A combination of spectroscopic and molecular docking techniques to study interaction of bis(indolyl)methane with bovine milk α-casein

ABSTRACT

Spectroscopic and molecular docking techniques were used to study the interaction of bis(indolyl)methane (BIM), poorly soluble in aqueous solutions, with bovine α-casein, as an efficient drug carrier. The results of Fourier transform infrared and circular dichroism spectroscopy demonstrated that α-casein interacts with BIM molecule mainly via hydrophobic and hydrophilic interactions, resulting changes in the secondary structure of α-casein. The fluorescence quenching measurements at 293–310 K revealed that the quenching mechanism was static, and the binding site of BIM to α-casein was singular. Thermodynamic analysis, along with the negative value of and positive value of , indicates that the hydrophobic interaction, hydrogen bonds, and van der Waals forces played major roles in the complex formation process. The distance between donor and acceptor, obtained according to fluorescence resonance energy transfer and docking studies, indicated that BIM induced a change from a hydrophobic environment around the Trp-193 of α-casein to an aqueous or hydrophilic environment.

KEYWORDS:

• α-Casein
• Bioefficacy
• Bis(indolyl)methane
• Docking
• Fluorescence quenching

Introduction

Different routes of drug administration possess various levels of advantages and drawbacks. For example, oral and nasal deliveries result in high levels of drug in the blood with comparably poor release profiles; aerosol preparations are complex and problematic with regard to the load of drug. The lack of targeting, with possible damage to healthy cells, is one of the drawbacks of transdermal delivery. In order to overcome the delivery challenges, “targeted drug delivery” has been developed in the last few decades^[1].

Indole derivatives have been focused for their prominent role in various commercial applications for centuries. Bis(indolyl)methane (BIM), one of the key derivatives of indole, exhibits a wide range of important biological activities^[2]. In animal models, BIM also has shown efficacy in treatments for a range of carcinomas, including prostate,^[3] breast,^[4] pancreatic,^[5] bladder,^[6] renal cell,^[7] lung,^[8] and colon cancers^[9,10]. BIM exhibits antimicrobial, antifungal, and antibacterial activities^[11,12]. The oxidized form of BIM is utilized as a dye^[13] and as a colorimetric sensor^[14]. The major problem with BIM is its extremely low solubility in aqueous solution and poor biocompatibility.

Milk proteins may form structures useful for delivery of some bioactive compounds. These proteins have surface activity, resulting in their easy adsorption at interfaces. Therefore, they have been considered as a medium for delivering certain bioactive molecules that normally have low bioaccessibility^[15,16]. Milk proteins are classified into two well-defined groups: casein and whey or serum protein^[17,18]. In contrast to the whey proteins, caseins exist in particles containing many thousands of individual protein molecules, known as the casein micelles^[15]. Casein micelles contain four main phosphoproteins: (38%), (10%), (40%), and (12%) casein, with molecular weights between 19 and 25 kDa. They contain a large number of proline residues, making them rheomorphic structures. The content of α-helix or β-sheet structures is low in caseins due to high levels of proline^[17,19]. All the caseins also possess a high content (35–45%) of polar amino acids (Val, Leu, Ile, Phe, Tyr, and Pro) and therefore, low solubility in aqueous systems may be expected. However, the high content of phosphate groups, low level of sulfur-containing amino acids, and high carbohydrate content influence the polar amino acids. One of the structural differences between and is that while contains no cysteine, contains two cysteines, which normally exist as intermolecular disulfide bonds; that is, under non-reducing conditions, –casein exists as a disulfide-linked dimer. The number of phosphate groups in the –casein is 8, occasionally 9, and in–casein it may be 10, 11, 12, or 13^[17].

In recent years, researches have shown that casein can be used to overcome the limitations related to the low solubility of polyphenol,^[20,21] resveratrol,^[22,23] curcumin,^[24,25] vitamin D₂ and A,^[26,27] and lipids^[28] in water. In this work, the interaction between BIM with bovine milk α-casein was studied using Fourier transform infrared (FTIR), circular dichroism (CD), and fluorescence spectroscopy. The relative interaction strength and binding mode were measured by fluorescence spectroscopy. Some conformational changes of protein can be revealed by synchronous spectra. Besides, the interaction of BIM and α-casein was investigated using molecular docking technique.

