Study of interaction of methylene blue with DNA and albumin

Poghos O. Vardevanyan*, Ara P. Antonyan, Marine A. Parsadanyan, Mariam A.

Shahinyan, Nara H. Petrosyan

Department of Biophysics, Faculty of Biology, Yerevan State University, Yerevan, Armenia 0025

*Corresponding author: Phone: (+374 60) 710 522; Fax: (+374 10) 554 641; E-mail:
p.vardevanyan@ysu.am

Short title – Methylene blue interacting with DNA and HSA

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Study of interaction of methylene blue with DNA and albumin

The interaction of thiazine dye methylene blue (MB) with Calf thymus DNA and human
blood serum albumin (HSA) has been studied. MB was revealed to stabilize the native structure
of DNA and HSA, since the melting temperature of the complexes is shifted to higher values in
relation to that of both macromolecules in pure state. It was also revealed that the absorption and
fluorescence spectra of the MB-DNA complexes change significantly, while those of MB-
albumin complexes do not change noticeably. Analysis of the obtained data allows to conclude
that MB binds to DNA by two modes, including intercalation and electrostatic mechanisms. In
the case of HSA, the main binding mode of MB, conditioning the stabilization of the protein
native structure, is the electrostatic mechanism.

Keywords: Methylene blue; DNA; human serum albumin; absorption and fluorescence spectra;
denaturation curve

Introduction:
Studies on the interaction of various ligands, especially photosensitizers (methylene blue

(MB), acridine orange (AO), Bengal Rose (BR) etc.), with biomacromolecules are scientifically

interesting, which comes from their important biological properties. Being associated with a

macromolecule, both photophysics and photochemistry of a photosensitizer can be changed,

which in turn can modify the photo-oxidation mechanism of the macromolecule – a phenomenon

that is important for albumins, taking into account their high physiological concentrations and

binding capacity. Complex-formation of the dye molecule with protein may lead to the

alternative mechanism of photo-oxidation, which means that through the interaction with the

dye, the protein can acquire a site-specific II type mechanism of photo-oxidation (Alarcon, et al.,

2010; Steinmetzet, & Reinert, 1998; Ahmadi, Mahmoodian-Moghadam, Mokaberi, Saberi, &

Chamani, 2015; Xie, Sang, Tao, & Liu, 2013; Xie, Tao, & Liu, 2013). From this point of view

acridine dyes, including MB, have certain value (fig. 1). These ligands usually possess a wide

spectrum of biological action (Kumar, Kaur, & Kumari, 2012; Patel, Mali, & Patel, 2010;

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Cholewinski, Dzierzbicka, & Kolodziejczyk, 2011; Silva, et al., 2018). MB is a thiazine dye and

contains a sulfur atom in a central ring of chromophore.

MB is photo-sensitive as well and binding to macromolecules, results in formation of

activated singlet oxygen, which in turn invokes photo-oxidative damages in macromolecules.

Luminescence measurements of singlet oxygen showed the dependence of quantum yield on

concentration of DNA complexes with this ligand. Despite the fact that this ligand is an

intercalator, the molecular mechanisms of MB interaction with DNA hitherto remain as a

discursive topic. Particularly, hypotheses of MB binding to DNA are based on the spectroscopy

methods that are linear and circular dichroic methods (Kumar, Kaur, & Kumari, 2012).

Spectroscopic studies have shown that MB binds to AT- and GC-regions of DNA by various

modes, though these methods do not explain the binding differences (Orth, Beck, Genze, &

Ruck, 2000).

Proteins, as nucleic acids (NA) are known to be important biomacomolecules and

contribute to all viability processes practically. Blood serum albumin is one of the most

important proteins in multi-cellular animals, especially in warm-blooded animals (Baler, et al.,

2014; Vardevanyan, Antonyan, Shahinyan, & Mikaelyan, 2016). Role of this protein is relevant

that is why its quantitative content is equal about 40-50 mg/ml in blood plasma (60% of all

proteins). This protein is multi-functional, because among macromolecules, it plays the main role

in establishment of blood osmotic pressure (Li, Zhang, Sun, Zhang, & Liu, 2013; Singh, &

Mitra, 2017; He, et al., 2015; Grigoryan, Zatikyan, & Shilajyan, 2020). Meanwhile, the chief role

of albumin is connected to storage and transport of multiple endogenous and exogenous

compounds, most of which are being transported through the circulatory system in complex with

albumin. Albumin is found in inter-tissue fluids and liquid body secretions as well (content of

this protein in the whole protein component of extravascular fluid is equal about 60%) (Baler, et

al., 2014; Vardevanyan, Antonyan, Shahinyan, & Mikaelyan, 2016). Human serum albumin

