In this work the study of the effect of millimeter range electromagnetic waves (MM EMW) on methyl violet interaction with human serum albumin (HSA) has been carried out, using the UV-denaturation, fluorescence spectroscopy and CD spectroscopy methods. It was revealed that MM EMW irradiation leads to weakening of the stability of HSA and decreasing of interaction force between HSA and methyl violet (MV). It was also shown that MM EMW irradiation by water non-resonant frequency, affecting immediately the structure of HSA, changes its secondary structure, while the irradiation by water-resonant frequency does not invoke structural changes, but weakens the stability more. The last insistence results from the water cluster reconstruction, due to which the structured water affects the conformation of HSA.
Research Article
Study of the Effect of Mm Emw on Methyl Violet Interaction With Human Serum Albumin
https://doi.org/10.21203/rs.3.rs-1753011/v1
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millimeter range electromagnetic waves
denaturation parameters
fluorescence intensity
quenching constant
ellipticity
Nowadays the intensity of radiofrequency electromagnetic waves in environment is growing and the protection topic of living material from this physical factor is becoming a huge problem, needing deep study and elaboration of measures against its possible dangers [1, 2]. Millimeter range electromagnetic waves (MM EMW) take a special place among radiofrequency waves. It is due to their wide application in various spheres of our life, such as high-speed wireless networks [3, 4], radars [5], nondestructive testing [6], security screening in airports [7], nonlethal crowd control weapons [8], etc. On the other hand, the natural background of these waves on The Earth is not high [9], which means that the adaptation resources of biological systems are not developed during evolution to properly protect them from probable damages. From the beginning of the studies of this physical factor effect on biosystems it was maintained that MM EMW have a potential dangerous effect on different tissues of animals – skin [10], cornea [11], nervous system [12], etc. At the present MM EMW are also used in physiotherapeutic procedures, such as treatment of different cancers, cardiovascular diseases, diabetes, ulcers, leucopenia, skin disorders, bronchial asthma and wound healing [13–15]. Furthermore, literature data indicate that MM EMW-therapy increases the level of immune resistance, changes activity of enzymes, destroys microorganisms and changes DNA and protein thermostability [16–18]. Along with the mentioned prominent problems it is very important to study the effect of MM EMW on biomacromolecules, particularly, on their complex-formation with small molecules – ligands. As a biomacromolecule – protein, albumin is very important, due to its multiple functions in organism. Albumin is the most abundant protein in blood of vertebrates and in blood plasma it composes 40–50 mg/ml, which is almost 60% of the total plasma protein content. On the other hand, it is one of the most studied proteins [19, 20]. In the present work human serum albumin (HSA) was taken. HSA has a molecular weight of 66.5 kDa, composed of 585 amino-acids. The tertiary structure of HSA consists of three domains (I-III), each of which, in turn, contains two sub-domains (A and B), stabilized by 17 disulfide bridges. These two sub-domains represent an interest for some ligand binding to HSA [21, 22]. HSA is valued from the pharmaceutical point of view, due to its ability to bind and carry drugs, endogenous and exogenous substances [18, 23]. Because of the main transporting function, albumins, including HSA, are widely used in model studies, to reveal the peculiarities of protein-drug interactions. Among ligands, being drugs and various physiological substances, methyl violet (MV) is worthwhile to be studied, which is resulted from its several properties. MV, known also as crystal violet (molecular formula – C25H30CIN3, molecular mass – 408 Da), is a triphenylmethane dye (Fig. 1), being used as a biological stain, fungicide in the agriculture, exterior drug for skin diseases [24, 25]. It also possesses good sterilization, little toxicity and hormesis [26]. It was shown earlier that interacting with HSA, MV forms a complex [24, 26]. Herein the study of MV interaction with HSA under the influence of MM EMW has been carried out using spectral methods. Spectral methods are the prominent tools to research the topology of both binding sites and conformational changes, taking place at ligand binding to macromolecule [27].
Figure 1 near here
Human serum albumin (1% solution) (“Sigma”, USA), methyl violet (“Sigma”, USA) and physiological solution were used in experiments. Concentration of MV was determined spectrophotometrically, using the following value of the extinction coefficient: ε590 = 87000 M− 1cm− 1. For each measurement 5 samples of studied solutions were taken. Measurements were carried out in thermostating cells, using quartz cuvettes with 1 cm optic pathway, 3 ml volume and hermetically closing caps. In all experiments the measurement error does not exceed 5–10%.
