Binding of hemoglobin to ultrafine carbon nanoparticles: a spectroscopic insight into a major health hazard

Abstract

Carbon nanoparticles (CNPs) are light and easily absorb into different parts of organs of the human body. They are suspended particulate matters of respirable sizes. In the atmosphere, ultrafine CNPs are known to be generated mainly from the combustion of fuels and have been reported to be a major contributor to the induction of cardiopulmonary diseases. In third world countries, these diseases are more prevalent because of the higher abundance of ultrafine CNPs in the air. Different nanostructured materials, when exposed to the human body, can easily enter into the body through the lungs or other organs and tissues. In the laboratory, ultrafine carbon nanoparticles were synthesized and their structure was confirmed by DLS experiments, TEM and AFM imaging studies. Their interactions with hemoglobin (Hb) and myoglobin (Mb) were studied using fluorescence spectroscopy. The results indicate a remarkably strong interaction between carbon nanoparticles and Hb (or Mb). Temperature dependent steady state fluorescence spectroscopy showed exothermic binding of Hb to CNPs, which is favored by enthalpy and entropy changes. A circular dichroism study also indicated significant change in the protein secondary structure and a partial unfolding of the helical conformation. These findings are highly important for understanding the interactions between CNPs and Hb (or Mb), which might help to better clarify the potential risks and undesirable health hazards associated with carbon nanoparticles.

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Introduction

The domain of nanotechnology is rapidly increasing and today there are more than 1500 nanoproducts in the market.^1,2 The different nanostructured materials such as nanotubes,³ nanoparticles,² nanocages,⁴ nanopowders,⁵ nanowires,⁶ nanoshells,⁷ nanorods,⁸ nanofibers,⁹ quantum dots,¹⁰ fullerenes,¹¹ liposomes,¹² neosomes,¹³ nanoclusters,¹⁴ nanomeshes,¹⁵ nanocrystals,¹⁶ nanofilms¹⁷ and nanocomposites¹⁸ are internationally produced in bulk quantities because of their wide potential applications in skincare and consumer products, healthcare, photonics, electronics, biotechnology, engineering products, pharmaceuticals, drug delivery, agriculture, etc. When different nanostructured materials are exposed to the human body they can easily enter into the body through the lungs or other organs and tissues such as the brain, liver, kidney, heart, colon, bone and blood via food, drink and medicine.^19–22 These nanomaterials may cause cytotoxic effects, e.g., the deformation and inhibition of cell growth leading to various diseases in humans and animals. The toxicity and interactions of these nanomaterials with biological systems largely depend on their physicochemical properties such as size, concentration, solubility, chemical composition and stability.^23–26

Carbon nanoparticles (CNPs) are popular nanomaterials because of their interesting properties, which are desirable in many industrial purposes.²⁷ CNPs have physicochemical properties that are already in use in the commercial, environmental and medical sectors.^28–32 However, with diameters in the nanoscale range these extremely small particles may constitute a health risk.³³

Particle emission studies from a steady burning single paraffin wax candle found the diameter of the produced particles to be around 30 nm.³⁴ The concentrations of the particles emitted from a pure wax candle and a scented candle in a chamber were reported to be 240 000 particles per cm³ and 69 000 particles per cm³, respectively.³⁵ CNPs are very light and easily enter in the working environment as suspended particulate matter of respirable sizes.³⁶ Therefore, these CNPs could pose an occupational inhalation exposure hazard. CNPs of sizes <2.5 micron are found in the combustion streams of methane, propane, butane and natural gas flames of typical stoves. Both indoor and outdoor fine particle samples contain a significant fraction of CNPs. It has already been reported that CNPs are more toxic than quartz and cause occupational health hazard if chronically inhaled.³⁷ Environmental fine CNPs are known to be generated mainly from the combustion of fuels, and has been reported to be a major contributor to the induction of cardiopulmonary diseases caused by pollutants.³⁸ Therefore, CNPs from manufactured and combustion sources in the environment could have adverse effects on human health.

