Introduction

Most of the anti-cancer agents are metallo-drugs [1, 2] and the serum proteins play a role for the transport of metallo-drugs. The binding of these metallo-drugs with serum will affect the delivery, distribution and local concentration of the drug at the disease site [3]. Therefore the study of interaction of metallo-drugs with blood serum proteins receives importance [3]. Human serum albumin (HSA), the most abundant plasma protein in the humans, [4, 5] plays a variety of roles in our body like controlling blood pressure, metabolism, radical deactivation and transport including drugs [6, 7]. The interaction of HSA with drugs may lead to a change in the drug stability and toxicity during the chemotherapy [4]. Since bovine serum albumin (BSA) has the 80 % structural similarity with HSA, BSA has been used as model by many group of researchers [4-8]. Though BSA is a dietary protein, the most important function of BSA is transportation [8-12]. This heart shaped protein, BSA has three homologous domains I–III [13] and each domain is made up of two subdomains, A and B, having unique binding properties [14]. BSA has two tryptophan residues, Trp-134 in the first domain and Trp-212 in the second domain. Trp-212 is located within a hydrophobic binding pocket and Trp-134 is on the surface of the molecule [14]. Trp-134 is available for the interaction with the binding molecules such as drugs [15]. The emission property of Trp-134 and Trp-212 depends on the microenvironment of the protein.

Another protein that has been taken for the present study is ovalbumin which is the major content of avian egg white [16]. Ovalbumin is widely used in the food industry and is also present in many biological systems [17]. Ovalbumin has slightly elongated ellipsoid structure with effective spherical diameter of 5 nm [18]. The formation of amyloid type cross-β structure is also possible in ovalbumin which is confirmed by its interaction with thioflavin T [19]. Both BSA and ovalbumin can be used as drug carriers due to their biocompatibility and low cost [20-23].

The major anticancer agents used in the cancer chemotherapy are platinum compounds like cisplatin, and its second-generation derivatives, carboplatin and oxaliplatin [1, 2, 24, 25]. But these platinum complexes show high toxicity and are inactive against some tumors [26-28]. Among the other metal complexes, ruthenium(II)/(III) complexes are efficient alternatives for platinum [29-32]. Several studies show that organometallic complexes containing ruthenium(II) have promising anticancer characteristics [29] and some are also in clinical trials [31, 32]. Ruthenium(II)-polypyridyl complexes are also useful for the cellular uptake studies due to their stability in aqueous solutions and luminescencent properties [33-35]. Protein mediated transport and generation of activated (excited) Ru(II) species within tumors leads to the generation of reactive oxygen species responsible for the cytotoxic effect in photodynamic therapy (PDT) [36-40]. Binding of these metal complexes with proteins can modulate the activity of proteins [41]. Dendrimer type ruthenium(II)-polypyridyl complexes have also been reported for their surface binding ability with proteins [42, 43]. Metal complexes having large surface area and multivalency are expected to be useful for protein binding studies [44]. Increasing the hydrophobicity of the ruthenium drugs also controls the release of drug and cellular uptake [45].

In the present study para-tert-butylcalix[4]arene moiety is attached to ruthenium(II)-polypyridyl complex to increase the hydrophobicity. There are several reports on the surface binding studies of calixarenes [46-49]. Cellular uptake studies of calixarene derivatives show that contrary to the behavior of cyclodextrin, the uptake of calixarene leads to little deformation of the cell structure [50, 51]. Because of these advantageous properties the binding of calixarene receptors with proteins received importance [52]. Ungaro et. al. [53] have reviewed the application of multivalent calixarene ligands on lectin binding and inhibition, DNA condensation and cell transfection. Neri et. al. [54] have developed a histone deacetylase (HDAC) surface binding agent and established the surface binding properties of calixarene derivatives. Because of these interesting luminescent and binding properties of ruthenium(II) complexes and calixarenes pertaining to the biological system we have investigated the interaction of ruthenium(II)-calixarene system with proteins and important findings reported.

