Research article Received: 7 August 2014,
Accepted: 2 December 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2847
Interaction of digestive enzymes with tunable light emitting quantum dots: a thorough Spectroscopic investigation Vaishnavi Ellappan,a,b Manibalan Kesavan,a Parameshwari Ramalingam,c Jeganathan Kulandaivelc and Renganathan Rajalingama* ABSTRACT: In this article, we have examined the direct spectroscopic and microscopic evidence of efficient quantum dotsα-chymotrypsin (ChT) interaction. The intrinsic fluorescence of digestive enzyme is reduced in the presence of quantum dots through ground-state complex formation. Based on the fluorescence data, quenching rate constant, binding constant, and number of binding sites are calculated under optimized experimental conditions. Interestingly, fluorescence quenching method clearly illustrated the size dependent interaction of MPA-CdTe quantum dots. Conformational change of ChT was traced using synchronous fluorescence measurements, circular dichroism and FTIR spectroscopic methods. Furthermore, the AFM results revealed that the individual enzyme molecule dimensions were changed after interacting with quantum dot. Consequently, this result could be helpful for constructing safe and effective utilisation of QDs in biological applications. Copyright © 2015 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: MPA-CdTe quantum dots; chymotrypsin; atomic force microscopy; circular dichroism
Introduction Semiconductor quantum dots (QDs) are nanomaterials, endowed with unique size-dependent optical properties such as high quantum yield, and good photostability (1,2). Exclusive properties of these nanoparticles make them an excellent candidate for numerous applications from bioimaging to drug delivery (3–6). Knowledge of protein–nanoparticles interactions is important in order to understand how nanoparticles function in a biological environment. The focus of recent research is on the interactions between these small molecules with proteins (7). Such findings are significant as they can help to obtain a better understanding about the adsorption and distribution of these small molecules in living systems. Jhonsi et al. investigated the interaction of oxide nanoparticles (ZnO and TiO2) with serum albumins (8,9). In addition, some pioneering work on the semiconductor colloidal cadmium chalcogenoid QDs namely starch-capped Cadmium sulphide (CdS) with bovine serum albumin (BSA), CdSe-GSH with Human serum albumin (HSA) and MPA-CdTe with denatured BSA has also been carried out (10–13). Major interactions involved in protein adsorption can be classified as electrostatic, hydrogen-bonding, hydrophobic, etc. An intensive survey of research shows that QDs are now widely used in biotechnology and medical applications, implying the interactions of water-soluble semiconductor QDs with functional biomolecules. Yilin Wang et al. investigated the interaction of pepsin with CdSe (14) and papain with CdTe QDs (15). Despite these studies, the interaction mechanism of QDs with chymotrypsin is still unexplored. A clear investigation of this problem is important for the safe and effective utilization of QDs in biomedical research. Fluorescence quenching measurement has been used to obtain adequate information about the
Luminescence 2015
structure and dynamics of biologically important macromolecular systems such as proteins. It can reveal the accessibility of quenchers to protein’s fluorophore group, and help in understanding the essential binding phenomenon. This area has been actively studied for decades because of its importance in the wide range of biomedical applications, immunomagnetic cell separation (16), immobilized enzymes or catalysts (17), biosensors (18,19), drug delivery systems (20), and so on. In the present work, we have chosen α-chymotrypsin (ChT) as a target biomolecule. Chymotrypsin is an ellipsoidal-shaped protein and belongs to the serine protease family. It functions as a digestive enzyme in mammals, containing an α-helix at the C-terminal end (residues 230–245) and several β-sheets domains (21). Being an extensively characterized protein with well defined geometry it is an excellent system to study the protein surface recognition. The effect of the core and core/shell QDs on the intrinsic tryptophan fluorescence quenching has also been explored. We also followed
* Correspondence to: Rajalingam Renganathan, School of Chemistry, Bharathidasan University, Tiruchirappalli 620 024, India. Tel: +91 431 2407053; Fax: +91 431 2407045. E-mail: [email protected] a
School of Chemistry, Bharathidasan University, Tiruchirappalli 620 024, India
b
Department of Chemistry, Sri G. V. G Visalakshi College for Women, Udumalpet 642 128
c
Centre for Nanoscience and Nanotechnology, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India Abbreviations: AFM, atomic force microscopy; BSA, bovine serum albumin; CD, circular dichroism; SAED, selected area electron diffraction; TEM, transmission electron micrographs.
Copyright © 2015 John Wiley & Sons, Ltd.
V. Ellappan et al. alterations in the secondary and tertiary structures of the enzyme by means of synchronous fluorescence, circular dichroism (CD) measurements and characterized by using steadystate fluorescence, Fourier transform infrared spectra (FT-IR), and atomic force microscopy (AFM) techniques under physiological conditions.
solution was then heated at 50°C until black Te powder was fully disappeared and white sodium tetraborate precipitate settled at the bottom of the flask (about 45 min), the resulting purple NaHTe in clear supernatant was separated out and injected by syringe into 24 mL of N2 saturated pure water to produce a 0.01 M of NaHTe solution.
Experimental
Synthesis of quantum dots
Cadmium chloride (CdCl2.2.5H2O), tellurium (Te), sodium borohydride (NaBH4), mercaptopropionic acid (MPA) and tetraethylorthosilicate (TEOS) were purchased from Sigma-Aldrich. α-Chymotrypsin was dissolved in double-distilled water to prepare stock solutions (1 × 104 M) and stored at 0–4°C. All measurements were performed at ambient temperature. ChT (1 × 106 M in phosphate buffer at pH 7.5) was titrated by successive addition of mercaptopropionic acid capped CdTe QDs stock solution prepared at various time intervals. Titrations were manually done by using a micropipette for the addition of QDs. The samples were carefully purged by using pure nitrogen gas for 10 min. Quartz cells with high vacuum Teflon stopcocks were used for purging. The excitation wavelength of ChT was fixed as 280 nm. All measurements were done in ambient temperature (25°C). The fluorescence intensities were corrected for absorption of the exciting light and reabsorption of the emitted light to decrease the inner filter effect by using the following relationship:
Mercaptopropionic acid-coated CdTe QDs (MPA-CdTe) were synthesized using wet chemical route. MPA was dissolved in 100 mL water in a three-necked flask. The pH of the solution was adjusted by adding 0.5 mol L1 NaOH and it was saturated with nitrogen for 20 min. Subsequently, freshly prepared NaHTe solution was injected into the solution under vigorous stir. The CdTe precursors were formed at this stage, accompanied by a faint yellow colour formation. Afterward, the solution was kept at 100°C refluxing temperature for a specified period. To monitor the growth of the QDs, aliquots of reaction solution were taken at various time intervals. To obtain MPAcoated CdTe QDs precipitate, the QDs solution was added in acetone, then precipitated QDs were separated by centrifugation at 15,000 rpm for 10 min and were repeated several times to remove unreacted mercaptopropionic acid (23). The sedimentation was dried in a vacuum drying chamber for IR spectroscopy analysis. For overcoating the CdTe core with SiO2, 20 mL of ethanol, 0.5 mL of MPA-CdTe aqueous solution, and 0.25 mL of TEOS where added into RB and mixed under stirring (24). Scheme 1 shows the schematic representation of silica-coated CdTe QDs. The particle size of the prepared CdTe QDs was determined from the first absorption maximum according to equation (2) mentioned below (25):
Fcor ¼ Fobs e
A
exc=2
þ Aem=2
(1)
where, Fcor and Fobs are the fluorescence intensities corrected and observed, respectively, and Aexc and Aem are the absorption of the systems at the excitation and the emission wavelength, respectively. The fluorescence intensity used in this paper is the corrected intensity. The spectra were recorded using a spinning cell holder as an accessory and of native α-ChT in 50 mM sodium phosphate buffer, pH 7.5. α-Chymotrypsin (1 mg mL–1) solution in the aqueous buffer was used for recording the spectrum. Fourier transform infrared spectra were obtained using a Perkin-Elmer Spectrum RXI FT-IR Spectrometer at room temperature in the range of 4000–400 cm1. The samples were kept in a liquid cell between two windows (CaF2). Mirror velocity was 0.3 cm/sec and number of co-added scans are four then total collection time was less than 2 min. AFM images of ChT and QDs adsorbed on ChT were obtained with a multimode AFM (Agilent 5500). Transmission electron micrographs (TEM) were recorded using a FEI TECNAI 3010 electron microscope operating at 300 kV (Cs = 0.6 mm, resolution 1.7 Å). Circular dichroism (CD) measurements were carried out on a Jasco spectropolarimeter (J-715), calibrated with D-10-camphor sulphonic acid. Concentration of the chymotrypsin sample was adjusted within the range of 10–5 to 10–6 M.
D ¼
9:8127 x 107 λ3 1:7147 x 103 λ2 þ ð1:0064Þ λ 194:84
(2)
Where D (nm) is the particle size of MPA-CdTe QDs and λ (nm) is the wavelength of the first excitonic absorption peak of the corresponding MPA-CdTe QDs. The results showed that the particle diameters of the MPA-CdTe QDs were around 2.03, 3.17, 3.6 and 4.23 nm (photograph of QDs are illustrated in Fig. 1d), corresponding to the first absorption maximum. The particle size of the red emissive QDs was confirmed by TEM measurement and the result obtained was illustrated in Fig. 1(a). From TEM results, we identified that the fine and uniform particle size was around ~4.52 nm. It was observed that the particle sizes determined from both the calculation as well as TEM measurements were matched well. The selected area electron diffraction (SAED) pattern shows a set of rings instead of spots due to the random orientation of the nanoparticles (Fig. 1b).
Preparation of sodium hydrotelluride (NaHTe) NaHTe was prepared according to the earlier reported procedure with some modifications (22). Briefly, a mixture of 0.0284 g of NaBH4 and 0.0197 g of Te powder (molar ratio of Te to NaBH4 is 1:3) was added to a small one-necked flask, containing 1 mL of N2 saturated double-deionized water. A small outlet was connected to the flask to release resulting hydrogen. The
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Scheme 1. Shows the schematic route for synthesizing CdTe-SiO2.
Copyright © 2015 John Wiley & Sons, Ltd.
Luminescence 2015
Spectroscopic evidence for Chymotrypsin and quantum dots interaction
Figure 1. (a) High resolution TEM image; and (b) SAED pattern of red emissive CdTe quantum dots. (c) Size-dependent emission behaviour of green, yellow, orange and –7 red MPA-CdTe (3 × 10 M) and CdTe-SiO2. (d) Photograph showing the image of emission of MPA-CdTe under ultraviolet (UV) light and the increase in particle diameter is indicated by an arrow.