Carbon nanotubes (CNTs) are known to have a wide range of biomedical applications but the importance of nanoparticle–protein interactions are not stressed enough,³⁹ although systemic interactions of CNTs with various plasma proteins have been reported. However, despite the fact that CNPs can penetrate the plasma membrane, knowledge regarding the effect of CNPs on blood corpuscular protein, i.e. haemoglobin, is still obscure.^40–44 Inhalation of CNPs from the environment directly exposes them to our blood circulatory system.^42–44 Therefore, knowledge of the interaction between CNP and the oxygen transporter protein, hemoglobin, is essential to clarify the impact of CNPs on human health. In the current paper we present a detailed study on the interaction of hemoglobin with CNPs, using fluorescence spectroscopy and circular dichroism spectroscopy (CD). For a better understanding, we have used myoglobin alongside hemoglobin, as this can be considered as a monomeric unit or hemoglobin. Myoglobin is functionally similar to hemoglobin and shares a similar structural fold, despite the dissimilarities in amino acid composition, thus, it provides a deeper insight into the effect of CNPs on hemoglobin.

Materials and methods

Materials

Human hemoglobin and myoglobin were purchased from Sigma-Aldrich (USA). Carbon soot, acetone, TrisHCl and nitric acid were also purchased from Sigma-Aldrich. All the samples were prepared in 20 mM Tris–HCl buffer of pH 7.4 for CNP–protein interaction studies. Deionized and triple distilled water was used for preparing a buffer solution that was passed through 0.22 μm pore size Millipore filters (Millipore India Pvt. Ltd., Bangalore, India). A carbon coated copper grid for the TEM study was purchased from Allied Scientific Product, USA. ASTM V1 Grade Ruby Mica sheet for AFM study was purchased from Micafab India Pvt. Ltd., Chennai, India.

Synthesis of carbon nanoparticles (CNPs)⁴⁵

100 mg carbon soot was added to 60 mL of 6 M Nitric acid in a 25 mL three necked flask. The reaction mixture was then refluxed at 100 °C for 10 hours under stirring. Afterwards, a black solution was obtained. The solution was then centrifuged at 2000 rpm for 15 minutes to separate out the unreacted carbon soot. Subsequently, the aqueous solution was mixed with acetone and then centrifuged at 30 000 rpm for 20 minutes and a black precipitate was obtained. This precipitate was washed in 5–10 mL water to remove the excess nitric acid. Then, the precipitate was dried and its weight was measured. This was then dissolved in an appropriate amount of buffer to obtain the desired concentration. The pH of the final suspension was adjusted to 7.4 and this suspension was used for further experiments.

Dynamic light scattering (DLS)-based zeta-potential measurements

To determine the size distribution of the CNPs, DLS experiments (model: Zetasizer Nano Z, Malvern Instruments Ltd, United Kingdom) were carried out with the aqueous suspension. The scattered lights were collected at a 90° angle. Data were acquired and analyzed by a Precision Deconvolve program. For a typical DLS experiment, 200 μL of a sample solution was slowly pipetted into a clean quartz micro-cuvette.

TEM sample preparation and imaging

For TEM imaging, 20 μL of the nanoparticle solution was placed on a 300-mesh carbon coated copper grid and the excess samples were removed cautiously by a tissue paper. This was finally dried and the images of the resulting nanoparticle solutions were recorded on a Tecnai G2 Spirit Bio TWIN (Type: FP5018/40) at an acceleration voltage of 80 kV.

AFM sample preparation and imaging⁴⁵

Twenty microliters of the nanoparticle solution was deposited onto a freshly cleaved muscovite Ruby mica sheet for 5 to 10 min. After 10 min, the sample was dried using a vacuum dryer. An AAC-mode atomic force microscopy was performed using a Pico Plus 5500 AFM (Agilent Technologies, Inc., Santa Clara, CA, USA) with a piezo scanner at a maximum range of 9 μm. Microfabricated silicon cantilevers of 225 μm in length with a nominal spring force constant of 21–98 N m⁻¹ were used from nanosensors. The cantilever oscillation frequency was tuned to the resonance frequency. The cantilever resonance frequency was 150–300 kHz. The images (512 × 512 pixels) were captured with a scan size between 0.5 and 5 μm at a scan speed rate of 0.5 rpm. The images were processed by flattening using Pico view software (Molecular Imaging Inc., Ann Arbor, MI, USA). The image presented in this paper was derived from the original data. Length, height and width were measured manually using Pico view software.