Experimental

The metal complexes, Rubc2 and Rubc3 (structure shown in Chart 1), are synthesized using the literature procedure [55] and characterized using HR-MS technique (The details are given in the supplementary material, Figs. S1 and S2). The mass values are very close to the literature values [55]. The BSA procured from Ocimum Bioscience and ovalbumin from Sigma-Aldrich are used without further purification. The double distilled deionized water is used in the preparation of phosphate buffer and this buffer is used throughout the study. The UV-visible absorption spectral studies are carried out using Analytik Jena Specord S100 spectrophotometer. The emission spectrum is recorded using Jasco FP 6300 spectrofluorometer. The circular dishroism (CD) measurements are carried out using Jasco polarimeter J180 at 2 cycles, 50 mm path length, 1 nm band width and 1 s response.

Fig. 1
figure 1

Luminescence enhancement of (a) Rubc2 and (b) Rubc3 (1 × 10−5 M) on addition of increasing concentration of BSA (1 × 10−6–9 × 10−6 M)

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Fig. 2
figure 2

Luminescence quenching of ovalbumin (1 × 10−5 M) in the presence of increasing concentration of (a) Rubc2 and (b) Rubc3 (1 × 10−5–9 × 10−5 M)

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Fig. 3
figure 3

Luminescence decays of (a) Rubc2 and (b) Rubc3 in the absence (Black square) and in the presence of BSA 1 × 10−5 M (Red circle) and 3 × 10−5 M (Blue triangle)

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Fig. 4
figure 4

CD Sepctra of BSA (3 × 10−7 M) in the absence (1) and in the presence of (a) Rubc2 at 1 × 10−7 M (2) and (b) Rubc3 at 3 × 10−7 M (3)

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Fig. 5
figure 5

Overlap integral of normalized emission spectrum of BSA with normalized absorption spectrum of (a) Rubc2 and (b) Rubc3

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Fig. 6
figure 6

Emission spectra of 1:1 mixture of (a) BSA and Rubc2 (b) BSA and Rubc3 excited at 285 nm

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Fig. 7
figure 7

Cavity of HSA (model to BSA) and ovalbumin using CASTp server

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Fig. 8
figure 8

Docking of Rubc3 with HSA (a) full view (b) closer view of the Ru(II) complex docked in protein

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Chart 1
figure 9

Structure of the ruthenium(II)-bipyridyl-calixarene complexes used in this study

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Excited state lifetime measurements were made with laser flash photolysis technique using an Applied Photophysics SP-Quanta Ray GCR-2(10) Nd:YAG laser as the excitation source. The time dependence of the luminescence decay is observed using a Czerny-Turner monochromator with a stepper motor control and a Hamamatsu R-928 photomultiplier tube. The production of the excited state on exposure to light of wavelength 355 nm was measured by monitoring (pulsed xenon lamp of 250 W) absorbance change. Here the wave length is fixed at the λmax of emission in steady state method and the decay is measured. The natural log of the decay values is plotted against time which gives decay rate constant k. The inverse gives lifetime, τ of the complex.

Determination of Binding Constants

The binding constant of Rubc2 and Rubc3 with proteins is determined from the emission spectral data using Scatchard and Hills equations.

Scatchard Equation [56]

For emission measurements the concentration of Rubc2 and Rubc3 is fixed at 1 × 10−5 M and of protein is varied from 1 × 10−6 M to 9 × 10−6 M. We have used Scatchard equation for finding the cooperativity of the binding of Rubc2 and Rubc3 with proteins. The Scatchard equation shown in Eq. (1) is used to calculate the binding constant, Ka.

$$ \upsilon /{{\text{C}}_{\text{F}}} = {\text{n}}{{\text{K}}_{\text{a}}} - \upsilon {{\text{K}}_{\text{a}}} $$
(1)

In Eq. (1) υ—ratio of the concentration of bound ligand to total available binding sites, CF—number of binding sites per protein molecule, n—binding stoichiometry. The analysis of the data using the plot υ/CF vs. υ shows that the plot is non-linear indicating the cooperative binding [56].