Figure S1 illustrates the TEM image of synthesised CdTe-SiO2. The concentration of prepared QDs was calculated from the absorption spectrum using LambertBeer’s law, equation (3), (3,23): A ¼ εC1
(3)
In equation (3), A is the absorbance at the peak position of the first excitonic absorption peak for CdTe, C is the molar concentration of the QDs of the same sample, l is the path length of the radiation beam used for recording the absorption spectrum, and ε is the molar extinction coefficient (for CdTe QDs, ε = 10,043 (D)2.12 in which D is the particle size of CdTe QDs) (26). Based on equation (3) concentration of green (G), yellow (Y), orange (O) and red (R) CdTe QDs are 1 × 105 M.
features (figure not shown) proves the successful chemical attachment of MPA onto the QDs surface. For MPA, the peak at 2600– 2500 cm1 is assigned to S–H stretching vibration. The peak at 3500–3000 cm1 was produced by the stretching vibration of O– H. Especially, the presence of the S–H stretching vibration at 2600–2500 cm1 for the QD has not been found. This reflects that –SH in MPA of the QDs was covalently bound on the QDs surface. More –COOH exposure onto the QDs surface can be illustrated by a much narrower absorption band around 3400 cm1, a decreased absorption at 1675 cm1, a blue-shifted from 1609 cm1 to 1673 cm1. Peaks at 1250 cm–1 and 954 cm–1 illustrated the Si–O–Si absorption and bending vibration of Si–OH and at 471 cm–1 corresponded to the bending vibration of Si–O–Si. Thus the IR spectral features clearly indicate that the SiO2 layers are successfully coated onto the surface of MPA-CdTe (27) (figure not shown).
Results and discussion Spectral characterization of CdTe QDs In order to evaluate the influence of both the core and core/shell QDs on ChT, we finally selected a set of various sizes of MPACdTe and CdTe-SiO2 QDs (graphical abstract). Generally, colloidal QDs have a broad absorption and narrow emission spectra in the visible region. Their narrow emission is attributed to the radiative recombination of electron and hole pairs. Monodispersed QDs with narrow size distributions have been synthesized; Fig. 1(c) shows the size-dependent emissive behaviour of green, yellow, orange and red MPA-CdTe QDs. To identify the conjugation mode between mercaptopropionic acid and CdTe QDs coated by mercaptopropionic acid, the IR spectra of pure MPA and the QDs were measured. The resemblance in both IR spectral
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Absorption characteristics of ChT-CdTe quantum dot interactions Before performing the excited state reactions between ChT and QDs, it is essential to know the type of interaction between them in the ground state. The peak centred at ~280 nm in the ChT absorption spectrum is due to aromatic amino acids (primarily tryptophan and tyrosine). With gradual addition of CdTe-R to the ChT solution, the absorption spectrum undergoes a hyperchromic effect without any noticeable shift, indicating that QDs affect the band corresponding to the tryptophan and tyrosine residues of the ChT (Fig. 2a). In contrast, addition of a similar concentration of CdTe-G affects the absorbance spectrum of ChT to a greater extent. A
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Absorbance
V. Ellappan et al. 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
Kapp ¼
ChT+ CdTe-R ChT
230
330
430
Absorbance
(4)
½ChT……CdTe ½ChT· ½CdTe
(5)
The Kapp value was calculated by the method of Benesi and Hildebrand (29) using the following equation (6): 530
630
Aobs ¼ ð1 αÞ C0 εChT1 þ αC0 εc 1
Wavelength (nm) 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
Kapp
⇌ ChT…CdTe
ChT þ CdTe
a)
(6)
where Aobs is the absorbance of the ChT solution containing different concentrations of MPA-capped CdTe at 280 nm, α is the degree of association between ChT and capped CdTe, εChT and εc are the molar extinction coefficients at the defined wavelengths for ChT and the formed complex respectively, C0 is the initial concentration of ChT and ‘l’ is the optical path length, which is taken as unity. Equation (6) can be expressed by equation (7), where the A0 and Ac are the absorbance of ChT and the complex at 280 nm, with the concentration C0:
b) ChT+ CdTe-G
ChT
Aobs ¼ ð1 αÞA0 þ αΑc 230
330
430
530
(7)
630
1/[Aobs-A0]
Wavelength (nm) 18
[CdTe-G]
16
[CdTe-Y]
14
[CdTe-O]
12
[CdTe-R]
10
[CdTe-SiO2]
c)
At high concentrations of QDs, α can be equated to (Kapp [capped CdTe])/(1 + Kapp[QDs]). In this case, equation (7) can be expressed as equation (8):
600
8
a
500
6 4 0 0
0.2
0.4
0.6
0.8
1
1.2
1/[Q]
Intensity
400
2
300 200
–5
Figure 2. UV-vis absorption spectra of ChT dotted line (conc. ~1 × 10 M). (a) In the presence of CdTe-R; and (b) in the presence of CdTe-G; at ambient temperature –6 in buffer solution. The concentrations of QD were (0) 0; (1) 7.7 × 10 M; (2) 1.54 × –5 –5 –5 –5 10 M; (3) 2.3 × 10 M; (4) 3.1 × 10 M; and (5) 3.8 × 10 M. (c) The straight line dependence of 1/(Aobs – A0) on the reciprocal concentration of different size QDs.
100 0 300
320
340
360
380
400
420
440
Wavelength (nm) 600
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b 500 400
Intensity
similar experiment was also conducted in the presence of yellow, orange and CdTe-SiO2. The above results can be rationalized in terms of a strong interaction between QDs and ChT in the ground state through complex formation, as confirmed by earlier studies. (28) In contrast, addition of even lower concentrations of green emissive QDs leads to abnormal changes in ChT, as shown in Fig. 2(b). The absorption spectra of ChT in the absence and presence of yellow and orange CdTe are shown in Fig. S2(a) and Fig. S2(b). The addition of CdTe-SiO2 led to a gradual increase in ChT absorption with a red shift (higher wavelength) of 2 nm, as shown in Fig. S2(c). These observations indicated that there was a structural change (microenvironment) in ChT which occured upon interaction with the surface of MPA-CdTe. Importantly, the equilibrium for the formation of a complex between ChT and CdTe is defined by equation (4) where Kapp is the apparent association constant:
300 200 100 0 300
320
340
360
380
400
420
440
Wavelength (nm) Figure 3. Fluorescence quenching of ChT (black line) by a) CdTe-G b) CdTe- SiO2 –6 of various concentration ranging from 05 × 10 (λexi = 280: λemi = 340 nm) and dotted line emission behaviour of CdTe-G and CdTe-SiO2 respectively, excitated at similar wavelength.
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Luminescence 2015
Spectroscopic evidence for Chymotrypsin and quantum dots interaction 10000
1 1 1 ¼ þ Aobs A0 Ac A0 Kapp ðAc -A0 Þ½CdTe
a
Prompt
(8)
ChT
Counts
1000
Therefore, if the enhancement of absorbance at 280 nm was due to absorption of the complex, one would expect a linear relationship between 1/(Aobs-A0) and the reciprocal concentration of CdTe with a slope equal to 1/Kapp(Ac – A0) and an intercept equal to 1/(Ac – A0) (shown in the Fig. 2c). The values of Kapp obtained from such plots are depicted in Table S1.
ChT+CdTe-G
100
10
1 0
10
20
Fluorescence spectral analysis of quantum dots with α-chymotrypsin
(9)
where F0 and F are the fluorescence intensities of ChT in the absence and presence of MPA-CdTe, respectively. KSV is the Stern–Volmer constant and [MPA-CdTe] is the concentration of respective quencher. From the Ksv Table 1, we have calculated the value of quenching rate constant by using the lifetime (τ) of ChT (4.02 ns, from time resolved measurement). In many instances, the fluorophore can be quenched both by collision and by complex formation with the same quencher. The gradual deviation from linearity of the Stern–Volmer plots (Fig. S3) with continuous addition of CdTe-G suggests the existence of more than one binding site with lower binding affinity and different accessibilities, it also indicated the occurrence of combined quenching. In contrast, a linear Stern–Volmer plot obtained for other QDs is shown in Fig. S3. In this case, the values of kq are in the order of 1012, consequently we confirm that the quenching is mainly a static quenching process. For static quenching, increased temperature reduces the stability of the complex formed, resulting in a reduced quenching constant and the results are shown in Table 1. Stern–Volmer quenching constant of the ChTquantum dot system at T = 298 and 308 K Ksv × 106 M–1 System
kq M–1 s–1 × 1012
298.15 K
308.15 K
298.15 K
308.15 K
0.3623 0.2976 0.301 0.253 0.1723
0.3545 0.2689 0.1631 0.132 0.0963
90.00 73.97 74.81 63.13 42.82
88.11 66.84 40.54 22.82 23.935
CdTe-G CdTe-Y CdTe-O CdTe-R CdTe-SiO2
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10000
40
b
Prompt ChT
1000
Counts
The interaction of ChT with MPA-CdTe was studied using a spectrofluorometer at room temperature. Chymotrypsin was dissolved in phosphate-buffered medium and the fluorescence spectrum was recorded at 340 nm. The solution of ChT (1 × 106 M) was titrated with increasing concentrations of (0–5 × 106 M) QD solution, as shown in Fig. 3(a, b). The MPA-CdTe also has luminescent properties but its excitation (450 nm) and emission (522 nm) wavelengths are much further away from the ChT absorption and emission (280 and 340 nm respectively), so it may not interrupt the interaction study. While increasing the concentration of QDs the emission intensity of ChT was found to decrease progressively with the red shift of around 5 nm. A Stern–Volmer plot was utilised to analyse the quenching phenomenon. The ratios F0/F were calculated and plotted against quencher concentration according to equation (9): F0 =F ¼ 1 þ Ksv ½MPA-CdTe
30
Time (ns)
ChT+CdTe-R
100
10
1 0
10
20
30
40
Time (ns) Figure 4. Fluorescence quenching of ChT (black line) by CdTe-SiO2 at various concen–6 trations ranging from 05 × 10 M (λexi = 280: λemi = 340 nm) and dotted line emission behaviour of CdTe-SiO2 excited at similar wavelengths. Fluorescence decay of ChT 6 (1 × 10 M) in the absence and presence of (a) CdTe-G; (b) CdTe-R in the concentration 6 range of 5 × 10 M. The blue coloured decay is the prompt used for calibration.
