The Interactions of Glutathione-Capped CdTe Quantum Dots with Trypsin
- First Online:
- 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
- 240 Views
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.
KeywordsQuantum dotsTrypsinToxic interactionMulti-spectroscopic techniquesFluorescence spectroscopy
Quantum dots (QDs) are semiconductor nanocrystals in the size range of 1–10 nm . 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 [2–4]. With the extensive application of QDs, the potential adverse effects of QDs on human health and the environment cause the concerns of researchers [5–7]. 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 . Few studies are specifically designed for toxicological assessment .
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 . 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.
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 . 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.
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.).
Trypsin Activity Assay
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.
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
UV–VIS Absorption Investigation
Synchronous Fluorescence Spectroscopy Investigation
Fluorescence Emission Spectroscopy
The Fluorescence Quenching Mechanism
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) . Consequently, we confirm that the quenching is mainly a static quenching process.
Binding Constant and the Number of Binding Sites
The Interaction Forces Between CdTe QDS and Trypsin
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.
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.