(HSA) is consisted of three domains that form heart-shaped form via 17 disulfide bonds, which

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stabilize domains. This shape of the albumin is established at relatively neutral values of pH and

is called N-isoform (Baler, et al., 2014). Crystallographic analyses of HSA indicate that this

protein is consisted of 585 amino-acidic residues, comprises three homological -helices (I-III)

and single tryptophan residue (Trp-214) (Hu, Li, Liu, Dong, & Qu, 2005; Ziyarat, Asoodeh,

Barfeh, Pirouzi, & Chamani, 2014). Dimensional form of albumin practically plays a key role in

the transport of different compounds, including drug preparations, since it is responsible for their

reversible binding to HSA. Transport of exogenous compounds via albumin has a clinical and

pharmacological value, since it can reason the pharmacokinetics of drug preparations (Xu, et al.,

2015; Sienko, et al., 2014).

To estimate the distribution mechanism of ligands on the target molecules in organism,

their interaction with albumin should be carried out, since albumins are abundant ones in

circulatory system and act as transporters of metabolites and xenobiotics. Therefore, the studies

of low-molecular weight compounds with proteins and NA provide information about

pharmacokinetics and pharmacodynamics of their bioavailability (Nafisi, Panahyab, & Sadeghi,

2012).

Figure 1.

Structure of HSA (from left) and MB (from right).

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 Thus, in this work the comparative study of MB interaction with DNA and HSA has been

carried out using spectroscopic techniques (UV-melting and UV-VIS and fluorescence

spectroscopy) and the affinity of MB to them was examined.

Materials and methods:

Calf thymus DNA, human serum albumin, methylene blue (“Sigma”, USA),

physiological solution, bi-distilled water were used in experiments. The preparations were used

without additional purification. Concentrations of MB and DNA were determined

spectrophotometrically, using the following coefficients of extinction: 664=76000 M-1cm-1 for

MB and 260=6600 M-1cm-1 for calf thymus DNA. Concentration of stack solution of albumin was

equal to 1% (calculated by Malb.=67000 Da). Working solutions were diluted by 7.7 times, by

physiological solution (optic absorption was about 0.4 in cuvettes with pathway length 1 cm).

Melting of MB complexes with DNA and HSA was carried out on double-beam

spectrophotometer PYE Unicam SP-8-100 (England). Heating of thermostating cells was

realized using program device SP 876 Series 2. Absorption measurements of the complexes was

registered at the wavelength =260 nm for DNA and =280 nm for HSA. MB intensively

absorbs in the interval 220300 nm, though this fact does not significantly affect the analysis

of the obtained data. These data were taken on the PC using the program software LabVIEW.

Absorption spectroscopy studies of the interaction of MB with DNA and HSA were

carried out on spectrophotometer Perkin Elmer Lambda 365 (USA). Data of melting studies

were analyzed by program software Microsoft Excel 2010. The absorption spectra of MB and its

complexes with DNA and HSA were obtained in the interval 500750 nm. Fluorescence

spectra of MB and its complexes with DNA and HSA were obtained on the spectrofluorometer

Cary Eclipse (Australia), excitation of the sample solutions was carried out at wavelength =620

nm, fluorescence spectra were registered in the interval 600800 nm. Error of the

experimental data does not exceed 5%.

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Results:

Absorption spectra of the complexes DNA-MB and HSA-MB were obtained and

presented in fig. 2 (a and b respectively). From the presented figure it is obvious that the

absorption spectra of MB complexes with DNA decrease in maxima and take red shift by about

5-6 nm. A pseudo-isosbestic point is formed in the spectra of the complexes, which indicates that

MB molecules are in bound and free states simultaneously at the concentrations of DNA much

less, than MB concentrations. At higher concentrations of DNA all molecules of the ligand are

practically transited to bound state. In the case of HSA, the decrease of the spectra of the

complexes protein-ligand is relevantly small. At the same time in contrast to DNA, an isosbestic

or pseudo-isosbestic point is not formed in the case of the protein. From the similarity of the

spectra for free and bound MB molecules one can assume that the binding takes place in protein

surrounding, available for water (Alarcon, et al., 2010).

It should be mentioned that we have obtained the absorption spectra in the region of

visible light to avoid the contribution of own absorption of molecules of DNA and protein (in the

interval of wavelength 220-300 nm). Such approach is explained by the fact that the changes in

UV region would not be precise for studying of DNA and HSA interaction peculiarities with

MB.