Table 1. Furthermore the type of interaction between ChT and CdTe was also confirmed by time-resolved spectroscopy. Henceforth, the fluorescence results indicated that QDs can effectively quench the fluorescence of ChT in a size-dependent manner. Lifetime measurements According to the results discussed in the above section, it is reasonable to mention that the complexes could be formed between ChT and QDs. Fig. 4(a, b) shows the fluorescence decay curves of ChT in the absence and presence of CdTe-G and CdTe-R respectively. Results clearly illustrate that there was no change in fluorescence lifetime of enzyme in the presence of both materials. Therefore, the result shows the absence of dynamic quenching between the enzyme and QDs. In Fig. 4(a), the decay traces of ChT in both the absence and presence of CdTe were actually plotted, however the lifetime of ChT remained the same under both conditions; henceforth, the merging of kinetic traces was observed (the plot looks like a single decay curve). These data in turn support the formation of a ground state complex between ChT and QDs. Similar results were obtained in the presence of other QDs (figures not shown). Binding constant and number of binding sites For a static quenching mechanism, we can deduce the binding constant (K) resulting from the formation of the ground state complex between fluorophore and the quencher. The relationship between fluorescence intensity and the concentration of quencher can be described by the double logarithmic plot.
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V. Ellappan et al. If it is assumed that there are similar and independent binding sites in the ChT, the relationship between the fluorescence intensity and the quencher medium can be deduced from the following equation (10), (30): nQ þ B → Qn…B
(10)
where B is the fluorophore, Q is the quencher, Qn…B is the postulated complex between fluorophore and n molecules of the quencher. The constant K is given by: K¼
½Qn…B ½Qn ½B
(11)
If the overall amount of biomolecules (bound or unbound with the quencher) is B0, then [B0] = [Qn…B] + [B], where [B] is the concentration of unbound biomolecules, then the relationship between fluorescence intensity and the unbound biomolecule as [B]/[B0] = F/F0 is: F0-F ¼ log K þ n log½Q (12) log F where K is the binding constant of ChT with MPA-CdTe, which can be determined from the plot of log [(F0 F)/F] versus log [Q] curve as shown in Fig. 5 and accordingly we obtained binding constant (K) which is given in Table 2. Number of binding sites (n) is obtained from intercept and slope of the plot respectively. 1 0.8 CdTe-SiO2
0.6
CdTe-G
log[F0-F/F]
0.4
CdTe-Y
0.2
CdTe-R
0
CdTe-O
-0.2 -0.4 -0.6 -0.8 -1 -5
-5.2
-5.4
-5.6
-5.8
-6
-6.2
log[Q] 6
Figure 5. Fluorescence decay of ChT (1 × 10 M) in the absence and presence of 6 (a) CdTe-G; (b) CdTe-R in the concentration range of 5 × 10 M. The blue-coloured decay is the prompt used for calibration. Double logarithmic plot of log[(F0 – F)/F] versus log[Q] for ChT-quantum dots.
Table 2. Binding constants (K), number of binding sites (n) and linear regression coefficient (R2) for ChT with MPA-CdTe QDs at 25°C System CdTe-G CdTe-Y CdTe-O CdTe-R CdTe-SiO2
K M–1 29.984 × 107 0.1890 × 106 0.0512 × 106 0.0974 × 106 0.6483 × 106
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These values of binding constant highlighted the validity of assumption proposed for the association between ChT and QDs. The number of binding sites that is close to 1 indicates that there is only one type of interaction between ChT and MPA-CdTe.
n
R2
1.4834 0.8853 0.8215 0.8326 1.1061
0.998 0.971 0.998 0.990 0.997
Thermodynamic parameters There are essentially four types of non-covalent interactions that could play a role in nanoparticles binding to proteins. These are hydrogen bonds, Van der Waals forces, electrostatic, and hydrophobic interactions (31). In order to obtain such information, the temperature was changed (15°C, 25°C and 35°C), and the reaction enthalpy change was recorded. The values of the thermodynamic parameters (ΔH, ΔG and ΔS) were calculated according to equation (13) and equation (14): ln K ¼ ΔH = RT þ ΔS = R
(13)
ΔG ¼ ΔH ΤΔS ¼ RT lnK
(14)
K is the binding constant at corresponding temperature and R is the gas constant. According to Ross et al. (32), the sign and the magnitude of the thermodynamic parameter determines the various individual kinds of interaction that may take place in protein association processes, as described here. From thermodynamic point of view, ΔH > 0 and ΔS > 0 implies a hydrophobic interaction; ΔH < 0 and ΔS < 0 suggest the van der Waals forces or hydrogen bond formation and ΔH ≈ 0 and ΔS > 0 reflects an electrostatic force. The binding constant values were calculated at three different temperatures and their results were depicted in Table S2. These values were utilised for calculating ΔH, ΔS and ΔG. Slope and intercept values from the linear plot of ln K and the reciprocal of absolute temperature provide ΔH and ΔS values of reaction (Fig. S4). Finally all the results were listed in Table S3. The negative value of ΔG revealed that the interaction process was spontaneous. From the view of thermodynamic theory, it can be deduced that the forces acting between ChT and CdTe-R, CdTe-O, CdTe-Y QDs were mainly due to van der Waals forces or hydrogen bond formation and the electrostatic interactions cannot be excluded (33). In the case of CdTe-G, the value of enthalpy was less than zero and the entropy exhibits a positive value. From this result we suggest that the intermolecular bond energies were decreasing and the disorder of the system was increasing (32). It was also proved by Liu’s results that the electrostatic interaction occurred between negatively charged nanoparticles and the protein. Analysis of conformation changes of chymotrypsin in the presence of quantum dots synchronous fluorescence and three-dimensional measurements. Binding of QDs to ChT is concluded from fluorescence quenching process, but it is still a puzzle about whether the binding affects the conformation and/or microenvironment of enzyme. Influences of QDs on the conformational changes of protein were assessed by synchronous fluorescence measurements. This method provides plenty of information about the molecular microenvironment in the vicinity of fluorophore functional groups. In principle, synchronous fluorescence spectra were obtained by simultaneous scanning of excitation and emission monochromators. According to early reports, if the difference between excitation and emission wavelength (Δλ) is 15 nm, synchronous fluorescence offers the characteristics of tyrosine residues, while when Δλ is
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Luminescence 2015
Spectroscopic evidence for Chymotrypsin and quantum dots interaction 240
a)
500
b)
220
400
200
Intensity
Intenisty
600
300
200
180
160 100 140
0 300
350 Wavelength (nm)
400
120 300
600
c)
500
310
315
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325
Wavelength (nm)
d)
210
400
Intensity
Intensity
305
260
300 200
160
110
100 60 0 300
350 Wavelength (nm)
400
10 300
305
310
315
320
325
Wavelength (nm) 6
Figure 6. Double logarithmic plot of log[(F0 – F)/F] versus log[Q] for ChT QDs. Synchronous spectra of ChT (1 × 10 M) in the absence and presence of (a) CdTe-R the 6 6 wavelength (05 × 10 M) in the wavelength difference of Δλ = 60 nm; (b) Δλ = 15 nm; (c) CdTe-G (05 × 10 M) in difference of Δλ = 60 nm; and (d) Δλ = 15 nm.
set at 60 nm, it provides the characteristic information of tryptophan residues (34). Figure 6(a, b) shows the synchronous fluorescence spectra of ChT gained at 60 nm and 15 nm upon gradual addition of CdTe-R. The fluorescence intensity of both tryptophan and tyrosine were decreased but the emission wavelength of tryptophan (340 nm) was red shifted (2 nm) with increasing concentration of CdTe-R (35). Comparing the emission wavelength of tryptophan, no significant changes were observed for tyrosine (Fig. 6b). It indicates that the interaction of CdTe-R with ChT does not affect the conformation of tyrosine micro-region. It was also shown in early reports that the polarity and the hydrophilicity around the tryptophan residues were increased (14). Identical responses were observed for yellow, orange and silica coated CdTe. In contrast, the presence of CdTe-G changed the emission behaviour of both tyrosine and tryptophan micro-regions as shown in Fig. 6 (c, d). A red shift may represent that the conformation of ChT is somewhat changed. CdTe-G affects the environment of both residues. Therefore, the result reveals that QD interaction solely depends on the size of QDs. Three-dimensional fluorescence techniques are a useful tool to confirm the conformational changes of protein. Fig. 7(a–d) represents the three-dimensional spectra of (a) ChT (1 × 10–6 M) alone; and in the presence of (b) CdTe-O; (c) CdTe-R; (d) CdTe-G and its corresponding contour maps recorded at pH= 7.50. Peak 1 indicates Rayleigh scattering (λex = λemi) and after the addition of QDs peak 1 was enhanced as a result of complex formation. Peak 2 (λex = 280 nm; λemi = 340 nm) was a characteristic emission peak obtained as a result of tryptophan, tyrosine residues but the fluorescence of phenylalanine residue was negligible. By analyzing the changes that occurred in peak 2, we were able to acquire knowledge about the characteristics of the polypeptide backbone structure of chymotrypsin. Peak 2 undergoes a gradual decrease in the presence of QDs and their stoke shift values are depicted in Table 3. Comparing the above phenomenon with
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UV/Vis and synchronous fluorescence results, we can conclude that there was specific interaction that was dependent upon the particle size. Thus the 3-D fluorescence measurement clearly reveals that the interaction of digestive enzyme and the QD induced some microenvironmental and conformational changes in chymotrypsin. Circular dichroism measurements. Modification in conformation of the enzyme was traced using circular dichromic spectrum (36). This method is non-destructive, relatively easy to operate, and requires a small amount of sample and few data collection. Additionally, data analyses are fast (37). Usually, the far-UV CD spectra (260–200 nm) were used to determine the secondary structure of the protein. The characteristic CD bands clearly reflected the content of secondary structure, such as α-helices and β-turns, of the enzyme with two minima at 208 and 222 nm. The concentration of ChT used for CD measurement was 1 × 10–6 M. Substracting the buffer and QDs contribution from the respective original protein and protein–nanostructure complex, the results were reported in terms of molar ellipticity [θ], based on mean amino acid residue weight (MRE): (38) MRE ¼
observed θðm degÞ 10Cpnl
a helix ð%Þ ¼
MRE208 400 33; 000 400
(15)
(16)
Upon the addition of all the QDs, the CD spectrum of the chymotrypsin reveals a significant increase in signal intensities. Henceforth, the results clearly indicated that the helical and sheet structure of chymotrypsin has been changed and this may be due to the formation of complex between the QDs and chymotrypsin. According to Fig. 8, in the presence of green QDs the spectra show more positive deviations compared with other QDs (39). In order to quantify the changes occurred in
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V. Ellappan et al.
6
6
Figure 7. Synchronous spectra of ChT (1 × 10 M) in the absence and presence of (a) CdTe-R the wavelength (05 × 10 M) in the wavelength difference of Δλ = 60 nm; 6 –6 (b) Δλ = 15 nm; (c) CdTe-G (05 × 10 M) in difference of Δλ = 60 nm; and (d) Δλ = 15 nm. Three-dimensional fluorescence spectra of (a) ChT (1 × 10 M) alone and in the presence of (b) CdTe-O; (c) CdTe-R; (d) CdTe-G pH = 7.50, temperature = 298 K.