Hills Equation [57]

Since the Scatchard plot is non-linear we have calculated the binding constant using Hills equation, Eq. (2).

$$ \log \left[ {\theta /\left( {1 - \theta } \right)} \right] = {\text{n}}\,\log \left[ {{\text{protein}}} \right] - \log \,{{{\text{K}}}_{{\text{d}}}} $$
(2)

Here θ—number of binding sites (CF in Scatchard equation), Kd—dissociation constant(the inverse of Ka), n—Hills coefficient of cooperativity which gives the nature of cooperativity. The value of n shows the nature of binding. n > 1 positive cooperative binding, n < 1 negative cooperative binding and n = 0 non-cooperative binding. From the plot of log[θ/(1–θ)] vs log [protein] the value of n and Kd are calculated.

Estimation of Quenching Constant, kq [58]

We have calculated the quenching constant (kq) by using the Stern-Volmer equation Eq. (3),

$$ {{\text{F}}_0}/{\text{F}} = 1 + {{\text{k}}_{\text{q}}}\tau \left[ {\text{Q}} \right] $$
(3)

Here F0 and F are the luminescence intensity of the luminophores in the absence and presence of quencher τ is the luminescence lifetime in the absence of quencher and [Q] is the concentration of quencher.

Measurement of Helicity of Proteins

For CD measurements the concentration of protein is fixed at 1 × 10-7 M and of Ru(II)-calixarene complex is varied from 1 × 10−5 to 3 × 10−5 M. The measurements were taken in Jasco polarimeter J180 at accumulation time 2 cycles, 50 mm path length, band width 1 nm, response 1 s. The helicity of the proteins is calculated by Eqs. (4) and (5) [59].

$$ {\text{Mean}}\,{\text{residual}}\,{\text{elipticity}}\,\left( {\text{MRE}} \right){\text{ = Observed}}\,{\text{CD/Cpnl}} \times {10} $$
(4)

where, Cp—molar concentration of protein, n—number of amino acid residues, l—path length.

$$ \alpha \left( {{\text{helix}}\,\% } \right) = \left( { - {\text{MRE}} - 4000} \right)/\left( {33000 - 4000} \right) \times 100 $$
(5)

In Eq. (5) 4000 is the MRE of the β–form and random coil conformation cross at the observed wavelength, and 33,000 is the MRE value of a pure α–helical at the observed wavelength.

Details of Docking Studies

The pdb (protein data bank) files of protein molecules have been downloaded from protein data bank (http://www.rcsb.org/pdb/home/home.do). Since there is no crystal structure available for BSA, the data from the crystal structure of HSA have been used for the study. Its file code is 1HA2 [60]. The pdb file used for the structure of ovalbumin is 1ugh [61]. The final model chosen is minimized using DISCOVER module of Insight II with 1,000 rounds of steepest descent and 1,000 rounds of conjugate minimization [62]. The cavity of the protein model has been picked by using Computed Atlas of Surface Topography of Proteins (CASTp) [63]. The docking towards the luminophores of proteins such as tryptophan and tyrosine has been done using GOLD [64]. For viewing the docked solutions and to generate pictures UCSF Chimera candidate version 1.5.3 is used [65].

Results and Discussion

Steady State Absorption Spectral Study

The ruthenium(II)-bipyridine-calixarene complexes, Rubc2 and Rubc3 (Chart 1 gives structures) have been synthesized by literature methods [55]. All the spectral studies are carried out in phosphate buffer at pH 7.4. The concentration of Rubc2 and Rubc3 is fixed at 1 × 10−5 M and the concentration of BSA and ovalbumin is varied from 1 × 10−6 M to 9 × 10−6 M. The UV-visible spectra of Rubc2 and Rubc3 are recorded in the absence and in the presence of different concentration of proteins. There is a considerable change in the intensity of absorption corresponding to the 1MLCT of Rubc2 and Rubc3. Due to the high molar extinction coefficient and experimental difficulties, higher concentration of protein has not been used i.e., the ratio of 1:10 is not maintained. Thus we have used lower concentration of proteins for the spectral study. With these concentrations we cannot apply Benesi-Hildebrand method for calculating binding constant. Therefore the binding constants are not calculated using absorption spectral technique. Absorption spectral technique is only used to show the enhancement of 1MLCT absorption on the addition of proteins. The spectral changes are shown in supplementary material (Figs. S3 and S4). The substantial increase in the intensity of MLCT absorption on increasing the protein concentration shows strong binding of Rubc2 and Rubc3 with proteins. It is important to point out that the proteins have no absorption at this wavelength (450 nm).