Materials and methods

Bovine α-casein (70%, Sigma-Aldrich, Germany) was dissolved in pH 7.0 phosphate buffer containing 80 mM NaCl, 5.55 mM Na₂HPO₄, and 3.15 mM NaH₂PO₄ with an ionic strength of 0.1 M. The protein concentration after dialysis was determined from the absorbance at 280 nm using a UV–visible spectrophotometer and adjusted to the required concentration according to an extinction coefficient of 1.09 ml mg^–1 cm^–1[29].

BIM (Scheme 1) was synthesized and purified by the method described previously^[30]. As BIM was insoluble in water, a stock solution (1 mM) of BIM in acetonitrile was first prepared and then diluted with buffer solution so that the acetonitrile content in the final solution did not exceed 6%. All solutions were prepared using double distilled water and used freshly following their preparation.

Scheme 1. Chemical structure of bis(indolyl)methane (BIM).

FTIR and CD spectroscopy measurements

In order to conduct FTIR measurements, a solution of BIM was added stepwise to the protein solution (0.25 mM) with constant stirring to ensure homogeneity of solution and to reach the target BIM concentrations of 0.125, 0.25, and 0.5 mM. The solutions were then freeze-dried in a Christ Alpha 1-2 LD freeze dryer at –40°C and 0.15 atm pressure for 4 h, the vacuum was broken at the end of the freeze-drying cycle. The pellets were prepared by mixing the freeze-dried sample with  KBr and compacted under vacuum for 3 min. FTIR measurements were carried out at room temperature by an ALPHA FTIR spectrometer (Bruker optics) using KBr pellet in the range of 4000–400 cm⁻¹. CD spectra of α-casein and its BIM complex were obtained using JASCO spectropolarimeter model j-810. For measurements in the far-UV region (195–245 nm), a quartz cell with a path length of 0.1 cm was used in nitrogen atmosphere. Protein concentration was kept constant (8.3 μM) and BIM concentration varied (8.30, 16.6, and 33.20 μM). The protein secondary structure was calculated using CDNN software, which indicates the different assignments of secondary structures.

Fluorescence spectroscopy

Emission spectra were recorded by a Cary eclipse spectrofluorimeter equipped with a thermostat, which controls the temperature of the cell compartment with a precision of ±0.1°C. For preparation of BIM–α-casein complex, a 1 mM stock solution of BIM in acetonitrile was prepared. α-Casein was dissolved in sodium phosphate buffered saline (PBS), pH 7.0. The entrapment of each BIM in casein nanoparticle at different BIM to α-casein molar ratios was performed by adding different volumes of BIM solution in acetonitrile to the α-casein solution in PBS with continuous stirring. The fluorescence excitation for α-casein was performed at ^[31].

Determination of synchronous fluorescence spectra

In order to verify the binding sites of BIM to α-casein molecules, the synchronous fluorescence spectra of α-casein solutions along with the increase in BIM concentrations were investigated in wavelength ranges from 245 to 350 nm for and .

Molecular docking

The α-casein structure was obtained according to the SWISS-MODEL; protein model was prepared based on the target-template alignment using Promod-II. The coordinates, which are conserved between the target and the template, are then copied from the template to the model. Insertions and deletions are remodeled using a fragment library. Side chains are rebuilt and the geometry of the resulting model is regularized using a force field^[32]. Docking experiments were carried out to visualize the binding site of BIM to α-casein. All the docking calculations were performed using Autodock 4.2 Tools^[33,34]. The macromolecule was kept rigid, while all the torsional bonds of ligands were set free to rotate. Geometry optimization was carried out with grid resolution of 0.375 Å and grids spacing of 80 Å × 80 Å × 80 Å. For the BIM ligand, 150 separate docking calculations were performed using the Lamarckian genetic algorithm (LGA) method. The structure with lowest free energy of binding in a highly populated cluster was chosen as the optimal docking pose. The ligand structure optimizing calculation was carried out at the 6–31 G** level by employing the Becke three-parameters Lee-Yang-Parr (B3LYP) hybrid density functional theory using the quantum chemistry software Gaussian 03.