Table 3. Three-dimensional fluorescence spectral characteristics of ChT in the absence and presence of QDs Fluorescence peak 2 System ChT CdTe-G CdTe-Y CdTe-O CdTe-R CdTe-SiO2
λexi/λemi (nm)
Δλ (nm)
280/340 280/350 280/346 280/345 280/343 280/343
60 100 66 65 63 63
Intensity 545 85 246 231 223 264
the amount of α-helix formation of ChT upon binding with QDs, changes in the intensity of the CD band of ChT have been
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analyzed by the above equations and α-helix content of ChT was obtained. It is noticed from the result, that reductions of the α-helix of ChT from 30.08 to 27.50% (on binding with CdTe-G), 29.47% (CdTe-Y), 28.45% (CdTe-O) and 28.48% (CdTe-SiO2) were observed. This observation strongly indicates that the binding of QD complexes to chymotrypsin induces significant conformational changes in ChT, although the secondary structure of ChT retains predominantly its α-helix character. Also it was noted from the above results that the CdTe-G QD changes the conformation of ChT more effectively than the respective QDs (40,41). FT-IR spectral analysis FT-IR spectroscopy is a powerful technique for monitoring conformational changes in the protein backbone. Figure S5 shows the FT-IR absorption spectra of the ChT in the absence and presence of
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Spectroscopic evidence for Chymotrypsin and quantum dots interaction 0 -2 ChT ChT+CdTe-G ChT+CdTe- Y ChT+CdTe-O ChT+CdTe-R ChT+CdTe-SiO2
θ(m deg)
-4 -6 -8
-10 -12 -14 200
210
220
230
240
250
260
Wavelength (nm)
peak at 1647 cm–1 authenticates the presence of the carboxyl group of amide I (Fig. S5). On exposure to CdTe-R and CdTe-O QDs the peak position of amide I was shifted to 1648 cm–1 and 1644 cm–1. Similar results were observed in the presence of CdTe-Y. When treated with QDs, there was a significant shift of amide II peak from 1538 cm–1 to 1543 cm–1–1539 cm–1. Unfortunately, in the presence of CdTe-G QDs, the intensity of the amide band was reduced with new peak formation at 1636 cm–1, suggesting their better efficiency in binding to ChT. These results show that the change in the secondary structure of the enzyme depends on the size of the QDs. Our next aim was to analyse the effect of core/shell nanostructure on the enzyme. Therefore, a similar experiment was carried out in the presence of CdTe-SiO2 and their amide I and II peaks were shifted to 1400 cm–1 and 1573 cm–1 respectively (figure was not shown) (45). As an overall conclusion from all the results we could suggest that the conformational changes occurred in the presence of QDs.
–6
Figure 8. Three-dimensional fluorescence spectra of (a) ChT (1 × 10 M) alone and in the presence of (b) CdTe-O; (c) CdTe-R; (d) CdTe-G pH = 7.50, temperature = –6 298 K. Far-UV CD spectra of chymotrypsin in phosphate buffer (1 × 10 M), pH 7.5. Symbols represent ChT ( ), ChT interacted with CdTe-G ( ), CdTe-Y (▲), CdTe-O (▼), CdTe-R ( ) and ChT interacted with CdTe-SiO2 ( ). (a) Chymotrypsin and ChT in the presence of (b) CdTe-G; (c) CdTe-O; (d) CdTe-R; and (e) CdTe-SiO2 quantum dots.
▪
QDs. In principle, the protein covers three major groups of infrared absorption bands. One is the amide II band, corresponding to the peptide NH bending vibration modes (1530–1550 cm–1). The second is the COO- antisymmetric stretching bands due to the Asp and Glu carboxylate groups (1565–1585 cm–1). The third is the broad amide I band corresponding to the peptide carbonyl stretching modes μ(CO) (1610–1700 cm–1) (42–44). Hence, it is expected that the positions of amide I 16001690 of C=O stretching, amide II 14801575 of CN stretching and NH bending reflect the information of changes in ChT secondary structure (33). A strong intense
AFM measurements In this work AFM images were obtained by using the noncontact mode in the air. This method facilitates additional Table 4. Representation of the mean height of the particle System
Mean height of the particle
ChT ChT + CdTe-G ChT+ CdTe-Y ChT + CdTe-O ChT + CdTe-R ChT + CdTe-SiO2
–6
~4.86 nm ~19.8 nm ~10.2 nm ~9.8 nm ~9.8 nm ~10 nm
▪
Figure 9. Far-UV CD spectra of chymotrypsin in phosphate buffer (1 × 10 M), pH 7.5. Symbols represent ChT ( ), ChT interacted with CdTe-G ( ), CdTe-Y (▲), CdTe-O (▼), CdTe-R ( ) and ChT interacted with CdTe-SiO2 ( ). (a) Chymotrypsin and ChT in the presence of (b) CdTe-G; (c) CdTe-O; (d) CdTe-R; and (e) CdTe-SiO2 quantum dots. AFM topographic images of a) ChT alone and ChT in the presence of b) CdTe-G c) CdTe-O d) CdTe-R e) CdTe-SiO2 quantum dots.
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V. Ellappan et al. information regarding the structural and surface changes that occur in biological molecules in the presence of other chemical environments. Several cases reported earlier that drying may not affect the aggregation and distribution of protein molecules on the mica surface. Here, we have adopted the AFM technique to investigate the interactions between ChT-QDs. The differences between structures of the ChT molecule with and without QDs on the mica surface (2-D image) were clearly shown in Fig. 9 (a–d). The distribution of the protein alone on the mica surface was not uniform (as shown in Fig. 9a). Whereas the interaction of ChT with QDs shows (Fig. 9b) an enormous modification as compared with Fig. 9(a). In order to verify true height profiles, cross-section analyses on all samples were performed (figures not shown). The AFM results revealed that the individual ChT molecules were adsorbed onto the mica surface and the mean height of the individual molecules was 4.86 ± 2.84 nm (shown in Fig. 9a). These dimensions are consistent with those previously reported results (7). These data clearly support the earlier mentioned binding modes in the section on ‘Binding constant and number of binding sites’. Binding affinity of the green emissive QDs is maximum when compared with the other emissive QDs, this is in turn reflected by measuring the height of the z-axis depicted in Table 4. The smaller the size of the QDs the higher its interaction affinity, resulting in complex molecules aggregated together. Similar to our study, results from nanoparticle interaction with serum albumin and myoglobin was obtained by Mandal et al. (28). Furthermore, Fig. 9(e) shows the image of the ChT-CdTe-SiO2 complex, here the circular structure indicates the presence of active adsorption of SiO2 onto the mica surface. In the presence of QDs, ChT molecules become swollen and the dimensions become larger after interaction with QDs. Additionally, the mean height of the enzyme reached 19.8 ± 4.50 nm (Fig. 9b) followed by aggregation. Thus, the morphological characteristics obtained by the AFM topographical study showed that a specific interaction occurs via complex formation of ChT with MPA-CdTe QDs. After
interaction with the MPA-CdTe, the microenvironment around the enzymes was changed. Therefore, these results revealed that an electrostatic interaction between ChT and QDs may occur. In summary, the studies on particle size variation revealed that the reactivity of QDs decreases as their size increases. Mechanism of interaction of quantum dots and ChT All the experimental results provided a good proof for the conformational change of chymotrypsin caused by QDs, therefore next we explored the interaction mechanism. In principle, the crystalline structure of ChT displays an elliptical shape (from protein data bank, ID: 4cha) (46) and we speculated that the size of the cavity determines the unusual size-dependent interactions. As shown in Fig. 10, we find that the size of the cavity on the top and the bottom of the ChT is about 1.7–2.5 nm while the entrance of this cavity is nearly 2.29 nm. Therefore, in comparison with the other emissive QDs, CdTe-G QDs with its
Scheme 2. Mode of interaction with quantum dots.
Scheme 3. Illustrating the possible mode of binding with MPA-CdTe.
Figure 10. Demonstrating the elliptical structure of chymotrypsin (PDB ID: 4cha).
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Spectroscopic evidence for Chymotrypsin and quantum dots interaction very smaller size approximately 2 nm can enter the ChT cavity and make unusual changes to the secondary structure. From these results we observed that the secondary conformation of ChT is seriously changed upon the addition of CdTe-G QDs. And this conformational changes ultimately led to the change in the tertiary structure. Consequently the cavity may be widened upon the addition of QDs, then it favours QDs with a mean size about 2 nm for binding, hence the sizes of the QDs are key factors and not be ignored when biological applications of nanomaterials are studied. Interaction of different sizes of QDs with chymotrypsin solely depends not only on pH and temperature but also on the size of the QDs chosen. It is conceivable that green QDs, due to their significantly smaller size can more readily interact with ChT (Scheme 2). These results are in good accordance with those obtained by other groups (47). From the above results, we inferred that the red emission QDs are more suitable for drug delivery. In order to minimise the toxicity caused by the capping ligand on the chymotrypsin, silica capped QDs can be used. Core-shell CdTe is more preferable owing to its less toxic interaction (48). As a consequence, these functionalized materials hold great promise for non-cytotoxic applications in site-directed surgeries, and in in vivo bioimaging. Scheme 3 illustrates the schematic representation of a possible mode of interaction between QDs and chymotrypsin. Under physiological neutral circumstances, the predominant QDs surface groups are negatively charged because of COO–, while the main ChT functional groups are R–COO– and R–NH3+. Thus, both van der Waals force (mentioned in the section on ‘Thermodynamic parameters’) and electrostatic force existed in the ChT QDs interaction. Furthermore, hydrogen bonds partly occurred between hydroxyl groups on the QDs surface and the protonated amino groups in the chymotrypsin (45,49).
Conclusions We have demonstrated the interaction of QDs with ChT using steady-state, time-resolved and synchronous fluorescence spectroscopic measurements. Chymotrypsin was adsorbed onto the surface of colloidal QDs through van der Waals interactions. The results from fluorescence spectroscopy indicated that the probable quenching mechanism was based on a static quenching procedure. These results clearly indicated that the QDs quench the fluorescence of chymotrypsin through complex formation. The quenching rate constant, binding constant, and number of binding sites were calculated according to the relevant fluorescence data. The AFM image clearly showed the changes in morphology of ChT and thus supported the complex formation. From the synchronous fluorescence spectra, it was established that the conformational changes in chymotrypsin occurred especially in the tryptophan micro-region. FT-IR and CD measurements were successful employed to describe the type of interaction. The importance of using CdTe-SiO2 colloidal quantum dots over MPA-CdTe QDs and the applications for these studies in biology were discussed.
Acknowledgements EV and RR thank DST and the Government of India (Ref: SR/NM/ NS-26/2013, dt.: 05–06–14) and (Ref: SR/S1/PC-12/2011, 20.9.2011) for the project. EV thanks UGC SAP Fellowship for Meritorious Students.
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Supporting information Additional supporting information may be found in the online version of this article at the publisher’s web site.