Fluorescence spectra of MB complexes with DNA and HSA are presented in fig. 3 (a and

b respectively). As it is obvious from fig. 3 fluorescence quenching of the ligand occurs while

interacting with DNA and HSA, since the fluorescence intensity of MB decreases along with

concentration increasing both of DNA and of protein. It is also obvious from fig. 3 that the

fluorescence intensity quenching of MB is higher in the case of DNA, than HSA.

Melting of MB complexes with DNA and HSA at various ratios r=ligand/macromolecule

was carried out as well. The melting curves were obtained and presented in fig. 4. From the

presented figure it is shown that stabilization of both DNA and HSA takes place in the presence

6
of MB, as a result of which the melting curves of the complexes were shifted toward higher

temperatures in relation to the denaturation curves of pure DNA or HSA.

Figure 2. Absorption spectra of MB (1) and its complexes with DNA (2-11) (a) and HSA (2-37)

(b).

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 a

Figure 3. Fluorescence spectra of MB (1) and its complexes with DNA (2-16) (a) and HSA (2-8)

(b).

Discussion:

MB is in cationic form; it is a heterocyclic aromatic compound that depending on

concentration can be in solution both as a monomer and as a dimer or excimer, which is reflected

on spectral properties of this ligand (Nafisi, Panahyab, & Sadeghi, 2012). Aggregation of MB in

dimer and excimer forms in the solution results from the fact that intra-molecular transition of

electron by donor-acceptor groups can take place inside its molecule. Consequently, studying the
8
interaction of MB with biomacromolecules (NA, proteins) this fact should be taken into account.

At low concentrations, MB mainly is in monomeric form (Nafisi, Panahyab, & Sadeghi, 2012).

1-
a

t, 0C

1-
b

t, 0C

Figure 4. Melting curves of the complexes DNA-MB (a) and HSA-MB (b) at ratios

r=MB/macromolecule: 1 –0, 2 – 0.02, 3 – 0.05 and 4 – 0.167 for DNA; 1 – 0, 2 – 0.05, 3 – 0.1, 4

– 0.2 and 5 – 1.0 for HSA.

Interaction peculiarities of MB monomeric form with DNA indicate that this ligand binds

by more, than one mode: one mode is intercalation mechanism, another – electrostatic (Liu, Wu,

& Yang, 2012; Changlun, Zhou, & Jianmin, 2010; Nafisi, Saboury, Keramat, Neault, & Tajmir-

Riahi, 2007; Hossain, Giri, & Kumar, 2008; Rohs, & Sklenar, 2004; Vardevanyan, Antonyan,

Parsadanyan, Shahinyan, & Hambardzumyan, 2013; He, et al., 2015). Consequently, one can

state that this ligand binds to DNA specifically (intercalation mechanism) and non-specifically

(electrostatic mechanism). From this point of view the experimentally obtained data in this work

indicate that for HSA a specific binding to MB can be excluded. Particularly, this fact is

9
maintained by little changes appeared in the absorption and fluorescence spectra of MB at

interaction with protein, although in the case of DNA these changes are pronounced. Actually, in

the bound state with albumin MB has practically the same spectral characteristics, as in free one;

while in the case of DNA, the optic properties of the ligand bound molecules relevantly differ

from those in free state. It results from the fact that at the intercalation binding mode the

heterocyclic aromatic molecule of MB is transferred from the solution to hydrophobic inter-

nucleotide space and enters into stacking interaction with DNA azotic bases. Taking into account

that it leads to significant loss of chromophore freedom degree, a weakening of oscillation force

of the ligand molecules occurs, due to which the absorption intensity of MB, bound to DNA,

decreases, but the maxima are shifted to the longer wavelengths. As a result of this fact a pseudo-

isosbestic point in the absorption spectra of DNA-MB complexes is formed, which means that at

this point the free and bound molecules of DNA have sufficiently close values of the absorption.

In the case of interaction with HSA, the optic characteristics of MB free and bound molecules

differ less, which means that the local surrounding of MB molecules, bound to the protein, is

practically the same, as that of free molecules. It is indicated by the results of fluorescence

measurements as well. Numerous experimental data show that the fluorescence of MB is

intensively quenched at the complex-formation with DNA (Liu, Wu, & Yang, 2010). Analogous

effect is revealed at the interaction of MB with HSA as well, though the fluorescence quenching

of MB is more pronounced, than the absorption decrease and indicates formation of the

complexes between MB and HSA, which leads to changing of the ligand molecule environment,

being in bound state (Grigoryan, Zatikyan, & Shilajyan, 2020; Hu, Li, Liu, Dong, & Qu, 2005).

Fluorescence quenching of MB at the interaction with DNA and HSA was determined by the

method of Stern-Volmer (Liu, Wu, & Yang, 2010).