Results and discussion

Structural changes revealed by FTIR and CD analysis

The BIM interaction with α-casein was analyzed by FTIR spectroscopy. The wave number and intensity of α-casein were changed (Fig. 1a; α-casein (1)) for C=O stretching (amide I band) at 1642 cm^–1 and C–N stretching participant with N–H bending modes (amide II band) at 1548 cm^–1, due to hydrophilic contacts between BIM and α-casein^[21]. The results of BIM interaction with α-casein, at low BIM concentration, 0.125 mM (Fig. 1a; α-casein–0.125 mM BIM (2)), showed a partial increase in intensity. A spectral shift was witnessed for the protein amide I band at 1642 cm^–1 and amide II band at 1550 cm^–1. As BIM concentration increased to 0.25 mM (Fig. 1a; α-casein–0.25 mM BIM (3)), a decrease in intensity was observed for amide I and II bands at 1642 and 1550 cm^–1. On the other hand, when the BIM concentration reached 0.5 mM (Fig. 1a; α-casein–0.5 mM BIM (4)) two variations were observed for amide I and II bands at 1632 and 1533 cm^–1. When the BIM concentration was further increased, the intensity was decreased, suggesting an altering of the secondary structure of the protein with increasing BIM concentrations.

Figure 1. (a) FTIR spectra of (1) free α-casein 0.25 mM, (2) α-casein–0.125 mM BIM, (3) α-casein–0.25 mM BIM, and (4) α-casein–0.5 mM BIM; and (b) far-UV CD spectra of (1) free α-casein 8.3 µM, (2) α-casein–8.3 µM BIM, (3) α-casein–16.6 µM BIM, and (4) α-casein–33.2 µM BIM.

The hydrophobic contacts in the BIM–α-casein complex were investigated in the 2800–3000 cm^–1 range of FTIR spectra via CH₂ anti-symmetric and symmetric stretching vibrations. Upon the increase of 0.125 mM BIM to α-casein, CH₂ vibrations at 2876, 2929, and 2996 cm^–1 were shifted to 2880, 2933, and 2966 cm^–1, respectively. Protein anti-symmetric and symmetric CH₂ stretching vibration shifts suggest the presence of hydrophobic interactions between BIM and α-casein^[35]. Based on the results obtained presently, it was demonstrated that α-casein can be used to overcome the limitations related to the low solubility and bioefficacy of BIM.

CD spectroscopy was used to analyze conformation changes in α-casein during interactions with BIM. CD spectra were measured in the range of 195–245 nm; further investigation of α-casein conformational changes in the presence of BIM is shown in Fig. 1b. The results indicate that the presence of BIM in the range of 8.3–33.2 μM induces changes in the secondary structure of α-casein. The featured spectrum with increase of BIM, twin negative peaks at ~202 nm, and a shoulder at ~222 nm suggested that the decrease in unordered structures of α-casein is accompanied by an increase in the α-helix content (Fig. 1b and Table 1)^[36]. The CD data show that α-casein can be used as carrier, because the addition of BIM introduces more stability to the secondary structure of the protein.

Table 1. Secondary structure of free α-casein (CD spectra) and their BIM complexes at pH 7 calculated by CDNN software.

Conformational components	Free α-casein (8.3 μM)	α-casein–8.3 μM BIM	α-casein–16.6 μM BIM	α-casein–33.2 μM BIM
% α-helix	13.9	15.7	15.6	15.6
% β-sheet	15.8	15.3	15.2	15.1
% Unordered structures	70.3	69.0	69.2	69.3

Fluorescence measurements

In order to describe the binding interactions of α-casein with BIM, the intrinsic fluorescence of the protein was measured in the presence of various amounts of BIM. The intrinsic fluorescence of α-casein may change and therefore the intensity of intrinsic fluorescence of protein can be decreased by addition of the quencher (BIM). The fluorescence emission spectra of α-casein upon the addition of BIM are shown in Fig. 3a. Since α-casein has two tryptophan residues located in a primarily hydrophobic domain of the α-casein micelle, they can provide valuable information about the interaction and formation of the BIM–α-casein complex. It can be seen that BIM has quenched the fluorescence of α-casein without any shift in the wavelength of emission peak maximum. This may be due to an insignificant dielectric change in the local environment of tryptophan residue^[26]. Both static and dynamic processes can be described by the well-known Stern–Volmer equation as follows^[37,38]:

(1)

Figure 2. Fluorescence quenching of (a) in PBS of pH 7, at 25°C by BIM concentration: 0, 0.33, 2, 4, 6, 8, 10, 15, 20, 30, and 40 μM, inset: the variation of F_o/F versus [BIM]; and (b) Stern–Volmer plots of the interaction between BIM and α-casein at 20°C (▲), 25°C (∆), 30°C (●), and 37°C (○).