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Accepted: 2 December 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2847
Interaction of digestive enzymes with tunable light emitting quantum dots: a thorough Spectroscopic investigation Vaishnavi Ellappan,a,b Manibalan Kesavan,a Parameshwari Ramalingam,c Jeganathan Kulandaivelc and Renganathan Rajalingama* ABSTRACT: In this article, we have examined the direct spectroscopic and microscopic evidence of efficient quantum dotsα-chymotrypsin (ChT) interaction. The intrinsic fluorescence of digestive enzyme is reduced in the presence of quantum dots through ground-state complex formation. Based on the fluorescence data, quenching rate constant, binding constant, and number of binding sites are calculated under optimized experimental conditions. Interestingly, fluorescence quenching method clearly illustrated the size dependent interaction of MPA-CdTe quantum dots. Conformational change of ChT was traced using synchronous fluorescence measurements, circular dichroism and FTIR spectroscopic methods. Furthermore, the AFM results revealed that the individual enzyme molecule dimensions were changed after interacting with quantum dot. Consequently, this result could be helpful for constructing safe and effective utilisation of QDs in biological applications. Copyright © 2015 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: MPA-CdTe quantum dots; chymotrypsin; atomic force microscopy; circular dichroism
Introduction Semiconductor quantum dots (QDs) are nanomaterials, endowed with unique size-dependent optical properties such as high quantum yield, and good photostability (1,2). Exclusive properties of these nanoparticles make them an excellent candidate for numerous applications from bioimaging to drug delivery (3–6). Knowledge of protein–nanoparticles interactions is important in order to understand how nanoparticles function in a biological environment. The focus of recent research is on the interactions between these small molecules with proteins (7). Such findings are significant as they can help to obtain a better understanding about the adsorption and distribution of these small molecules in living systems. Jhonsi et al. investigated the interaction of oxide nanoparticles (ZnO and TiO2) with serum albumins (8,9). In addition, some pioneering work on the semiconductor colloidal cadmium chalcogenoid QDs namely starch-capped Cadmium sulphide (CdS) with bovine serum albumin (BSA), CdSe-GSH with Human serum albumin (HSA) and MPA-CdTe with denatured BSA has also been carried out (10–13). Major interactions involved in protein adsorption can be classified as electrostatic, hydrogen-bonding, hydrophobic, etc. An intensive survey of research shows that QDs are now widely used in biotechnology and medical applications, implying the interactions of water-soluble semiconductor QDs with functional biomolecules. Yilin Wang et al. investigated the interaction of pepsin with CdSe (14) and papain with CdTe QDs (15). Despite these studies, the interaction mechanism of QDs with chymotrypsin is still unexplored. A clear investigation of this problem is important for the safe and effective utilization of QDs in biomedical research. Fluorescence quenching measurement has been used to obtain adequate information about the
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structure and dynamics of biologically important macromolecular systems such as proteins. It can reveal the accessibility of quenchers to protein’s fluorophore group, and help in understanding the essential binding phenomenon. This area has been actively studied for decades because of its importance in the wide range of biomedical applications, immunomagnetic cell separation (16), immobilized enzymes or catalysts (17), biosensors (18,19), drug delivery systems (20), and so on. In the present work, we have chosen α-chymotrypsin (ChT) as a target biomolecule. Chymotrypsin is an ellipsoidal-shaped protein and belongs to the serine protease family. It functions as a digestive enzyme in mammals, containing an α-helix at the C-terminal end (residues 230–245) and several β-sheets domains (21). Being an extensively characterized protein with well defined geometry it is an excellent system to study the protein surface recognition. The effect of the core and core/shell QDs on the intrinsic tryptophan fluorescence quenching has also been explored. We also followed
* Correspondence to: Rajalingam Renganathan, School of Chemistry, Bharathidasan University, Tiruchirappalli 620 024, India. Tel: +91 431 2407053; Fax: +91 431 2407045. E-mail: [email protected] a
School of Chemistry, Bharathidasan University, Tiruchirappalli 620 024, India
b
Department of Chemistry, Sri G. V. G Visalakshi College for Women, Udumalpet 642 128
c
Centre for Nanoscience and Nanotechnology, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India Abbreviations: AFM, atomic force microscopy; BSA, bovine serum albumin; CD, circular dichroism; SAED, selected area electron diffraction; TEM, transmission electron micrographs.
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V. Ellappan et al. alterations in the secondary and tertiary structures of the enzyme by means of synchronous fluorescence, circular dichroism (CD) measurements and characterized by using steadystate fluorescence, Fourier transform infrared spectra (FT-IR), and atomic force microscopy (AFM) techniques under physiological conditions.
solution was then heated at 50°C until black Te powder was fully disappeared and white sodium tetraborate precipitate settled at the bottom of the flask (about 45 min), the resulting purple NaHTe in clear supernatant was separated out and injected by syringe into 24 mL of N2 saturated pure water to produce a 0.01 M of NaHTe solution.
Experimental
Synthesis of quantum dots
Cadmium chloride (CdCl2.2.5H2O), tellurium (Te), sodium borohydride (NaBH4), mercaptopropionic acid (MPA) and tetraethylorthosilicate (TEOS) were purchased from Sigma-Aldrich. α-Chymotrypsin was dissolved in double-distilled water to prepare stock solutions (1 × 104 M) and stored at 0–4°C. All measurements were performed at ambient temperature. ChT (1 × 106 M in phosphate buffer at pH 7.5) was titrated by successive addition of mercaptopropionic acid capped CdTe QDs stock solution prepared at various time intervals. Titrations were manually done by using a micropipette for the addition of QDs. The samples were carefully purged by using pure nitrogen gas for 10 min. Quartz cells with high vacuum Teflon stopcocks were used for purging. The excitation wavelength of ChT was fixed as 280 nm. All measurements were done in ambient temperature (25°C). The fluorescence intensities were corrected for absorption of the exciting light and reabsorption of the emitted light to decrease the inner filter effect by using the following relationship:
Mercaptopropionic acid-coated CdTe QDs (MPA-CdTe) were synthesized using wet chemical route. MPA was dissolved in 100 mL water in a three-necked flask. The pH of the solution was adjusted by adding 0.5 mol L1 NaOH and it was saturated with nitrogen for 20 min. Subsequently, freshly prepared NaHTe solution was injected into the solution under vigorous stir. The CdTe precursors were formed at this stage, accompanied by a faint yellow colour formation. Afterward, the solution was kept at 100°C refluxing temperature for a specified period. To monitor the growth of the QDs, aliquots of reaction solution were taken at various time intervals. To obtain MPAcoated CdTe QDs precipitate, the QDs solution was added in acetone, then precipitated QDs were separated by centrifugation at 15,000 rpm for 10 min and were repeated several times to remove unreacted mercaptopropionic acid (23). The sedimentation was dried in a vacuum drying chamber for IR spectroscopy analysis. For overcoating the CdTe core with SiO2, 20 mL of ethanol, 0.5 mL of MPA-CdTe aqueous solution, and 0.25 mL of TEOS where added into RB and mixed under stirring (24). Scheme 1 shows the schematic representation of silica-coated CdTe QDs. The particle size of the prepared CdTe QDs was determined from the first absorption maximum according to equation (2) mentioned below (25):
Fcor ¼ Fobs e
A
exc=2
þ Aem=2
(1)
where, Fcor and Fobs are the fluorescence intensities corrected and observed, respectively, and Aexc and Aem are the absorption of the systems at the excitation and the emission wavelength, respectively. The fluorescence intensity used in this paper is the corrected intensity. The spectra were recorded using a spinning cell holder as an accessory and of native α-ChT in 50 mM sodium phosphate buffer, pH 7.5. α-Chymotrypsin (1 mg mL–1) solution in the aqueous buffer was used for recording the spectrum. Fourier transform infrared spectra were obtained using a Perkin-Elmer Spectrum RXI FT-IR Spectrometer at room temperature in the range of 4000–400 cm1. The samples were kept in a liquid cell between two windows (CaF2). Mirror velocity was 0.3 cm/sec and number of co-added scans are four then total collection time was less than 2 min. AFM images of ChT and QDs adsorbed on ChT were obtained with a multimode AFM (Agilent 5500). Transmission electron micrographs (TEM) were recorded using a FEI TECNAI 3010 electron microscope operating at 300 kV (Cs = 0.6 mm, resolution 1.7 Å). Circular dichroism (CD) measurements were carried out on a Jasco spectropolarimeter (J-715), calibrated with D-10-camphor sulphonic acid. Concentration of the chymotrypsin sample was adjusted within the range of 10–5 to 10–6 M.
D ¼
9:8127 x 107 λ3 1:7147 x 103 λ2 þ ð1:0064Þ λ 194:84
(2)
Where D (nm) is the particle size of MPA-CdTe QDs and λ (nm) is the wavelength of the first excitonic absorption peak of the corresponding MPA-CdTe QDs. The results showed that the particle diameters of the MPA-CdTe QDs were around 2.03, 3.17, 3.6 and 4.23 nm (photograph of QDs are illustrated in Fig. 1d), corresponding to the first absorption maximum. The particle size of the red emissive QDs was confirmed by TEM measurement and the result obtained was illustrated in Fig. 1(a). From TEM results, we identified that the fine and uniform particle size was around ~4.52 nm. It was observed that the particle sizes determined from both the calculation as well as TEM measurements were matched well. The selected area electron diffraction (SAED) pattern shows a set of rings instead of spots due to the random orientation of the nanoparticles (Fig. 1b).
Preparation of sodium hydrotelluride (NaHTe) NaHTe was prepared according to the earlier reported procedure with some modifications (22). Briefly, a mixture of 0.0284 g of NaBH4 and 0.0197 g of Te powder (molar ratio of Te to NaBH4 is 1:3) was added to a small one-necked flask, containing 1 mL of N2 saturated double-deionized water. A small outlet was connected to the flask to release resulting hydrogen. The
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Scheme 1. Shows the schematic route for synthesizing CdTe-SiO2.
Copyright © 2015 John Wiley & Sons, Ltd.
Luminescence 2015
Spectroscopic evidence for Chymotrypsin and quantum dots interaction
Figure 1. (a) High resolution TEM image; and (b) SAED pattern of red emissive CdTe quantum dots. (c) Size-dependent emission behaviour of green, yellow, orange and –7 red MPA-CdTe (3 × 10 M) and CdTe-SiO2. (d) Photograph showing the image of emission of MPA-CdTe under ultraviolet (UV) light and the increase in particle diameter is indicated by an arrow.