Sample exposure by MM EMW: The solution of HSA was irradiated by MM EMW with 41.8 and 64.5 GHz frequencies. The choice of these two frequencies resulted from the following ideas: the irradiation with 41.8–42.2 GHz frequencies significantly affects the biological objects [28, 29]; 64.5 GHz frequency is a resonant one for water [30]. The exposure time was 1 hour, which is justified by our early experiments [18, 31]. After the irradiation of HSA solution, the complex of HSA-MV was prepared for UV-denaturation measurements, then the solution of MV was titrated by irradiated HSA solution for fluorescent study and the third – the irradiated HSA solution was titrated by MV solution for CD spectra obtaining. The source of MM EMW was the generator G4-142 (“Istok”, Fryazino, Russia), with working interval 53.57–78.33 GHz and power flux density ≈50 µW/cm2 at an output power of 8 mW. The given value of the power flux density was registered at the sample place. The output power of the generator was measured by thermistor head M5-49 and wattmeter M3-10A (“Istok”, Fryazino, Russia). The output signal frequency was controlled by CH2-25 wave-meter (“Istok”, Fryazino, Russia). The frequency stability of the generator in continuous wave mode was equal to ±15 MHz. The exposure of the sample solution to MM EMW was conducted in the far-field zone of cone-shaped antenna with an aperture of 32⋅32 mm at a distance of 250 mm from the radiating plane of the antenna. Electromagnetic field was homogeneous. The sample was irradiated from the top of glassy container with 6 cm diameter, where the thickness of the solution was equal to 1 mm, which permits the rays completely penetrating into the solution volume. Sample irradiation was carried out in Petri dishes, covered by thin permeable cellophane layer to ban water evaporation during the irradiation. Specific absorption rate (SAR), calculated by the method described in [32], was equal to almost 20 µW/kg, which makes impossible to induce any thermal effect. Simultaneously, the control solutions, to which the irradiated samples were compared, were sham-exposed, being placed into the exposure zone, while the generator was turned on, but the output power was attenuated to zero.
UV-denaturation measurements. The exposed and sham-exposed (non-irradiated) HSA complexes with MV were placed in UV-VIS spectrophotometer Perkin Elmer Lambda 365 (Netherlands) and the temperature of the solutions was being increased in cuvettes by temperature controlling equipment up to 900C, with the increasing rate 0.50C/min. The complexes were prepared, adding the irradiated and non-irradiated HSA solutions to MV solution. The concentration ratio MV/HSA of the complexes was equal to 1/10. Thermal denaturation started from 500C up to 900C. At each increment (after each 1 minute) the values of the temperature and respective absorption were fixed on PC. The absorption value was fixed at the wavelength λ = 280 nm. Obtaining the total data, the denaturation curves were constructed – dependence of denaturation degree (1-θ) on temperature (t, 0C). The methodology of denaturation curve construction is described in [33]. From the denaturation curves the denaturation parameters were determined – denaturation degree (Tm) and denaturation interval width (ΔT). From the change of these values one can judge about the thermostability alteration of the complexes.
Fluorescence spectroscopy measurements. Fluorescence measurements were carried out on spectrofluorometer Cary Eclipse (Australia). Sample excitation was done at the wavelength 590 nm. Fluorescence spectra were registered in interval 650≤λ≤720 nm. This interval was chosen to avoid the simultaneous absorption of ligand and HSA. After registration of fluorescence spectrum of the pure ligand, the ligand solution was titrated by non-irradiated and irradiated solutions of HSA at concentration ratios ligand/HSA = 1/2 − 1/10.
CD spectroscopy measurements. Circular dichroic measurements were carried out on Olis™ DSM20 CD spectrophotometer (USA). The CD spectra of HSA solution were registered in interval 200≤λ≤350 nm. After registration of pure irradiated and non-irradiated HSA spectra the solution of the protein was titrated by the solution of MV. The concentration ratio ligand/HSA varied from 1/10 to 1/2.
The denaturation curves of irradiated and non-irradiated HSA with MV are presented in Fig. 2. It is seen from Fig. 2 that MM EMW irradiation destabilizes the complex, though the destabilization degree depends on the affecting frequency.