Steady State Emission Spectral Study

The emission spectra of Rubc2 and Rubc3 show peaks at 653 nm and 658 nm respectively in the absence of proteins, upon using an excitation wavelength corresponding to their 1MLCT absorption. When the protein is added to Rubc2 and Rubc3 we observe an increase in the emission intensity corresponding to emission of both complexes (Fig. 1). The enhancement in emission intensity of Rubc2 and Rubc3 in the presence of proteins indicates that there is binding of ruthenium(II) complex with proteins. The concentration of Rubc2 and Rubc3 is fixed at 1 × 10−5 M and the concentration of BSA and ovalbumin varied from 1 × 10−6 M to 9 × 10−6 M and the emission spectra of Rubc2 and Rubc3 are recorded in the presence of different concentrations of proteins. The emission spectral changes are shown in Fig. 1 and Fig. S5 (in supplementary material). Enhancement in the emission intensity at 650 nm is observed with an increase in [protein]. We have tried to use the Schatchard equation (Eq. (1)) to calculate the binding constant values of Rubc2 and Rubc3 with proteins. The Schatchard plots are shown in supplementary material (Figs. S6 and S7). The Scatchard plots in Figs. S6 and S7 show an upward curvature which indicates a negative-cooperative binding of the complexes with protein. So the binding constants cannot be calculated by these plots. Therefore the Hills equation (Eq. (2)) is used for the calculation of binding constant. The Hills plots are shown in in supplementary material (Figs. S8 and S9). The binding constant (K) and cooperativity (n) values calculated from the Hills plot are collected in Table 1. From these binding constant values (105–106 M−1) it is clear that the binding of Rubc2 and Rubc3 with proteins is very strong and comparable with the other Ru(II)-bipyridine complexes (105 M−1) with cytochrome C [41] and Ru(II)-dendrimers (106 M−1) with other proteins (chymotrypsin and lectin) [42, 43]. Ru(II)-calixarene complexes exhibit more efficient binding towards BSA compared to ovalbumin. Among the two Ru(II) complexes chosen Rubc3 exhibits more efficient binding than Rubc2.

Table 1 Binding constants (Ka) and quenching constants (kq) and cooperativity (n) of the ruthenium(II)-bipyridine-calixarene complexes with proteins
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The proteins, BSA and ovalbumin, contain fluorescent amino acids, such as phenyl alanine, tyrosine and tryptophan in their structure. The predominant fluorophore is tryptophan and its emission maximum, λmax, is around 348 nm. Hence, the emission intensity of protein is also monitored to understand more on the tryptophan environment. The concentration of the proteins is fixed at 1 × 10−5 M and the concentration of Rubc2 and Rubc3 is varied from 1 × 10−5–9 × 10−5 M and the excitation wavelength is 280 nm. The results of the titration of Rubc2 and Rubc3 with BSA and ovalbumin are shown in Fig. 2 and Fig. S10 (in supplementary material) respectively. Since the molar extinction coefficient (∈) of the BSA and ovalbumin (43,000 mol−1 cm−1and 58,800 mol−1 cm−1 respectively) is very high compared to that of Rubc2 and Rubc3 (2,351 mol−1 cm−1and 1,721 mol−1 cm−1 respectively) at 280 nm the quantum of light absorbed by Ru(II) complexes at this wavelength is negligible. Therefore the observed luminescence quenching process is predominantly due to the energy transfer from the proteins to the Ru(II)-complexes, not due to the absorption of a part of light quanta by the Ru(II)-complexes at 280 nm. From this titration, quenching constants are calculated by using Stern-Volmer equation (Eq. (3)). The Stern-Volmer plots are shown in supplementary material (Figs. S11 and S12) and the quenching constant values are collected in Table 1. The average excited state lifetime of BSA and ovalbumin, 6.7 ns and 7.7 ns respectively have been used for the determination of kq values [66, 67].