Figure S1 illustrates the TEM image of synthesised CdTe-SiO2. The concentration of prepared QDs was calculated from the absorption spectrum using LambertBeer’s law, equation (3), (3,23): A ¼ εC1
(3)
In equation (3), A is the absorbance at the peak position of the first excitonic absorption peak for CdTe, C is the molar concentration of the QDs of the same sample, l is the path length of the radiation beam used for recording the absorption spectrum, and ε is the molar extinction coefficient (for CdTe QDs, ε = 10,043 (D)2.12 in which D is the particle size of CdTe QDs) (26). Based on equation (3) concentration of green (G), yellow (Y), orange (O) and red (R) CdTe QDs are 1 × 105 M.
features (figure not shown) proves the successful chemical attachment of MPA onto the QDs surface. For MPA, the peak at 2600– 2500 cm1 is assigned to S–H stretching vibration. The peak at 3500–3000 cm1 was produced by the stretching vibration of O– H. Especially, the presence of the S–H stretching vibration at 2600–2500 cm1 for the QD has not been found. This reflects that –SH in MPA of the QDs was covalently bound on the QDs surface. More –COOH exposure onto the QDs surface can be illustrated by a much narrower absorption band around 3400 cm1, a decreased absorption at 1675 cm1, a blue-shifted from 1609 cm1 to 1673 cm1. Peaks at 1250 cm–1 and 954 cm–1 illustrated the Si–O–Si absorption and bending vibration of Si–OH and at 471 cm–1 corresponded to the bending vibration of Si–O–Si. Thus the IR spectral features clearly indicate that the SiO2 layers are successfully coated onto the surface of MPA-CdTe (27) (figure not shown).
Results and discussion Spectral characterization of CdTe QDs In order to evaluate the influence of both the core and core/shell QDs on ChT, we finally selected a set of various sizes of MPACdTe and CdTe-SiO2 QDs (graphical abstract). Generally, colloidal QDs have a broad absorption and narrow emission spectra in the visible region. Their narrow emission is attributed to the radiative recombination of electron and hole pairs. Monodispersed QDs with narrow size distributions have been synthesized; Fig. 1(c) shows the size-dependent emissive behaviour of green, yellow, orange and red MPA-CdTe QDs. To identify the conjugation mode between mercaptopropionic acid and CdTe QDs coated by mercaptopropionic acid, the IR spectra of pure MPA and the QDs were measured. The resemblance in both IR spectral
Luminescence 2015
Absorption characteristics of ChT-CdTe quantum dot interactions Before performing the excited state reactions between ChT and QDs, it is essential to know the type of interaction between them in the ground state. The peak centred at ~280 nm in the ChT absorption spectrum is due to aromatic amino acids (primarily tryptophan and tyrosine). With gradual addition of CdTe-R to the ChT solution, the absorption spectrum undergoes a hyperchromic effect without any noticeable shift, indicating that QDs affect the band corresponding to the tryptophan and tyrosine residues of the ChT (Fig. 2a). In contrast, addition of a similar concentration of CdTe-G affects the absorbance spectrum of ChT to a greater extent. A
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Absorbance
V. Ellappan et al. 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
Kapp ¼
ChT+ CdTe-R ChT
230
330
430
Absorbance
(4)
½ChT……CdTe ½ChT· ½CdTe
(5)
The Kapp value was calculated by the method of Benesi and Hildebrand (29) using the following equation (6): 530
630
Aobs ¼ ð1 αÞ C0 εChT1 þ αC0 εc 1
Wavelength (nm) 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
Kapp
⇌ ChT…CdTe
ChT þ CdTe
a)
(6)
where Aobs is the absorbance of the ChT solution containing different concentrations of MPA-capped CdTe at 280 nm, α is the degree of association between ChT and capped CdTe, εChT and εc are the molar extinction coefficients at the defined wavelengths for ChT and the formed complex respectively, C0 is the initial concentration of ChT and ‘l’ is the optical path length, which is taken as unity. Equation (6) can be expressed by equation (7), where the A0 and Ac are the absorbance of ChT and the complex at 280 nm, with the concentration C0:
b) ChT+ CdTe-G
ChT
Aobs ¼ ð1 αÞA0 þ αΑc 230
330
430
530
(7)
630
1/[Aobs-A0]
Wavelength (nm) 18
[CdTe-G]
16
[CdTe-Y]
14
[CdTe-O]
12
[CdTe-R]
10
[CdTe-SiO2]
c)
At high concentrations of QDs, α can be equated to (Kapp [capped CdTe])/(1 + Kapp[QDs]). In this case, equation (7) can be expressed as equation (8):
600
8
a
500
6 4 0 0
0.2
0.4
0.6
0.8
1
1.2
1/[Q]
Intensity
400
2
300 200
–5
Figure 2. UV-vis absorption spectra of ChT dotted line (conc. ~1 × 10 M). (a) In the presence of CdTe-R; and (b) in the presence of CdTe-G; at ambient temperature –6 in buffer solution. The concentrations of QD were (0) 0; (1) 7.7 × 10 M; (2) 1.54 × –5 –5 –5 –5 10 M; (3) 2.3 × 10 M; (4) 3.1 × 10 M; and (5) 3.8 × 10 M. (c) The straight line dependence of 1/(Aobs – A0) on the reciprocal concentration of different size QDs.
100 0 300
320
340
360
380
400
420
440
Wavelength (nm) 600
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b 500 400
Intensity
similar experiment was also conducted in the presence of yellow, orange and CdTe-SiO2. The above results can be rationalized in terms of a strong interaction between QDs and ChT in the ground state through complex formation, as confirmed by earlier studies. (28) In contrast, addition of even lower concentrations of green emissive QDs leads to abnormal changes in ChT, as shown in Fig. 2(b). The absorption spectra of ChT in the absence and presence of yellow and orange CdTe are shown in Fig. S2(a) and Fig. S2(b). The addition of CdTe-SiO2 led to a gradual increase in ChT absorption with a red shift (higher wavelength) of 2 nm, as shown in Fig. S2(c). These observations indicated that there was a structural change (microenvironment) in ChT which occured upon interaction with the surface of MPA-CdTe. Importantly, the equilibrium for the formation of a complex between ChT and CdTe is defined by equation (4) where Kapp is the apparent association constant:
300 200 100 0 300
320
340
360
380
400
420
440
Wavelength (nm) Figure 3. Fluorescence quenching of ChT (black line) by a) CdTe-G b) CdTe- SiO2 –6 of various concentration ranging from 05 × 10 (λexi = 280: λemi = 340 nm) and dotted line emission behaviour of CdTe-G and CdTe-SiO2 respectively, excitated at similar wavelength.
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Luminescence 2015
Spectroscopic evidence for Chymotrypsin and quantum dots interaction 10000
1 1 1 ¼ þ Aobs A0 Ac A0 Kapp ðAc -A0 Þ½CdTe
a
Prompt
(8)
ChT
Counts
1000
Therefore, if the enhancement of absorbance at 280 nm was due to absorption of the complex, one would expect a linear relationship between 1/(Aobs-A0) and the reciprocal concentration of CdTe with a slope equal to 1/Kapp(Ac – A0) and an intercept equal to 1/(Ac – A0) (shown in the Fig. 2c). The values of Kapp obtained from such plots are depicted in Table S1.
ChT+CdTe-G
100
10
1 0
10
20
Fluorescence spectral analysis of quantum dots with α-chymotrypsin
(9)
where F0 and F are the fluorescence intensities of ChT in the absence and presence of MPA-CdTe, respectively. KSV is the Stern–Volmer constant and [MPA-CdTe] is the concentration of respective quencher. From the Ksv Table 1, we have calculated the value of quenching rate constant by using the lifetime (τ) of ChT (4.02 ns, from time resolved measurement). In many instances, the fluorophore can be quenched both by collision and by complex formation with the same quencher. The gradual deviation from linearity of the Stern–Volmer plots (Fig. S3) with continuous addition of CdTe-G suggests the existence of more than one binding site with lower binding affinity and different accessibilities, it also indicated the occurrence of combined quenching. In contrast, a linear Stern–Volmer plot obtained for other QDs is shown in Fig. S3. In this case, the values of kq are in the order of 1012, consequently we confirm that the quenching is mainly a static quenching process. For static quenching, increased temperature reduces the stability of the complex formed, resulting in a reduced quenching constant and the results are shown in Table 1. Stern–Volmer quenching constant of the ChTquantum dot system at T = 298 and 308 K Ksv × 106 M–1 System
kq M–1 s–1 × 1012
298.15 K
308.15 K
298.15 K
308.15 K
0.3623 0.2976 0.301 0.253 0.1723
0.3545 0.2689 0.1631 0.132 0.0963
90.00 73.97 74.81 63.13 42.82
88.11 66.84 40.54 22.82 23.935
CdTe-G CdTe-Y CdTe-O CdTe-R CdTe-SiO2
Luminescence 2015
10000
40
b
Prompt ChT
1000
Counts
The interaction of ChT with MPA-CdTe was studied using a spectrofluorometer at room temperature. Chymotrypsin was dissolved in phosphate-buffered medium and the fluorescence spectrum was recorded at 340 nm. The solution of ChT (1 × 106 M) was titrated with increasing concentrations of (0–5 × 106 M) QD solution, as shown in Fig. 3(a, b). The MPA-CdTe also has luminescent properties but its excitation (450 nm) and emission (522 nm) wavelengths are much further away from the ChT absorption and emission (280 and 340 nm respectively), so it may not interrupt the interaction study. While increasing the concentration of QDs the emission intensity of ChT was found to decrease progressively with the red shift of around 5 nm. A Stern–Volmer plot was utilised to analyse the quenching phenomenon. The ratios F0/F were calculated and plotted against quencher concentration according to equation (9): F0 =F ¼ 1 þ Ksv ½MPA-CdTe
30
Time (ns)
ChT+CdTe-R
100
10
1 0
10
20
30
40
Time (ns) Figure 4. Fluorescence quenching of ChT (black line) by CdTe-SiO2 at various concen–6 trations ranging from 05 × 10 M (λexi = 280: λemi = 340 nm) and dotted line emission behaviour of CdTe-SiO2 excited at similar wavelengths. Fluorescence decay of ChT 6 (1 × 10 M) in the absence and presence of (a) CdTe-G; (b) CdTe-R in the concentration 6 range of 5 × 10 M. The blue coloured decay is the prompt used for calibration.
Table 1. Furthermore the type of interaction between ChT and CdTe was also confirmed by time-resolved spectroscopy. Henceforth, the fluorescence results indicated that QDs can effectively quench the fluorescence of ChT in a size-dependent manner. Lifetime measurements According to the results discussed in the above section, it is reasonable to mention that the complexes could be formed between ChT and QDs. Fig. 4(a, b) shows the fluorescence decay curves of ChT in the absence and presence of CdTe-G and CdTe-R respectively. Results clearly illustrate that there was no change in fluorescence lifetime of enzyme in the presence of both materials. Therefore, the result shows the absence of dynamic quenching between the enzyme and QDs. In Fig. 4(a), the decay traces of ChT in both the absence and presence of CdTe were actually plotted, however the lifetime of ChT remained the same under both conditions; henceforth, the merging of kinetic traces was observed (the plot looks like a single decay curve). These data in turn support the formation of a ground state complex between ChT and QDs. Similar results were obtained in the presence of other QDs (figures not shown). Binding constant and number of binding sites For a static quenching mechanism, we can deduce the binding constant (K) resulting from the formation of the ground state complex between fluorophore and the quencher. The relationship between fluorescence intensity and the concentration of quencher can be described by the double logarithmic plot.