Figure 2 near here
In the Table 1 the values of denaturation temperature and denaturation interval width are presented. As it is presented in the Table 1, the MM EMW irradiation leads to decreasing of the denaturation temperature.
| Complexes | Tm (0C) | ΔT (0C) |
|---|---|---|
| HSA-MV | 84.5±0.1 | 10.1±0.4 |
| Irradiated by 41.8 GHz HSA-MV | 83.8±0.2 | 10.3±0.5 |
| Irradiated by 64.5 GHz HSA-MV | 80±0.2 | 9.1±0.5 |
Table 1 near here
To reveal the stabilization of the formed complex, the fluorescent studies were carried out. In the Fig. 3 the fluorescence spectra of MV and its complexes with HSA were presented. As it is obvious from the spectra, the fluorescence intensity of MV decreases while titrating by HSA solution. In the Fig. 4 the fluorescence spectra of MV and its complexes with irradiated HSA by 41.8 GHz (A) and 64.5 GHz (B) were presented.
Figure 3 near here
Figure 4 near here
It is obvious from Fig. 4 that the fluorescence quenching again occurs, but in this case the quenching is less, than for non-irradiated complexes. To reveal these changes quantitatively the Stern-Folmer curves were constructed, according to the respective equation:
1
where F0 and F are fluorescence intensities of MV in the absence and presence of HSA respectively. KSV is Stern-Folmer quenching constant, C is quencher concentration (for this case HSA concentration). The Stern-Folmer binding curves are presented at the temperature 250C (Fig. 5).
Figure 5 near here
To establish whether there is a conformational change of the protein or not, the experiments of MV-HSA complexes were carried out by CD spectroscopy as well. It is known that CD technique is very sensitive in establishment of macromolecule conformational changes during its interaction with ligand [34]. The main optical active structures in protein are peptide bonds of polypeptide chain, aromatic amino-acid residues and disulfide bonds [26]. In the Fig. 6 the CD spectra of HSA (A), irradiated HSA by 41.8 GHz (B) and 64.5 GHz (C) frequencies and their complexes with MV are presented in the far ultraviolet region.
Figure 6 near here
Previously we have shown that interaction of MV with HSA leads to stabilization of the latter, as compared to pure protein [35]. In the Fig. 2 it is presented that MM EMW irradiation destabilizes the complex and the destabilization degree differs depending on the affecting frequency. Nonetheless, it is important to note that the destabilized MV-HSA complexes are, in any case, stable, than the pure protein [35]. The destabilization degree can be judged on the basis of the values of denaturation parameters. From the Table 1 it is obvious that the MM EMW irradiation leads to decreasing of the denaturation temperature. Meanwhile, the MM EMW irradiation by 64.5 GHz frequency makes the protein-ligand complex denature in lower temperature, than at the irradiation by non-resonant frequency – 41.8 GHz. At the same time the value of the denaturation interval width of the MV-HSA complex, irradiated by 41.8 GHz does not significantly differ from that of the control sample. Though, the denaturation interval width of MV-HSA complex, irradiated by 64.5 GHz varies from the previous two examples. This fact is connected to the effect of MM EMW, mediated by water. Affecting through the water, MM EMW with frequency 64.5 GHz destabilizes the structure of HSA, which results in protein denaturation at low temperature and comparatively rapidly. To examine whether the interaction strength between MV and HSA changes or not, we focused on fluorescence spectra. As it is obvious from the spectra (Fig. 3), the fluorescence intensity of MV decreases while titrating by HSA solution. It means that HSA quenches the MV intensity and along with HSA concentration increasing, the ligand fluorescence intensity decreases. The fluorescence decrease occurs from the value 601 a.u. to 510 a.u. The decrease of maximal fluorescence intensity composes 90 a.u. HSA, interacting with MV, forms a complex, which leads to screening of fluorophore from excitation light and MV fluorescence intensity decreases. It is obvious from Fig. 4 that the fluorescence quenching again occurs, but in the case of irradiation the quenching is less, than for non-irradiated complexes. Thus, in the case of HSA irradiation by 41.8 GHz frequency the fluorescence intensity of MV decreases from 640 a.u. to 580 a.u. – by 60 a.u. Moreover, for HSA irradiation by 64.5 GHz the quenching has these values: from 600 a.u. to 560 a.u., which means that the decrease of the fluorescence intensity is weaker. HSA irradiation by 41.8 GHz results in protein structure alteration, therefore interaction between HSA and MV becomes weaker. It indicates that the fluorophore