Figure 3. (a) Modified Stern–Volmer plots, (b) double logarithm plots of the interaction between BIM and α-casein at 20°C (▲), 25°C (∆), 30°C (●), and 37°C (○).

where and are fluorescence intensities in the presence and absence of quencher, is the concentration of quencher, is Stern–Volmer constant, is the bimolecular quenching rate constant, and is the lifetime of the fluorophore in the absence of quencher; 4.53 ns for α-casein^[35]. The plot of versus for α-casein is presented in the inset of Fig. 2a. The plot in lower concentrations of BIM (up to 20 μM) is linear; for its higher concentrations (above 20 μM) it becomes curved. This result suggests that the type of quenching of α-casein in low concentrations of BIM follows single mechanism kinetics, whereas for higher concentrations of BIM, there is combined quenching, that is, both static and dynamic quenching^[39]. In order to differentiate static from dynamic quenching, the temperature effect on the quenching constant was examined. The plots of versus at four temperatures are shown in Fig. 3b. values were obtained from the slopes of the plots. The results indicate the occurrence of a static quenching mechanism in formation of the BIM–α-casein complex at lower concentrations of . This conclusion comes from the fact that the quenching constant decreases as the temperature increases from 293 to 310 K (up to 20 μM) (Fig. 2b and Table 2)^[40]. The between 293 and 310 K is shown in Table 2.

Table 2. Quenching constants ( and ), number of substantive binding sites , and binding constant () for interaction of BIM with α-casein micelle obtained from the fluorescence measurements in phosphate buffer of pH 7 at different temperatures.

T (K)	KSV × 10^–Citation5 (L mol^–Citation1)	R^2	Kq × 10^–Citation13 (L mol^–Citation1)	n	Ka × 10^–Citation5 (L mol^–1)	R^2
	1.04 ± 0.02	0.9970	3.30 ± 0.05	0.96 ± 0.03	1.06 ± 0.04	0.9915
	1.01 ± 0.02	0.9970	2.23± 0.05	1.14 ± 0.03	0.93 ± 0.06	0.9955
	0.97 ± 0.02	0.9964	2.13 ± 0.05	1.07 ± 0.03	0.83 ± 0.08	0.9920
	0.88 ± 0.02	0.9980	1.95± 0.03	1.10 ± 0.03	0.73 ± 0.08	0.9883

The modified Stern–Volmer plot is useful when studying a protein that has tryptophans in different environments (buried environment or surface environment) or fluorescence is heterogeneous and residues do not have an identical accessibility to the quencher. Therefore, the quenching data were analyzed according to the modified Stern–Volmer Eq. (2):

(2)

where and are the same parameters as in the Stern–Volmer equation and is the effective quenching constant for the accessible fluorophores, which is analogous to associative binding constants for the quencher–acceptor system, and is the fraction of accessible fluorescence. The plots of versus , represent the slope and the intercept of the straight line^[41,42]. Thus, the ratio of the ordinate and the slopes gives. The values of for α-casein in the presence of the BIM at 293, 298, 303, and 310 K were found to be 1.00, 1.04, 1.06, and 1.08, respectively. The plots of versus are shown in Fig. 3a. The obtained values of (Table 2) show that increasing the temperature from 293 to 310 K decreases the binding constant. For static quenching, the number of sites can be obtained from following equation, if there are independent binding sites to a set of equivalent sites on macromolecules^[43,44]:

(3)

The plots of versus are presented in Fig. 3b, where is the slope of straight line. The number of BIM molecules bond per α-casein () from 293 to 310 K are shown in Table 2 and Fig. 3b. This figure illustrates that BIM binds to α-casein with 1:1 stoichiometry. The binding studies were carried out in the temperature range of 293–310 K. Thermodynamic parameters of the interaction between BIM with α-casein were calculated and analyzed. Equilibrium features of the BIM–α-casein complex can be conveniently characterized by three thermodynamic parameters, that is, standard molar Gibbs free energy changes the standard molar enthalpy changes () and the standard molar entropy changes (. can be calculated from the standard equilibrium constant and using the familiar relationship (4):

(4)

where is analogous to the associative binding constants at the corresponding temperature , and and refer to the gas constant and the absolute temperature, respectively. The van’t Hoff equation gives a linear plot of versus , if the heat capacity change for the reaction is essentially zero^[44]:

(5)

From a plot of versus (Fig. 4a), can be calculated from the slope of the straight line and was computed by Eq. (6):

(6)

Figure 4. (a) van’t Hoff plot for the interaction of BIM and α-casein. (b) Spectral overlaps of UV–vis absorption (dashed lines) of BIM with the fluorescence emission (solid lines) of α-casein in PBS buffer of pH 7, and .