Fluorescence spectroscopy

The steady-state fluorescence spectra were recorded with a Perkin Elmer LS-45 spectrofluorophotometer at 25 °C. The samples were excited at 280 nm wavelength and the emission spectra were recorded over the emission range of 300–450 nm. In all the cases, the excitation and emission slit widths were maintained at 5 nm each. The intrinsic fluorescence of protein was measured in the presence and the absence of the CNPs. Intrinsic fluorescence of Mb and Hb originated from both the Trp and Tyr. Hb contains 6 Trp and 12 Tyr whereas Mb contains 2 Trp and 2 Tyr. The fluorescence of the protein was quenched in the presence of the CNPs. The quenching experiment was carried out simply by adding small aliquots of concentrated CNP solution to the hemoglobin or myoglobin solution taken in a 1 cm path length quartz cuvette. The protein concentration was maintained at 0.5 μM and the CNP concentration was varied from 0.5 μM to 5 μM. The optical density of the solution at the excitation wavelength was kept below 0.05. Small errors due to dilution upon the addition of the CNPs were neglected. Fluorescence intensities at the emission maximum were recorded as a function of ligand concentration. To derive the binding parameters, the obtained data were analyzed using a modified Stern–Volmer equation (eqn (1)).

F₀/ΔF = 1/fK[Q] + 1/f (1)

where F₀ is the fluorescence intensity in the absence of an external quencher, ΔF is the difference in fluorescence in the absence and in the presence of the quencher at concentration [Q], K is the Stern–Volmer quenching constant and f is a fraction of the initial fluorescence which is accessible to the quencher. The binding dissociation constant, K_d, was measured as the reciprocal of K.

Temperature dependent fluorescence quenching was performed to obtain the K_d values as a function of temperature, and then the thermodynamic parameters of binding were determined from it by fitting van't Hoff's equation (eqn (2)) to the data.

ln K = −ΔH°/RT + ΔH°/R (2)

where K is the equilibrium constant (here the Stern–Volmer quenching constant) of binding at a corresponding temperature T and R is the gas constant. The equation gives the standard enthalpy change (ΔH°) and standard entropy change (ΔS°) on binding. The free energy change (ΔG°) has been estimated from the following relationship (eqn (3)):

ΔG° = ΔH° − TΔS° (3)

CD spectroscopy

The conformational changes in the secondary and tertiary structures of hemoglobin and myoglobin on the binding of CNPs were studied using a Jasco J-810 spectrometer at 25 °C. The CD spectra of Hb and Mb were finally obtained by averaging five successive scans recorded at a scan speed of 50 nm min⁻¹ and subtracting the appropriate blanks (tris buffer) from these spectra. For Hb and Mb proteins, the changes in the far UV-CD spectra (200–250 nm) provide insights about the changes in the secondary structure of the proteins. The protein concentrations were 0.75 μM for Hb and 3.0 μM for Mb. The results are expressed in terms of mean residual ellipticity (MRE) in ° cm² dmol⁻¹ according to the equation given below.⁴⁶
where C_p is the molar concentration of the protein, n is the number of amino acid residues (n = 574 for Hb and 154 for Mb) and l is the path length (here l = 0.1 cm, i.e. the path length of the cuvette used). The helical content of free and bound protein was finally evaluated from the MRE values at 208 nm using the equation given below.⁴⁶

Results and discussion

DLS study

The results of the DLS experiment showed that the mean particle size of the CNPs is 20.17 nm. The zeta potential distribution of CNPs are negative charged to −39.1 mV in water, which is sufficient to keep the particles from interacting with each other; therefore, maintains a stable particle size of the sample. The resulting negative charges of CNPs that have been tagged are attributed to the negative surface charge on them.

TEM and AFM imaging

The transmission electron microscopy (TEM) images of the prepared CNPs have a spherical morphology with a relatively regular diameter of ∼20.0 nm (Fig. 1A). They are almost uniform in nature. The atomic force microscopy (AFM) study was in good agreement with the TEM results, which showed that the average sizes have a spherical morphology with a relatively regular diameter of ∼22.0 nm (Fig. 1B).

Fig. 1 TEM (A) and AFM (B) image of synthesized ultrafine CNPs.

