Spectroscopic investigation on the interaction of some surfactant-cobalt(III) complexes with serum albumins

Journal of Luminescence 145 (2014) 269–277

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Spectroscopic investigation on the interaction of some surfactant-cobalt(III) complexes with serum albumins Gopalaswamy Vignesh, Selvan Nehru, Yesaiyan Manojkumar, Sankaralingam Arunachalam n School of Chemistry, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India

art ic l e i nf o

a b s t r a c t

Article history: Received 20 April 2013 Received in revised form 12 July 2013 Accepted 16 July 2013 Available online 2 August 2013

The interaction of HSA/BSA with single and double chain surfactant-cobalt(III) complexes, cis-[Co(phen)2(UA) Cl](ClO4)2
2H2O (1), cis-[Co(phen)2(UA)2](ClO4)3
2H2O (2), cis-[Co(en)2(UA)Cl](ClO4)2
2H2O (3), cis-[Co (en)2(UA)2](ClO4)3
2H2O (4), were investigated by steady state fluorescence, UV–vis absorption, synchronous, three dimensional fluorescence and circular dichroism spectroscopy. The results reveal that the quenching of HSA/BSA by all the four complexes takes place through static mechanism. The binding constant, binding sites and thermodymamic parameter were calculated. The results illustrate that the double chain surfactant-cobalt (III) complexes bind more strongly than the corresponding single chain complexes. The distance between donor (HSA/BSA) and acceptor (surfactant-cobalt(III) complexes) was obtained according to FRET. The results of synchronous, three dimensional and circular dichroism spectroscopy studies show that all the complexes caused considerable amount of conformational and some amount of environment changes in HSA/BSA. & 2013 Elsevier B.V. All rights reserved.

Keywords: Human serum albumin Bovine serum albumin Surfactant-cobalt(III) complexes Circular dichroism Three dimensional fluorescence

1. Introduction Serum albumins are the most abundant and important biomacromolecles in the living systems and play a crucial role in the all the biological processes. They play an important role in the transport and deposition of endogenous and exogenous ligands present in the blood [1]. In recent years the nature of interaction between serum albumins (HSA and BSA) and many compounds like drugs, metal complexes, polymers, surfactants and dendrimers has been investigated successfully. These interaction studies elucidate the properties of drug–protein complex, as they provide useful information of the structural features that determine the therapeutic effectiveness of drugs [2–6]. Therefore, it has become an important research field in life sciences, chemistry, and clinical medicine. The primary structure of these transport proteins has about 580 amino acid residues and is characterized by low content of tryptophan along with a high content of cystine, stabilizing a series of nine loops. The secondary structure of serum albumins has 67% helix of six turns and 17 disulfide bridges. The tertiary structure is composed of three domains I, II and III and each domain can be subdivided into two sub domains, A and B. Bovine and human serum albumins (BSA and HSA) display approximately 80% sequence homology and repeating pattern of disulfides. Crystal structure analyses have revealed that HSA contains 585

n

Corresponding author. Tel.: +91 431 2407053. E-mail address: [email protected] (S. Arunachalam).

0022-2313/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jlumin.2013.07.050

amino acid residues with 17 tyrosyl residues and only one tryptophan (Trp) located at position 214 along the chain (subdomain IIA); whereas, BSA contains 582 amino acid residues with 20 tyrosyl residues and two tryptophans located at positions 134 and 212, in which Trp-134 at the surface of the molecule and Trp212 located in domain IIA [7–11]. The main binding sites on HSA and BSA are located in hydrophobic cavities in subdomains IIA and IIIA [12,13]. Studies on the nature of interaction between surfactants and proteins have attracted considerable interest in the past decades due to manifold applications of protein-surfactant systems eg. food industry, pharmaceutical industry, analytical biochemistry and physiological systems. [14–16]. Surfactants are amphiphilic substances having both hydrophilic and hydrophobic groups and their interaction with proteins also change the conformations and the biological functions of proteins. They are being used in inducing unfolding of proteins and in some cases, stabilizing proteins at very low concentrations [17]. Surfactant metal complexes are a new class of materials in which polar head groups of the surfactant molecules contain a metal ion surrounded by ligands coordinated to the metal (Fig. 1). There are only a few reports on the interaction between such surfactant–metal complexes and proteins [18]. In spite of the greatest effort and success in the study of surfactant-cobalt(III) complexes, such complexes still attract much attraction due to the relative simplicity of their synthesis and their interesting properties. In this work we explore the effect of the head group and additional alkyl chain of some surfactant-cobalt(III) complexes on the structure of HSA/BSA by