of MV is screened less from exciting light as a result of which the fluorescence intensity decreases less. The least fluorescence intensity quenching appears for MV interacted with HSA, which was irradiated by 64.5 GHz. This fact is explained by the water mediated structure change of HSA, due to which the interaction does not sufficiently cover the MV fluorophore and the fluorescence intensity does not decrease significantly. To evaluate the quenching degree Stern-Folmer curves were constructed. From the Stern-Folmer curves the quenching constants were calculated. It was found out that the quenching constant of MV-HSA is equal to KSV=6.5⋅103 l/mol, at the irradiation by 41.8 GHz frequency the quenching constant for MV-HSA complex is equal to KSV=3.8⋅103 l/mol and at the irradiation by 64.5 GHz frequency – KSV=2.6⋅103 l/mol. As it is obvious from the presented data, the quenching constant decreases in the presence of MM EMW irradiation, as compared to the sham-irradiated samples. Moreover, the irradiation by the water resonant frequency – 64.5 GHz causes higher decreasing of KSV in comparison to the irradiated sample by water non-resonant frequency – 41.8 GHz. These data are in accordance to the denaturation results and show that the irradiation decreases the protein stability, simultaneously weakening the interaction force between HSA and MV. It is worthy to pay attention to the fact that the irradiation by water-resonant frequency – 64.5 GHz causes more weakening, than that by water non-resonant frequency – 41.8 GHz. From CD spectroscopy data it is obvious (Fig. 6A) that along with addition of MV the CD spectra of HSA change, indicating that there is an interaction between HSA and MV. From Fig. 6B it is obvious that the CD spectra of MV-HSA complexes are different, as compared to pure HSA spectrum. In this case the HSA solution is irradiated by MM EMW with 41.8 GHz. The shape change indicates the conformational change, taking place in protein structure due to the irradiation. These data are in accordance with those obtained earlier, indicating that MM EMW by 41.8 GHz affects the protein structure immediately [33]. Nevertheless, in the Fig. 6C the CD spectra of MV-HSA complexes irradiated by 64.5 GHz are presented. As it is obtained the shape of CD spectra do not change, though the values of ellipticity increase, as compared to the irradiation absence.
Earlier it was found out that MV interacts with HSA by hydrogen bonds and van der Waals forces [36]. It means that MM EMW affects the mentioned bonds in the case of water non-resonant frequency immediately, in the case of water resonant frequency – in a mediated way by water. At the same time the effect of the frequency 41.8 GHz changes the secondary state of HSA, resulting in conformational state alteration in protein structure, indicated by CD spectra.
Thus, the obtained data by the methods of thermal denaturation, fluorescence and CD spectroscopies indicate that MM EMW has a pronounced effect on MV interaction with HSA, causing the significant changes. In the case of thermal denaturation MM EMW irradiation results in interaction weakening between MV and HSA, meanwhile the irradiation by water-resonant frequency – 64.5 GHz weakens it more, than that by water non-resonant frequency – 41.8 GHz. Fluorescence data reveal that again there occurs a weakening of MV interaction with HSA, since the irradiation by water-resonant frequency – 64.5 GHz leads to the smallest value of quenching constant. The CD spectra show that MM EMW irradiation by 41.8 GHz affects immediately the structure of HSA, because there is a change of secondary structure of HSA, while the irradiation effect by 64.5 GHz is mediated by water.
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The authors declare that the ethic side is preserved and they all participate in manuscript composing.
Consent for publication
The authors have read and approved the final manuscript for publication.
Competing interests
The authors have no relevant financial or non-financial interests to disclose.
The authors have no competing interests to declare that are relevant to the content of this article.
Author contributions
Authors Vardevanyan P. and Shahinyan M. have written the whole manuscript; Mikaelyan M. and Grigoryan S. have constructed the figures. All authors made substantial contributions to the conception and design of the work.
Funding
This work was supported by the Science Committee of RA (Grant number № 21T-1F063).
Availability of data and materials
The authors declare that the data supporting the findings of this study are available within the article.
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No competing interests reported.