Since the quenching constants are in the range of 1012–1013, it is clear that the major contribution for the quenching process is from static quenching. There is a strong ground state complex formation, confirmed by the UV-visible absorption spectral titrations between the receptor and the protein and the binding constant values are in the range of 105–107 M−1. The quenching constant value is comparatively higher for Rubc3 than Rubc2 for both proteins and is higher for BSA than ovalbumin for both complexes. It is interesting to note that similar results are observed with binding constants also. These large kq values calculated in the present study show that the quenching process through energy transfer occurs on very fast time scale. Such high quenching constants for energy transfer have already been reported [68, 69] and can emphasize the occurrence of FRET process.

Excited State Lifetime Studies

The excited state lifetime of Rubc2 and Rubc3 is measured using emission mode of laser flash photolysis technique using 1× 10−5 M Rubc2 and Rubc3. The details of measurement are given in the experimental section. The excited state lifetimes of Rubc2 and Rubc3, in the absence of proteins, are 428 ns and 437 ns respectively obtained by monitoring the emission at 650 nm and 653 nm. The lifetime decays are shown in Fig. 3 and Fig. S13 (in supplementary material). The excited state lifetime values are collected in Table 2.

Table 2 Lifetime of the ruthenium(II)bipyridine-calixarene complexes in the presence of proteins at pH 7.4
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The excited state lifetime of Rubc2 in the absence of proteins is 428 ns. The addition of 1 × 10−6 M of BSA and ovalbumin separately leads to a slight increase in the lifetime of Rubc2 to 432 and 435 ns respectively. On the other hand further increase in the protein concentration increases the lifetime of Rubc2 substantially to 460 and 492 ns respectively. Similar results are observed on the addition of BSA and ovalbumin with the other complex, Rubc3 as well. These results show that binding of Ru(II)-calixarene complexes with proteins results in the stabilization of these complexes in the excited state. It is important to recall that similar results are observed when luminescent ruthenium(II)-polypyridine complexes are added to DNA and surfactants [60-72].

Circular Dichroism Spectral Studies

To know about the structural changes of the proteins due to the binding of Rubc2 and Rubc3, the CD spectral studies have been carried out. The details of measurement are given under experimental section and the CD spectra are shown in Fig. 4 and Fig. S14 (in supplementary material). The CD spectral parameters are collected in Table 3.

Table 3 CD parameters of BSA (3 × 10−7 M) and Ovalbumin (9.8 × 10−7 M) with interaction of complexes
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The CD spectra of BSA in the absence and presence of Rubc2 and Rubc3 are shown in Fig. 4. When Rubc3 is added to BSA, considerable change is noted in the near UV–CD region 200–260 nm. This indicates that there is a significant alteration in the tertiary structure of proteins. From the changes in the CD spectrum, the α-helicity percentage can be calculated by Eq. 5. The native BSA has 66.8 % α-helicity. On the other hand addition of Rubc3 decreases the α-helicity substantially to 26.2 % and 14.9 % (Rubc3 1 × 10−7 M and 3 × 10−7 M, respectively). Addition of Rubc2 decreases the α-helicity to 53.8 % and 51.0 % (Rubc2 1 × 10−7 M and 3 × 10−7 M, respectively). In the case of native ovalbumin, it has 31.2 % α-helicity. The addition of Rubc2 decreases the α-helicity of ovalbumin to 29.0 % and 24.6 % (Rubc2 1 × 10−7 M, 3 × 10−7 M respectively) and addition of Rubc3 decreases the α-helicity to 24.0 %, and 21.1 % (Rubc3 1 × 10−7 M, 3 × 10−7 M respectively). The decrease in the α-helicity of both BSA and ovalbumin is to a greater extent in the presence of Rubc3 compared to Rubc2. Compared to ovalbumin the change in α-helicity is higher for BSA toward both Rubc2 and Rubc3. These observations also support that the binding efficiency is higher for Rubc3 than Rubc2.

