectra of trypsin in the presence of different concentrations of CdTe QDs. Conditions: (a) trypsin: 1 × 10−5 mol L−1; (b) CdTe QDs/×10−5 mol L−1: (a) 0, (b) 0.5, (c) 1, (d) 2. PBS buffer: 0.02 mol L−1, pH = 7.6; T = 298 K

Synchronous Fluorescence Spectroscopy Investigation

Synchronous fluorescence spectroscopy can give information about the molecular environment in the vicinity of the chromosphere molecules [29].When the wavelength interval (∆λ) is 60 or 15 nm, synchronous fluorescence spectra only show tryptophan residues or tyrosine residues of trypsin [30]. Trypsin contains four tryptophan and ten tyrosine residues. As seen in Fig. 3, the intensity of the synchronous fluorescence of trypsin at ∆λ = 15 nm was progressively reduced when different amounts of CdTe QDs were added. The emission peaks did not shift, indicating that CdTe QDs have little effect on the microenvironment of the tyrosine residues in trypsin. In Fig. 4, a gradual increase of the CdTe QDs concentration in the solutions of trypsin resulted in a decrease of synchronous fluorescence intensity and a slight red shift at the emission maxima, showing that the polarity around the tryptophan residues is increased and the hydrophobicity is decreased [31].
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Fig. 3

Synchronous fluorescence spectra of trypsin at ∆λ = 15 nm. Conditions: (a) trypsin: 5 × 10−6 mol L−1; (b) CdTe QDs/×10−6 mol L−1: (a) 0, (b) 4, (c) 8, (d)12, (e)16, (f) 20. PBS buffer: 0.02 mol L−1, pH = 7.6; T = 298 K

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Fig. 4

Synchronous fluorescence spectra of trypsin at ∆λ = 60 nm. Conditions: (a) trypsin: 5 × 10−6 mol L−1; (b) CdTe QDs/×10−6 mol L−1: (a) 0, (b) 4, (c) 8, (d)12, (e)16, and (f) 20. PBS buffer: 0.02 mol L−1, pH = 7.6; T = 298 K

Fluorescence Emission Spectroscopy

The Fluorescence Quenching Mechanism

Trypsin contains three intrinsic fluorophores: tryptophan, tyrosine, and phenylalanine. From Fig. 5, the fluorescence intensity of trypsin decreases when different amounts of CdTe QDs solution were added. Fluorescence quenching mechanisms are classified into dynamic quenching and static quenching which are usually analyzed according to the classical Stern–Volmer equation:
$$ {F_0}/F = 1 + {K_{\text{q}}}{\tau_0}\left[ Q \right] = 1 + {K_{\text{SV}}}\left[ Q \right] $$
(1)
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Fig. 5

Fluorescence emission spectroscopy. Conditions: (a) trypsin: 5 × 10−6 mol L−1; (b) CdTe QDs/×10−6 mol L−1: (a) 0, (b) 4, (c) 8, (d)12, (e)16, and (f) 20. PBS buffer: 0.02 mol L−1, pH = 7.6; T = 298 K

where F0 and F are the steady-state fluorescence intensities in the absence and presence of quencher, respectively; Kq is the quenching rate constant of the biological macromolecule; τ0 is the fluorescence lifetime of the system in the absence of quencher; Ksv is the Stern–Volmer quenching constant; and [Q] is the concentration of the quencher (CdTe QDs). The Stern–Volmer plots are shown in Fig. 6.
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Fig. 6

Stern–Volmer plots of fluorescence quenching for trypsin with CdTe QDs at different temperatures

For static quenching, increased temperature reduces the stability of the complex formed, resulting in a reduced quenching constant. In contrast, a higher temperature results in larger diffusion coefficients, and the dynamic quenching constants will increase when temperature rises. The Ksv values were calculated to be 3.5 × 104 and 2.7 × 104 L mol−1 at 288 and 298 K, respectively. It seems that this quenching process is static quenching.

Since the fluorescence lifetime of the biopolymer is 10−8 s, the quenching rate constant can be obtained by Kq = Ksv/τ0. This Kq value was observed to be 3.5 × 1012 and 2.7 × 1012 L mol−1 s−1 at 288 and 298 K, respectively. The quench constants are greater than the maximum scatter collision quenching constant of various quenchers with biopolymers (2.0 × 1010 L mol−1 s−1) [32]. Consequently, we confirm that the quenching is mainly a static quenching process.

Binding Constant and the Number of Binding Sites

For the static quenching interaction, the binding constant (Ka) and the number of sites (n) can be gotten by the following equation [33]:
$$ \lg \frac{{{F_{{0}}} - F}}{F} = { \lg }\;{\text{Ka}} + n\;{ \lg }\;{[}Q{]} $$
(2)
where F0, F, and [Q] are the same as in Eq. 1, Ka is the binding constant, and n is the number of binding sites. The binding constant and the number of binding sites at 288 and 298 K were calculated to be 1.98 × 106 L mol−1 and 1.37, and 6.43 × 104 L mol−1 and 1.09, respectively.

The Interaction Forces Between CdTe QDS and Trypsin

Generally speaking, the interaction forces between nanoparticles and biological macromolecules exist through four binding modes: hydrogen bond, hydrophobic interactions, van der Waals force, and electrostatic interactions, etc. [34]. Assuming the enthalpy change (ΔH°) does not vary over the temperature range, the value of enthalpy change (ΔH), entropy change (ΔS), and free energy change (∆G°) can be estimated from the equations below:
$$ {\text{In}}\frac{{{{({\text{Ka}})}_2}}}{{{{({\text{Ka}})}_1}}} = \frac{{\Delta H^\circ }}{R}\left( {\frac{{1}}{{{T_{{1}}}}} - \frac{{1}}{{{T_{{2}}}}}} \right) $$
$$ \Delta G = \Delta H - T\Delta S = - RT\;{\text{InKa}} $$
ΔH° and ΔS° were calculated to be −245 kJ/mol and −728 J mol−1 K 1G° were −35 and −27 kJ/mol when the temperatures were 288 and 298 K, respectively. The negative ΔH° and ΔS° reveal the predominance of hydrogen bonds and van der Waals' forces in the binding of CdTe QDs and trypsin. The negative sign for ΔG° means that the interaction process is spontaneous.

Conclusions

In conclusion, the interaction of glutathione-capped CdTe QDs with trypsin was studied using synchronous fluorescence spectroscopy, fluorescence emission spectra, and UV–vis absorption spectra under simulative physiological conditions. The results revealed that the microenvironment around trypsin was changed after CdTe QDs were added. Hydrogen bonds and van der Waals' forces played major roles in the interaction between CdTe QDs and trypsin. It is the structural changes of trypsin that strongly influence the activity of trypsin. Based on the relationship between structure and function, this paper establishes a new and simple strategy to investigate the interaction between QDs and trypsin at molecular level.

Acknowledgments

This work is supported by NSFC (20875055), the Cultivation Fund of the Key Scientific and Technical Innovation Project, and the Ministry of Education of China (708058), and the Key Science-Technology Project in Shandong Province (2008GG10006012) is also acknowledged.

Copyright information

© Springer Science+Business Media, LLC 2011