F0
=1+ K SV [macromolecule ]
F (1)

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where F0 and F are MB fluorescence intensities in the absence and presence of the quencher

respectively, KSV – Stern-Volmer quenching constant. Dependence curves of F 0/F on increasing

concentration of DNA (a) and HSA (b) are presented in fig. 5.

F0/F a

CDNA; mol/l
F0/F b

CHSA; mol/l

Figure 5. Dependence curves of MB fluorescence quenching by DNA (a) and HSA (b), at

pH=7.0, t=250C.

It is obvious from fig. 5 that the curves F 0/F show a linear dependence on the increasing

concentration of macromolecule, though the quenching constant for DNA is equal about

KSV=4.2104 Lmol-1 and for HSA – KSV=104 Lmol-1. It indicates that in both cases the quenching

should take place by either the static mechanism, including the ground state complex-formation

with DNA and HSA or the resonance quenching, promoted by MB binding to HSA nearby the

11
fluorescing part – Trp-214 (Hu, Li, Liu, Dong, & Qu, 2005; He, et al., 2015). Comparison of the

value of KSV to that of the association constant K a, at the interaction of MB with DNA or HSA,

reveals that these parameters practically coincide, which permits concluding that during the static

quenching processes, the value of KSV corresponds to the total binding constant of the quencher

to HSA in the case, when the quencher completely suppresses the intrinsic fluorescence of HSA.

Particularly, it has been shown that K a6.5105 M-1 at the interaction with calf thymus DNA

(Rohs, & Sklenar, 2004). In the case of HSA this value was equal to almost 10 4-105 M-1

(Alarcon, et al., 2010; Ding, Xie, Peng, & Peng, 2016).

However, the more reliable information about MB binding to DNA and HSA was

obtained by UV-melting method. From the received data it is revealed that the melting curves of

MB complexes with DNA and HSA are shifted to the higher temperatures (fig. 4). This fact

indicates that MB makes difficult thermo-denaturation of both DNA and HSA. MB, as many

ligands-intercalators, is a stabilizer of double-stranded (ds-) structure of NA that is why at the

binding of such molecules to ds-DNA, the temperature increase of NA transition to single-

stranded (ss-) state occurs (Vardevanyan, Antonyan, Parsadanyan, Torosyan, & Karapetian,

2016). From the melting curves it is also obvious that the stabilization of DNA ds-structure

depends on the ligand concentration and at the value of r=0.167 the melting temperature of the

complexes increases by 8-100, as compared to that of DNA.

Analogous shift occurs at the thermo-induced denaturation of HSA and its complexes

with MB as well. On the other hand, this ligand stabilizes the native form of HSA much less,

than of DNA, which becomes obvious from the denaturation curves (fig. 4). It has been shown

that HSA has two regions for the binding of drug preparations, which are localized in sub-

domains IIA and IIIA and are called sites 1 and 2 (Alarcon, et al., 2010; He, et al., 2015).

Though, despite the fact that site 1 is more hydrophobic, the preferable one for MB binding is

site 2 (Alarcon, et al., 2010).

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 However, this binding site in the case of MB cannot be considered specific one to which

MB shows a pronounced affinity. Based on this we assume that the main binding mechanism of

MB to HSA is electrostatic interaction (Hu, Li, Liu, Dong, & Qu, 2005; He, et al., 2015). In

DNA there also exist hydrophobic and hydrophilic regions that serve as adsorption centers for

ligands. In comparison to HSA, DNA hydrophobic adsorption centers (inter-plane spaces

between adjacent nucleotides or nucleotide pairs) for MB are specific binding sites this ligand

interacts with by the intercalation mechanism. Non-specific binding of MB to DNA is also

realized by the electrostatic mechanism, due to which the ligand molecules bind to sufficiently

hydrophilic sugar-phosphate skeleton of NA.

Conclusion:

The obtained data indicate that MB is a specifically binding ligand to DNA by several

modes. Meanwhile, this ligand can interact with proteins as well (Alarcon, et al., 2010; Hu, Li,

Liu, Dong, & Qu, 2005). Moreover, MB interaction with DNA occurs both by specific

(intercalation mechanism) and by non-specific (electrostatic mechanism) modes. For HSA the

binding specific mechanism is absent, while the electrostatic one is appeared as the main

interaction mode (Hu, Li, Liu, Dong, & Qu, 2005). This fact can have an important biological

value, since at both non-specific binding and transport of MB (as well as other drug or

biologically active compounds) via HSA, the possibility of their transfer to specific targets (for

instance, to NA) is facilitated.

“No potential conflict of interest was reported by the authors.”

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