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V. Ellappan et al. If it is assumed that there are similar and independent binding sites in the ChT, the relationship between the fluorescence intensity and the quencher medium can be deduced from the following equation (10), (30): nQ þ B → Qn…B
(10)
where B is the fluorophore, Q is the quencher, Qn…B is the postulated complex between fluorophore and n molecules of the quencher. The constant K is given by: K¼
½Qn…B ½Qn ½B
(11)
If the overall amount of biomolecules (bound or unbound with the quencher) is B0, then [B0] = [Qn…B] + [B], where [B] is the concentration of unbound biomolecules, then the relationship between fluorescence intensity and the unbound biomolecule as [B]/[B0] = F/F0 is: F0-F ¼ log K þ n log½Q (12) log F where K is the binding constant of ChT with MPA-CdTe, which can be determined from the plot of log [(F0 F)/F] versus log [Q] curve as shown in Fig. 5 and accordingly we obtained binding constant (K) which is given in Table 2. Number of binding sites (n) is obtained from intercept and slope of the plot respectively. 1 0.8 CdTe-SiO2
0.6
CdTe-G
log[F0-F/F]
0.4
CdTe-Y
0.2
CdTe-R
0
CdTe-O
-0.2 -0.4 -0.6 -0.8 -1 -5
-5.2
-5.4
-5.6
-5.8
-6
-6.2
log[Q] 6
Figure 5. Fluorescence decay of ChT (1 × 10 M) in the absence and presence of 6 (a) CdTe-G; (b) CdTe-R in the concentration range of 5 × 10 M. The blue-coloured decay is the prompt used for calibration. Double logarithmic plot of log[(F0 – F)/F] versus log[Q] for ChT-quantum dots.
Table 2. Binding constants (K), number of binding sites (n) and linear regression coefficient (R2) for ChT with MPA-CdTe QDs at 25°C System CdTe-G CdTe-Y CdTe-O CdTe-R CdTe-SiO2
K M–1 29.984 × 107 0.1890 × 106 0.0512 × 106 0.0974 × 106 0.6483 × 106
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These values of binding constant highlighted the validity of assumption proposed for the association between ChT and QDs. The number of binding sites that is close to 1 indicates that there is only one type of interaction between ChT and MPA-CdTe.
n
R2
1.4834 0.8853 0.8215 0.8326 1.1061
0.998 0.971 0.998 0.990 0.997
Thermodynamic parameters There are essentially four types of non-covalent interactions that could play a role in nanoparticles binding to proteins. These are hydrogen bonds, Van der Waals forces, electrostatic, and hydrophobic interactions (31). In order to obtain such information, the temperature was changed (15°C, 25°C and 35°C), and the reaction enthalpy change was recorded. The values of the thermodynamic parameters (ΔH, ΔG and ΔS) were calculated according to equation (13) and equation (14): ln K ¼ ΔH = RT þ ΔS = R
(13)
ΔG ¼ ΔH ΤΔS ¼ RT lnK
(14)
K is the binding constant at corresponding temperature and R is the gas constant. According to Ross et al. (32), the sign and the magnitude of the thermodynamic parameter determines the various individual kinds of interaction that may take place in protein association processes, as described here. From thermodynamic point of view, ΔH > 0 and ΔS > 0 implies a hydrophobic interaction; ΔH < 0 and ΔS < 0 suggest the van der Waals forces or hydrogen bond formation and ΔH ≈ 0 and ΔS > 0 reflects an electrostatic force. The binding constant values were calculated at three different temperatures and their results were depicted in Table S2. These values were utilised for calculating ΔH, ΔS and ΔG. Slope and intercept values from the linear plot of ln K and the reciprocal of absolute temperature provide ΔH and ΔS values of reaction (Fig. S4). Finally all the results were listed in Table S3. The negative value of ΔG revealed that the interaction process was spontaneous. From the view of thermodynamic theory, it can be deduced that the forces acting between ChT and CdTe-R, CdTe-O, CdTe-Y QDs were mainly due to van der Waals forces or hydrogen bond formation and the electrostatic interactions cannot be excluded (33). In the case of CdTe-G, the value of enthalpy was less than zero and the entropy exhibits a positive value. From this result we suggest that the intermolecular bond energies were decreasing and the disorder of the system was increasing (32). It was also proved by Liu’s results that the electrostatic interaction occurred between negatively charged nanoparticles and the protein. Analysis of conformation changes of chymotrypsin in the presence of quantum dots synchronous fluorescence and three-dimensional measurements. Binding of QDs to ChT is concluded from fluorescence quenching process, but it is still a puzzle about whether the binding affects the conformation and/or microenvironment of enzyme. Influences of QDs on the conformational changes of protein were assessed by synchronous fluorescence measurements. This method provides plenty of information about the molecular microenvironment in the vicinity of fluorophore functional groups. In principle, synchronous fluorescence spectra were obtained by simultaneous scanning of excitation and emission monochromators. According to early reports, if the difference between excitation and emission wavelength (Δλ) is 15 nm, synchronous fluorescence offers the characteristics of tyrosine residues, while when Δλ is
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Luminescence 2015
Spectroscopic evidence for Chymotrypsin and quantum dots interaction 240
a)
500
b)
220
400
200
Intensity
Intenisty
600
300
200
180
160 100 140
0 300
350 Wavelength (nm)
400
120 300
600
c)
500
310
315
320
325
Wavelength (nm)
d)
210
400
Intensity
Intensity
305
260
300 200
160
110
100 60 0 300
350 Wavelength (nm)
400
10 300
305
310
315
320
325
Wavelength (nm) 6
Figure 6. Double logarithmic plot of log[(F0 – F)/F] versus log[Q] for ChT QDs. Synchronous spectra of ChT (1 × 10 M) in the absence and presence of (a) CdTe-R the 6 6 wavelength (05 × 10 M) in the wavelength difference of Δλ = 60 nm; (b) Δλ = 15 nm; (c) CdTe-G (05 × 10 M) in difference of Δλ = 60 nm; and (d) Δλ = 15 nm.
set at 60 nm, it provides the characteristic information of tryptophan residues (34). Figure 6(a, b) shows the synchronous fluorescence spectra of ChT gained at 60 nm and 15 nm upon gradual addition of CdTe-R. The fluorescence intensity of both tryptophan and tyrosine were decreased but the emission wavelength of tryptophan (340 nm) was red shifted (2 nm) with increasing concentration of CdTe-R (35). Comparing the emission wavelength of tryptophan, no significant changes were observed for tyrosine (Fig. 6b). It indicates that the interaction of CdTe-R with ChT does not affect the conformation of tyrosine micro-region. It was also shown in early reports that the polarity and the hydrophilicity around the tryptophan residues were increased (14). Identical responses were observed for yellow, orange and silica coated CdTe. In contrast, the presence of CdTe-G changed the emission behaviour of both tyrosine and tryptophan micro-regions as shown in Fig. 6 (c, d). A red shift may represent that the conformation of ChT is somewhat changed. CdTe-G affects the environment of both residues. Therefore, the result reveals that QD interaction solely depends on the size of QDs. Three-dimensional fluorescence techniques are a useful tool to confirm the conformational changes of protein. Fig. 7(a–d) represents the three-dimensional spectra of (a) ChT (1 × 10–6 M) alone; and in the presence of (b) CdTe-O; (c) CdTe-R; (d) CdTe-G and its corresponding contour maps recorded at pH= 7.50. Peak 1 indicates Rayleigh scattering (λex = λemi) and after the addition of QDs peak 1 was enhanced as a result of complex formation. Peak 2 (λex = 280 nm; λemi = 340 nm) was a characteristic emission peak obtained as a result of tryptophan, tyrosine residues but the fluorescence of phenylalanine residue was negligible. By analyzing the changes that occurred in peak 2, we were able to acquire knowledge about the characteristics of the polypeptide backbone structure of chymotrypsin. Peak 2 undergoes a gradual decrease in the presence of QDs and their stoke shift values are depicted in Table 3. Comparing the above phenomenon with
Luminescence 2015
UV/Vis and synchronous fluorescence results, we can conclude that there was specific interaction that was dependent upon the particle size. Thus the 3-D fluorescence measurement clearly reveals that the interaction of digestive enzyme and the QD induced some microenvironmental and conformational changes in chymotrypsin. Circular dichroism measurements. Modification in conformation of the enzyme was traced using circular dichromic spectrum (36). This method is non-destructive, relatively easy to operate, and requires a small amount of sample and few data collection. Additionally, data analyses are fast (37). Usually, the far-UV CD spectra (260–200 nm) were used to determine the secondary structure of the protein. The characteristic CD bands clearly reflected the content of secondary structure, such as α-helices and β-turns, of the enzyme with two minima at 208 and 222 nm. The concentration of ChT used for CD measurement was 1 × 10–6 M. Substracting the buffer and QDs contribution from the respective original protein and protein–nanostructure complex, the results were reported in terms of molar ellipticity [θ], based on mean amino acid residue weight (MRE): (38) MRE ¼
observed θðm degÞ 10Cpnl
a helix ð%Þ ¼
MRE208 400 33; 000 400
(15)
(16)
Upon the addition of all the QDs, the CD spectrum of the chymotrypsin reveals a significant increase in signal intensities. Henceforth, the results clearly indicated that the helical and sheet structure of chymotrypsin has been changed and this may be due to the formation of complex between the QDs and chymotrypsin. According to Fig. 8, in the presence of green QDs the spectra show more positive deviations compared with other QDs (39). In order to quantify the changes occurred in
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V. Ellappan et al.
6
6
Figure 7. Synchronous spectra of ChT (1 × 10 M) in the absence and presence of (a) CdTe-R the wavelength (05 × 10 M) in the wavelength difference of Δλ = 60 nm; 6 –6 (b) Δλ = 15 nm; (c) CdTe-G (05 × 10 M) in difference of Δλ = 60 nm; and (d) Δλ = 15 nm. Three-dimensional fluorescence spectra of (a) ChT (1 × 10 M) alone and in the presence of (b) CdTe-O; (c) CdTe-R; (d) CdTe-G pH = 7.50, temperature = 298 K.