Fluorescence spectroscopy

The fluorescence spectra of Hb/Mb were measured in the presence and absence of CNPs. Hb shows a strong fluorescence with an emission peak at ∼353 nm (Fig. 2A). Mb showed a similar fluorescence behavior, however, with an emission peak at ∼330 nm. The CNPs showed no intrinsic fluorescence in the experimental buffer solution. However, the presence of CNPs in the solution effectively reduced the fluorescence yield of Hb/Mb. For both the Hb and Mb, the fluorescence intensity decreased gradually with increasing CNPs concentration, indicating effective quenching of the protein fluorescence. Fig. 2 shows the spectra in the presence of different concentrations of CNPs with Hb and Mb.

Fig. 2 Effect of the compounds on the intrinsic fluorescence of Hb and Mb. (A) The emission spectra of Hb (0.5 μM) as a function of CNPs with concentration varying from 0 μM to 4.5 μM (9 steps). (B) The emission spectra of Mb (0.5 μM) as a function of CNPs with concentration varying from 0 μM to 5.5 μM (11 steps). Emission maximum: 353 nm for Hb and 330 nm for Mb.

A temperature dependent experiment was performed to obtain the thermodynamic parameters of binding (Fig. 3). This experiment showed that the quenching constant for CNPs decreased with increasing temperature. Quenching decreased with increasing temperature, which suggests static quenching (Fig. 4). However, fluorescence lifetime measurements would be a definitive indicator. Binding constants and the thermodynamic parameters^47,48 of the binding are listed in Table 1. Here, we observe that the entropy of the system increases whereas enthalpy decreases; therefore, the process is enthalpy driven. We agree that the entropy change due to adsorption is negative. In this macromolecular system, when we consider the contribution of water exclusion/solvent release, the overall entropy of the system increases.⁴⁹

Fig. 3 Modified Stern–Volmer plot for the determination of K_d of Hb binding with CNPs (A), and Mb binding with CNPs (B).

Fig. 4 van't Hoff plot for the determination of the thermodynamics parameters for the binding of Hb with CNPs.

Table 1 Binding constant and the thermodynamic parameters of binding

	Kd (μM)	ΔG° (kJ mol^−1)	ΔH° (kJ mol^−1)	ΔS° (J mol^−1 K^−1)
Hb-CNP	0.29	−37.98	−32.19	19.10
Mb-CNP	0.25	NA	NA	NA

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Circular dichroism

Circular dichroism (CD) is a powerful technique to investigate the interaction of proteins with other molecules, including nanoparticles. In general, this technology is used to determine the protein conformation in solution or adsorbed onto other molecules or nanoparticles. Here the CD spectra were obtained in the wavelength range of 200–250 nm by preparing the aliquots of the samples, the results expressed in Fig. 5.

Fig. 5 Circular dichroism spectral changes of Hb (A), and Mb (B) on interaction with increasing concentration of CNPs.

The far ultraviolet CD spectra of native Hb and Mb (Fig. 5) have two minima at 208 (π–π*) and 223 nm (n–π*), which are characteristic of typical α-helical structures. The carbon nanoparticle is not CD active. For Hb and Mb, changes in the CD spectra for the secondary and tertiary structures have been noted upon CNP binding, but such changes are much greater for Hb compared to that of Mb. It has been found that the binding of CNP to Hb causes a reduction of α-helix of the protein secondary structure from 76% (free Hb) to 61% (Hb-CNP), whereas for Mb a reduction of α-helix of the protein secondary structure from 75% (free Mb) to 71% (Mb-CNP) has been observed. Such results suggest structural changes for both Hb and Mb upon binding to CNP.

Conclusion

The CNPs were synthesized and their interactions with Hb and Mb were studied. The results show a remarkably strong interaction between carbon nanoparticles with hemoglobin and myoglobin separately. TEM, AFM and DLS experiments have been performed to confirm the ultrafine nature of the synthesized CNPs. Temperature dependent fluorescence spectroscopy shows the exothermic binding of CNPs to Hb, which is favored by enthalpy and entropy changes. Circular dichroism study also reflects the significant disruption of the protein secondary structure and partial unfolding of the helical structure. These findings have given us information regarding the understanding of this strong interaction between carbon nanoparticles and hemoglobin and myoglobin. This might help to better clarify the potential risks and undesirable health hazards of carbon nanoparticles.

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