FRET from Proteins to Ru(II) Complexes

The FRET (fluorescence resonance energy transfer) finds extensive applications in life sciences to study the interaction of macromolecules especially with respective to their intra- and intermolecular interactions [73]. In clinical applications, FRET-based immunoassays are comparable with the radio immunoassays [74, 75]. As far as the present system is concerned the advantage is that the receptors, calixarenes, can bind with any molecule from small molecule like NO to very large molecule like proteins and even on the surface of a cell [76-78]. The importance of FRET in biochemistry is realized from its application that the efficiency of energy transfer can be used to evaluate the distance, r, between the probe and fluorophore residue in the protein [58]. According to the Forster’s non-radiative energy transfer theory, [79] the rate of energy transfer depends on (i) the relative orientation of the donor (proteins) and the acceptor (Ru(II) complexes) dipoles, (ii) the extent of overlap of emission spectrum of the donor with absorption spectrum of the acceptor and (iii) the distance between the donor and the acceptor. The efficiency of energy transfer, E, is calculated using Eq. (6).

$$ {\text{E = 1}} - {\text{F}}/{{\text{F}}_{{0}}} = {\text{R}}_{{0}}^{{6}}/\left( {{\text{R}}_{{0}}^{{6}} + {{\text{r}}^{{6}}}} \right) $$
(6)

Here F and F0 are the fluorescence intensities of proteins in the presence and absence of Rubc2 and Rubc3, r is the distance between the acceptor and the donor and R0 is the critical distance when the transfer efficiency is 50 % and the value of R0 is calculated by using Eqs. (7) and (8).

$$ {\text{R}}_{{0}}^{{6}} = {8}{.8} \times {1}{{0}^{{ - {25}}}}{\kappa^{{2}}}\phi {\text{J}}\,{{\text{N}}^{{ - {4}}}} $$
(7)
$$ {\text{J}} = \Sigma {\text{F}}\left( \lambda \right)\varepsilon \left( \lambda \right){{\lambda }^{4}}\Delta \lambda /\Sigma \,{\text{F}}\left( \lambda \right)\Delta \lambda $$
(8)

The term κ2 is the relative orientation of space of the transition dipole of the donor and acceptor, ϕ is the quantum yield of the donor in the absence of acceptor, F(λ) is the fluorescence intensity of the fluorescent donor at the wavelength λ and is dimensionless, ε(λ) the molar absorption coefficient of the acceptor at λ and its unit is cm−1 M−1.

Some interesting results are observed from the binding studies of Rubc2 and Rubc3 with proteins using emission spectral technique. When the emission spectra of Rubc2 and Rubc3 at the fixed concentration are recorded upon the addition of varying protein concentration, an enhancement of emission intensity is observed (Fig. 1 and Fig. S3 in supplementary material). On the other hand when we monitor the emission intensity at the fixed concentration of proteins upon varying the concentration of Rubc2 and Rubc3, quenching of emission intensity is observed (Fig. 2 and Fig. S10 in supplementary material). For the efficient energy transfer from the energy donor (proteins) to the acceptor (Rubc2 and Rubc3) the emission spectrum of the donor and absorption spectra of the acceptor must overlap. Figure 5 and Fig. S15 in supplementary material depict the overlap of the absorption spectrum of Rubc2 and Rubc3, respectively with the emission spectrum of BSA. These figures show that the overlap of absorption spectrum of Rubc2 and Rubc3 with the emission spectrum of BSA is efficient. Similar results are observed for complexes for the interaction of Rubc2 and Rubc3 with ovalbumin as well. To realize the presence of FRET we have taken a mixture of the protein and Rubc2/Rubc3 at the ratio 1:1, excited the solution at the excitation wavelength of protein (285 nm) and recorded the emission spectrum. The results are shown in Fig. 6 and Fig. S16 in supplementary material.

For this study the concentration of BSA and ovalbumin is fixed at 2 × 10−6 M and emission spectra are recorded from 300 to 800 nm using the excitation wavelength at 285 nm. The emission maxima, λmax, for BSA and ovalbumin are 339 nm and 331 nm respectively. The Rubc2 and Rubc3 are also excited separately at 285 nm and emission spectra recorded. The emission maxima, λmax, for Rubc2 and Rubc3 at the excitation wavelength 285 nm are 673 and 670 nm respectively. The 1:1 solution of BSA and Rubc2 is excited at 285 nm. The emission intensity at 339 nm of BSA is decreased but the λmax of Rubc2 is blue shifted from 673 to 641 nm and emission intensity at this wavelength is enhanced. Similar results are obtained, when 1:1 ratio of BSA and Rubc3 is used. The values corresponding to emission spectral changes are collected in Table 4.