The thermodynamic parameters for the interaction between α-casein and BIM were calculated and are shown in Table 3. The negative signs of at four temperatures indicate the spontaneity of the binding processes between α-casein and BIM. It is also observed from Table 3 that binding of BIM to α-casein is an exothermic process; however, the value of entropy change is positive. It is obvious that the process is driven by enthalpy change and entropy change. Under these circumstances, electrostatic interaction cannot be allowed, because BIM is not able to be ionized in these conditions. These results suggest that the hydrophobic interaction, hydrogen bond, and van der Waals forces play the major role in the formation of BIM–α-casein complex^[45]. These results also suggest that BIM stabilizes the secondary structure of α-casein. This is consistent with CD measurements discussed earlier, when an increase in the BIM concentration enhanced the α-helix of α-casein and reduced the random coil of its structure.

Table 3. Thermodynamic parameters for interaction of BIM with α-casein micelle obtained from the fluorescence measurements in phosphate buffer of pH 7 at different temperatures.

T (K)	ΔG° (kJ mol^–1)	ΔH° (kJ mol^–1)	ΔS° (J mol^–1 K^–1)
	–28.19 ± 0.02	–16.59 ± 0.10	39.59 ± 0.36
	–28.33 ± 0.02		39.39 ± 0.36
	–28.54 ± 0.03		39.43 ± 0.35
	–28.85 ± 0.03		39.56 ± 0.34

Energy transfer

In order to measure the distance between a ligand and macromolecules, fluorescence resonance energy transfer (FRET) is a reliable method. The Fӧrster distance is specified by the spectral overlap of the donor (α-casein) emission with the acceptor (BIM) absorption. Upon association, FRET will exhibit a decrease in the intensity of donor quenching, which can be used to calculate the distance between the donor and the acceptor^[37]. The energy transfer is then used for evaluation of the binding distance (r) between the BIM and two tryptophan residues in the α-casein. The efficiency of energy transfer (E) is calculated by Eq. (7):

(7)

where and are the same parameters in the Stern–Volmer equation, r is the distance between the donor (α-casein) and acceptor (BIM), and is the Fӧrster critical distance (nm) when the efficiency is 50%, which can be calculated by the following equation^[37]:

(8)

In the case of milk proteins, is a factor to describe the relative orientation of the transition dipoles of the donor and acceptor, is average refractive index of the medium (average) in the region of wavelength where spectral overlap is significant, is the fluorescence quantum yield of the donor, and is overlap integral, expresses the extent of overlap between the normalized fluorescence emission spectrum of donor and the acceptor absorption spectrum. It is noteworthy to indicate that J is given by Eq. (9)^[45,46]:

(9)

where is the fluorescence intensity of the fluorescent donor at , and is the molar absorption coefficient of the acceptor at a specific wavelength. Figure 4b shows the spectral overlap of fluorescence emission of solution and absorption spectrum of the BIM solution.

Eqs. (7)–(9) made it possible to calculate that , , . The distance between the donor and acceptor is less than 7 nm, indicating that the mechanism of energy transfer for quenching is non-radiative^[33,34]. The short distance values suggested a strong interaction between BIM and tryptophan residues in β-casein.

Synchronous fluorescence study

The synchronous fluorescence spectra provide significant information about the microenvironment near the chromophore molecules. In this study, synchronous fluorescence spectroscopy was used to get some information about structural changes and binding sites of BIM when it interacts with α-casein molecule. It has been reported that synchronized fluorescence spectrum is a valuable way for distinguishing the tryptophan (Trp) and tyrosine (Tyr) residues in protein^[47].

Figures 5a and 5b show synchronous fluorescence spectroscopy of α-casein with and nm, respectively. In Fig. 5a, for nm, a significant decrease in synchronous fluorescence intensities with a blue shift to 4.0 nm in their emission wavelength maximum (290–286 nm) was observed. It is suggested that the Tyr residues of α-casein might have moved into nonpolar hydrophobic cavities, resulting in the observed increase of hydrophobicity around the Tyr residues. According to Fig. 5b, for , when the concentration of BIM increases, a significant decrease is resulted in synchronous fluorescence intensities with a red shift to 5.0 nm in their emission wavelength maximum (282–287 nm). These results show that the hydrophobicity around the Trp residue of α-casein decreased, while the polarity is shifted to a higher value after binding with BIM, for which the Trp residues might be exposed to an aqueous or hydrophilic environment^[47,48].