Table 3. Three-dimensional fluorescence spectral characteristics of ChT in the absence and presence of QDs Fluorescence peak 2 System ChT CdTe-G CdTe-Y CdTe-O CdTe-R CdTe-SiO2
λexi/λemi (nm)
Δλ (nm)
280/340 280/350 280/346 280/345 280/343 280/343
60 100 66 65 63 63
Intensity 545 85 246 231 223 264
the amount of α-helix formation of ChT upon binding with QDs, changes in the intensity of the CD band of ChT have been
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analyzed by the above equations and α-helix content of ChT was obtained. It is noticed from the result, that reductions of the α-helix of ChT from 30.08 to 27.50% (on binding with CdTe-G), 29.47% (CdTe-Y), 28.45% (CdTe-O) and 28.48% (CdTe-SiO2) were observed. This observation strongly indicates that the binding of QD complexes to chymotrypsin induces significant conformational changes in ChT, although the secondary structure of ChT retains predominantly its α-helix character. Also it was noted from the above results that the CdTe-G QD changes the conformation of ChT more effectively than the respective QDs (40,41). FT-IR spectral analysis FT-IR spectroscopy is a powerful technique for monitoring conformational changes in the protein backbone. Figure S5 shows the FT-IR absorption spectra of the ChT in the absence and presence of
Copyright © 2015 John Wiley & Sons, Ltd.
Luminescence 2015
Spectroscopic evidence for Chymotrypsin and quantum dots interaction 0 -2 ChT ChT+CdTe-G ChT+CdTe- Y ChT+CdTe-O ChT+CdTe-R ChT+CdTe-SiO2
θ(m deg)
-4 -6 -8
-10 -12 -14 200
210
220
230
240
250
260
Wavelength (nm)
peak at 1647 cm–1 authenticates the presence of the carboxyl group of amide I (Fig. S5). On exposure to CdTe-R and CdTe-O QDs the peak position of amide I was shifted to 1648 cm–1 and 1644 cm–1. Similar results were observed in the presence of CdTe-Y. When treated with QDs, there was a significant shift of amide II peak from 1538 cm–1 to 1543 cm–1–1539 cm–1. Unfortunately, in the presence of CdTe-G QDs, the intensity of the amide band was reduced with new peak formation at 1636 cm–1, suggesting their better efficiency in binding to ChT. These results show that the change in the secondary structure of the enzyme depends on the size of the QDs. Our next aim was to analyse the effect of core/shell nanostructure on the enzyme. Therefore, a similar experiment was carried out in the presence of CdTe-SiO2 and their amide I and II peaks were shifted to 1400 cm–1 and 1573 cm–1 respectively (figure was not shown) (45). As an overall conclusion from all the results we could suggest that the conformational changes occurred in the presence of QDs.
–6
Figure 8. Three-dimensional fluorescence spectra of (a) ChT (1 × 10 M) alone and in the presence of (b) CdTe-O; (c) CdTe-R; (d) CdTe-G pH = 7.50, temperature = –6 298 K. Far-UV CD spectra of chymotrypsin in phosphate buffer (1 × 10 M), pH 7.5. Symbols represent ChT ( ), ChT interacted with CdTe-G ( ), CdTe-Y (▲), CdTe-O (▼), CdTe-R ( ) and ChT interacted with CdTe-SiO2 ( ). (a) Chymotrypsin and ChT in the presence of (b) CdTe-G; (c) CdTe-O; (d) CdTe-R; and (e) CdTe-SiO2 quantum dots.
▪
QDs. In principle, the protein covers three major groups of infrared absorption bands. One is the amide II band, corresponding to the peptide NH bending vibration modes (1530–1550 cm–1). The second is the COO- antisymmetric stretching bands due to the Asp and Glu carboxylate groups (1565–1585 cm–1). The third is the broad amide I band corresponding to the peptide carbonyl stretching modes μ(CO) (1610–1700 cm–1) (42–44). Hence, it is expected that the positions of amide I 16001690 of C=O stretching, amide II 14801575 of CN stretching and NH bending reflect the information of changes in ChT secondary structure (33). A strong intense
AFM measurements In this work AFM images were obtained by using the noncontact mode in the air. This method facilitates additional Table 4. Representation of the mean height of the particle System
Mean height of the particle
ChT ChT + CdTe-G ChT+ CdTe-Y ChT + CdTe-O ChT + CdTe-R ChT + CdTe-SiO2
–6
~4.86 nm ~19.8 nm ~10.2 nm ~9.8 nm ~9.8 nm ~10 nm
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Figure 9. Far-UV CD spectra of chymotrypsin in phosphate buffer (1 × 10 M), pH 7.5. Symbols represent ChT ( ), ChT interacted with CdTe-G ( ), CdTe-Y (▲), CdTe-O (▼), CdTe-R ( ) and ChT interacted with CdTe-SiO2 ( ). (a) Chymotrypsin and ChT in the presence of (b) CdTe-G; (c) CdTe-O; (d) CdTe-R; and (e) CdTe-SiO2 quantum dots. AFM topographic images of a) ChT alone and ChT in the presence of b) CdTe-G c) CdTe-O d) CdTe-R e) CdTe-SiO2 quantum dots.
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V. Ellappan et al. information regarding the structural and surface changes that occur in biological molecules in the presence of other chemical environments. Several cases reported earlier that drying may not affect the aggregation and distribution of protein molecules on the mica surface. Here, we have adopted the AFM technique to investigate the interactions between ChT-QDs. The differences between structures of the ChT molecule with and without QDs on the mica surface (2-D image) were clearly shown in Fig. 9 (a–d). The distribution of the protein alone on the mica surface was not uniform (as shown in Fig. 9a). Whereas the interaction of ChT with QDs shows (Fig. 9b) an enormous modification as compared with Fig. 9(a). In order to verify true height profiles, cross-section analyses on all samples were performed (figures not shown). The AFM results revealed that the individual ChT molecules were adsorbed onto the mica surface and the mean height of the individual molecules was 4.86 ± 2.84 nm (shown in Fig. 9a). These dimensions are consistent with those previously reported results (7). These data clearly support the earlier mentioned binding modes in the section on ‘Binding constant and number of binding sites’. Binding affinity of the green emissive QDs is maximum when compared with the other emissive QDs, this is in turn reflected by measuring the height of the z-axis depicted in Table 4. The smaller the size of the QDs the higher its interaction affinity, resulting in complex molecules aggregated together. Similar to our study, results from nanoparticle interaction with serum albumin and myoglobin was obtained by Mandal et al. (28). Furthermore, Fig. 9(e) shows the image of the ChT-CdTe-SiO2 complex, here the circular structure indicates the presence of active adsorption of SiO2 onto the mica surface. In the presence of QDs, ChT molecules become swollen and the dimensions become larger after interaction with QDs. Additionally, the mean height of the enzyme reached 19.8 ± 4.50 nm (Fig. 9b) followed by aggregation. Thus, the morphological characteristics obtained by the AFM topographical study showed that a specific interaction occurs via complex formation of ChT with MPA-CdTe QDs. After
interaction with the MPA-CdTe, the microenvironment around the enzymes was changed. Therefore, these results revealed that an electrostatic interaction between ChT and QDs may occur. In summary, the studies on particle size variation revealed that the reactivity of QDs decreases as their size increases. Mechanism of interaction of quantum dots and ChT All the experimental results provided a good proof for the conformational change of chymotrypsin caused by QDs, therefore next we explored the interaction mechanism. In principle, the crystalline structure of ChT displays an elliptical shape (from protein data bank, ID: 4cha) (46) and we speculated that the size of the cavity determines the unusual size-dependent interactions. As shown in Fig. 10, we find that the size of the cavity on the top and the bottom of the ChT is about 1.7–2.5 nm while the entrance of this cavity is nearly 2.29 nm. Therefore, in comparison with the other emissive QDs, CdTe-G QDs with its
Scheme 2. Mode of interaction with quantum dots.
Scheme 3. Illustrating the possible mode of binding with MPA-CdTe.
Figure 10. Demonstrating the elliptical structure of chymotrypsin (PDB ID: 4cha).
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Spectroscopic evidence for Chymotrypsin and quantum dots interaction very smaller size approximately 2 nm can enter the ChT cavity and make unusual changes to the secondary structure. From these results we observed that the secondary conformation of ChT is seriously changed upon the addition of CdTe-G QDs. And this conformational changes ultimately led to the change in the tertiary structure. Consequently the cavity may be widened upon the addition of QDs, then it favours QDs with a mean size about 2 nm for binding, hence the sizes of the QDs are key factors and not be ignored when biological applications of nanomaterials are studied. Interaction of different sizes of QDs with chymotrypsin solely depends not only on pH and temperature but also on the size of the QDs chosen. It is conceivable that green QDs, due to their significantly smaller size can more readily interact with ChT (Scheme 2). These results are in good accordance with those obtained by other groups (47). From the above results, we inferred that the red emission QDs are more suitable for drug delivery. In order to minimise the toxicity caused by the capping ligand on the chymotrypsin, silica capped QDs can be used. Core-shell CdTe is more preferable owing to its less toxic interaction (48). As a consequence, these functionalized materials hold great promise for non-cytotoxic applications in site-directed surgeries, and in in vivo bioimaging. Scheme 3 illustrates the schematic representation of a possible mode of interaction between QDs and chymotrypsin. Under physiological neutral circumstances, the predominant QDs surface groups are negatively charged because of COO–, while the main ChT functional groups are R–COO– and R–NH3+. Thus, both van der Waals force (mentioned in the section on ‘Thermodynamic parameters’) and electrostatic force existed in the ChT QDs interaction. Furthermore, hydrogen bonds partly occurred between hydroxyl groups on the QDs surface and the protonated amino groups in the chymotrypsin (45,49).
Conclusions We have demonstrated the interaction of QDs with ChT using steady-state, time-resolved and synchronous fluorescence spectroscopic measurements. Chymotrypsin was adsorbed onto the surface of colloidal QDs through van der Waals interactions. The results from fluorescence spectroscopy indicated that the probable quenching mechanism was based on a static quenching procedure. These results clearly indicated that the QDs quench the fluorescence of chymotrypsin through complex formation. The quenching rate constant, binding constant, and number of binding sites were calculated according to the relevant fluorescence data. The AFM image clearly showed the changes in morphology of ChT and thus supported the complex formation. From the synchronous fluorescence spectra, it was established that the conformational changes in chymotrypsin occurred especially in the tryptophan micro-region. FT-IR and CD measurements were successful employed to describe the type of interaction. The importance of using CdTe-SiO2 colloidal quantum dots over MPA-CdTe QDs and the applications for these studies in biology were discussed.
Acknowledgements EV and RR thank DST and the Government of India (Ref: SR/NM/ NS-26/2013, dt.: 05–06–14) and (Ref: SR/S1/PC-12/2011, 20.9.2011) for the project. EV thanks UGC SAP Fellowship for Meritorious Students.
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