Table 4 Wavelength of emission maximum and intensity of 1:1 solution of proteins and Rubc2/Rubc3
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The enhancement of emission intensity is substantial for Rubc3 with blue shift in λmax. Therefore the efficiency of energy transfer is more for Rubc3 than Rubc2. Similarly the enhancement of emission intensity and blue shift in λmax is more substantial for BSA than ovalbumin which shows that the energy transfer is high for BSA. The values of energy transfer efficiency, r and R0 values are collected in Table 5. These calculations show that the energy transfer efficiency of Rubc3 is higher than Rubc2 and of BSA is higher than ovalbumin.

Table 5 FRET parameters of Rubc2 and Rubc3 with proteins
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Docking Studies on the Binding of Rubc2 and Rubc3 with Proteins

From the above experimental results, it is clear that the complexes Rubc2 and Rubc3 bind efficiently with the two proteins. To gain more knowledge on the binding efficiency and the mode of binding we have carried out docking studies using GOLD software [70]. The details of the software and protein data bank (pdb) files are given in the experimental section. BSA has no crystal structure yet. Since HSA has 80 % similarity with the BSA we have downloaded HSA pdb file as model for BSA. The cavity of the proteins has been picked using an online tool, CASTp [69]. The cavities we have selected for HSA (model to BSA) and ovalbumin are shown in Fig. 7.

The hydrophobic surface area shown as red color in the cavity of the proteins is available for docking. Other cavities are also available but we have selected the biggest cavity for facile docking. The cavity volume for HSA (model to BSA) is 3587.5 Å3 and of ovalbumin is 397.3 Å3. Compared to the larger cavity available with HSA (model to BSA) for docking, ovalbumin has very small cavity which is also projecting outside of the protein. Since our study involves fluorescence as the technique and we are monitoring the fluorescence change experimentally, we identified the fluorophores (tryptophan, tyrosine and phenyl alanine) inside the cavity. Since the protein fluorescence is the collective fluorescence of tryptophan, tyrosine and phenyl alanine, we have chosen the TYR138 in HSA (model to BSA) and TYR125 in ovalbumin which are available inside the cavity as the site of binding. The docked structures are shown in Fig. 8 and Fig. S17 in supplementary material.

Figure 8 and Fig. S17 in supplementary material illustrate the role of calixarene moiety in the binding with proteins. The calixarene moiety finely fits inside the hydrophobic cavity. The ruthenium(II)-bipyridine moiety is projecting outside of the proteins. Figure 8 shows that the calixarene moiety is totally buried in the hydrophobic cavity of the HSA (model to BSA). Therefore it is clear that the presence of calixarene moiety facilitates the efficiency of binding with proteins. The amino acid residues in proteins interact hydrophobically with tert-butyl group of calixarene moiety of two complexes as shown in supplementary material (Figs. S18 and S19) for HSA (model to BSA) and ovalbumin respectively.

The distance between the interacting tert-butyl group of calixarene and the amino acid side chains of proteins is given in Table S1 in supplementary material. Table S1 shows that the distance between the tert-butyl group of calixarene in both Rubc2 and Rubc3 and side chains of proteins ranges from 2.6 Å to 5.1 Å. The side chains of the amino acid groups in the proteins interacting with tert-butyl moiety are also hydrophobic in nature. These results exhibit that the hydrophobic nature of the tert-butylcalix[4]arene moiety plays important role in the binding of Rubc2 and Rubc3 with proteins.

Conclusion

This study provides an excellent luminophore-receptor system for efficient binding with proteins. The importance of calixarenes as receptors for the binding of proteins and cell surface is already realized. In the present study the attachment of tert-butylcalix[4]arene group enhances the hydrophobicity of the Ru(II) complex, which increases the binding efficiency. The steady state emission and docking studies show that the efficient binding results in the efficient FRET a useful tool in the biological systems. The CD spectral studies establish that the binding is more efficient for BSA than ovalbumin and for Rubc3 than Rubc2. The docking studies reveal that the tert-butyl-calix[4]arene group serves as anchoring group for the binding. As already indicated this system promises wide applications in biological and clinical systems because of extensive binding of calixarene with a large number of molecules and the possibility of efficient FRET process.