Figure 5. Synchronous fluorescence spectra: (a) and (b) of α-casein solution by BIM in PBS of pH 7, at 25°C and [α with [BIM]= 0, 0.66, 2, 4, 6, 8, 15, 30, and 40 µM.

Docking study

The aim of this study was to use experimental data and computational docking studies for a better understanding of the interaction between BIM with α-casein (Fig. 6). Figure 6a demonstrates that BIM penetration into the structure of α-casein has a major role in the binding interaction of BIM–α-casein complex. In Fig. 6b, it is shown that BIM–α-casein is surrounded by Asn-2, His-6, Ala-81, Asn-83, Glu-84, Ile-85, Gln-87, Phe-88, and Leu-153, with an average binding distance between donor and acceptor atoms from 1.6 to 3.2 Å; and a free binding energy of –40.0 kJ mol^–1. The total number of hydrogen binding for BIM–α-casein is three. Figure 6 demonstrates that hydrogen bonds form between BIM and Ala-81, Glu-84, and Gln-87 of α-casein. A BIM molecule is located in the vicinity of hydrophobic and hydrophilic residues, resulting in van der Waals contacts and hydrogen binding. As noted previously, hydrophobic interaction, hydrogen bond, and van der Waals forces play major roles in the binding interaction between BIM and α-casein. From the docking study, the observed free binding energy for BIM–α-casein was –40.0 kJ mol^–1, which is more comparable to our experimentally determined free binding energy of –28.33 kJ mol^–1. The difference between experimental and theoretical results might be attributed to the absence of a solvent and the rigidity of receptor in docking study^[49]. These results demonstrate that α-casein is a useful vehicle for the solubilization and stabilization of BIM in aqueous solutions.

Figure 6. Best conformations for BIM docked to α-casein. The BIM molecule is violet. (a) Whole α-casein with BIM molecule; (b) binding site represented in sticks.

The distance between BIM and Trp-109 and Trp-193 in α-casein was calculated from docking study (Fig. 7). We found the distance between BIM and Trp-109 to be 15.6 Å and that between BIM and Trp-193 to be 21 Å. However, the distance between tryptophan and BIM obtained from FRET calculation in α-casein was found to be 27.8 Å. These results indicate that BIM most possibly causes the hydrophobicity around Trp-193 residue of α-casein. Consequence of these results are more comparable to our synchronous fluorescence study might be exposed Trp residue of α-casein after binding with BIM transferred to an aqueous or hydrophilic environment.

Figure 7. Distance between BIM and tryptophan residues of α-casein.

Conclusions

Based on the results of the present study, FTIR spectra shift in wavelength and change in intensity of absorbance indicate that α-casein interacts with BIM molecule mainly via hydrophobic and hydrophilic interactions. This is also accompanied by an increase in the solubility, bioavailability, and antitumor activity of BIM. Besides, FTIR spectra and the CD spectrum indicated that interaction of the BIM with α-casein could lead to conformational changes in α-casein and an increase in its α-helix content and a simultaneous decrease in the unordered structures of α-casein. Fluorescence quenching in the range of 293–310 K showed that BIM can quench the fluorescence intensity of α-casein, and based on measurements of and , the quenching mechanism is judged as a static process. Further fluorescence studies illustrated that BIM binds to α-casein with a 1:1 stoichiometry. The thermodynamic parameters obtained from fluorescence data showed a negative sign for enthalpy, while the entropy changes were positive. Therefore, hydrophobic interaction, hydrogen bonding, and van der Waals forces play important roles in the binding process. Synchronous fluorescence spectroscopy demonstrated that BIM most probably increases the hydrophobicity around the Trp residue of α-casein; in addition, Fӧrster energy transfer measurement and docking study showed that after addition of BIM, Trp-193 is transferred to an aqueous or hydrophilic environment. The results of docking study demonstrated that the interaction between BIM and α-casein is predominantly in the form of hydrogen bonds and hydrophobic contacts with the hydrophobic residues of α-casein. These results prove that α-casein displays a very good binding and entrapment capacity for this type of hydrophobic anticancer drug. Thus, it may serve as a useful vehicle for the solubilization and stabilization of the drug in aqueous preparations for oral delivery as an antitumor agent.

Acknowledgement

We gratefully acknowledge the Research Council of University of Guilan for supporting this work.