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2+ N N

N

N N

N

Co

H 2N

N

3+

=

Co

H2N

Cl

N

N

H 2N

NH2

N NH2

to the solution. A yellowish gray colored precipitate was separated out and it was filtered off and washed with alcohol followed by acetone and dried over fused calcium chloride and stored in a vaccum dessicator. 2.2.1.2. cis-[Co(en)2(UA)2](ClO4)3
3H2O (4). The complex cis-[Co (en)2(UA)2](ClO4)3
3H2O was synthesized by adopting the same procedure used for the synthesis of the above mentioned complex with an except, that the complex cis-[Co(en)2Cl2]Cl
3H2O [25] was used as precursor complex instead of cis-[Co(phen)2Cl2]Cl
3H2O. Caution: Perchlorate salts are potentially explosive and only small quantity was handled with care. 2.2.2. Cobalt(III) analysis Cobalt content in the complexes was determined by Kitson′s method. A known weight of the surfactant cobalt(III) complex was taken in a clean dried conical flask and 3 ml of con. HCl was added to it along with a small piece of tin metal. The solution was heated for an hour. The content was then transferred quantitatively to a 10 ml standard flask and makes up to the mark with concentrated hydrochloric acid. The cobalt concentration was found from the absorbance value at 691 nm by assuming the molar absorbance coefficient of [CoCl4]2+ as 561 M  1 cm  1 [26]. The percentage of cobalt thus obtained for complexes 2 and 4, are shown in Supplimentary Table 1.

N

N

Fig. 1. Representation of single and double chain surfactant-cobalt(III) complex.

using UV–vis, steady state fluorescence, synchronous three dimensional and circular dichroism spectroscopy.

2. Materials and methods 2.1. Materials BSA (lyophilized powder, essentially fatty acid free and globulin freeZ 99%), HSA (lyophilized powder, fatty acid free and globulin freeZ 99%) and undecylamine (UA) were purchased from Sigma Aldrich and used as supplied. The cobaltous chloride, 1, 10-phenanthroline and ethylenediamine were obtained from Rankem, India. Stock solutions of HSA/BSA were prepared by dissolving these in Tris–HCl buffer solutions ( 0.05 M Tris, 0.15 M NaCl) with pH ¼ 7.4. The concentrations of BSA and HSA were determined spectrophotometrically using the extinction coefficient of 43, 800 [19] and 36,500 M  1cm  1 [20] at 280 nm. The surfactant complexes, cis-[Co(phen)2(UA)Cl](ClO4)2
2H2O (1) and cis-[Co(en)2(UA)Cl](ClO4)2
2H2O (3), were known in our previous studies [21,22] and the complexes cis-[Co(phen)2(UA)2] (ClO4)3
2H2O (2) and cis-[Co(en)2(UA)2](ClO4)3
2H2O (4), were synthesized by slight modification in the reported procedure [23]. All other chemicals were of analytical grade and double distilled water was used in all the studies. 2.2. Methods 2.2.1. Synthesis of double chain surfactant-cobalt(III) complexes 2.2.1.1. cis-[Co(phen)2(UA)2] (ClO4)3
3H2O (2). To aqueous solution of cis-[Co(phen)2Cl2]Cl
3H2O (0.58 g) [24], undecylamine (0.34 g) in ethanol was added drop by drop over a period of 30 min. During the addition of undecylamine, the dark violet color solution gradually became yellowish gray. The mixture was kept at room temperature for 48 h until no further color change was observed. Afterwards, a saturated solution of sodium perchlorate was added

2.2.3. Critical micelle concentration The cmc values of the complexes were determined conductometrically using a specific conductivity meter as previously reported [20]. The conductivity cell was calibrated with KCl solution in the appropriate concentration range. The cell constant was calculated using molar conductivity data for KCl. Various concentrations of complexes were prepared in the range of 10  5–10  2 mol dm  3 in aqueous solution. The conductivities of these solutions were measured at 291, 296 and 301 K. The temperature of the thermostat was maintained constant within 70.01 K. The conductance was measured after thorough mixing and temperature equilibrating at each dilution. The establishment of equilibrium was checked by taking series of readings after 10 min interval until no significant change occurred. The cmc values of all the four complexes at three different temperatures (291, 296 and 301 K) are listed in the Supplementary Table 2. The conductivity measurements at three different temperatures were repeated and the accuracy of cmc values is found to be within 2%.

2.2.4. UV–vis absorption measurements The UV–vis absorption spectra were recorded at room temperature on a UV-1800 Shimazu UV spectrophotometer using cuvette with a 1 cm path length. The spectra were recorded between 200 and 800 nm.

2.2.5. Fluorescence experiments Fluorescence experiments were carried out on a JASCO FP650 spectrofluorometer (Japan) in a 1 cm quartz cell and a thermostat bath. The emission was measured from 290 nm to 450 nm with an excitation wavelength of 280 nm. The excitation and emission slits were fixed at 3 nm and 5 nm, scanning speed to 500 nm/min. The experiments were carried out at three different temperatures of (291, 296 and 301 K). The three dimensional fluorescence spectra were measured under the following conditions: the emission wavelength was recorded between 250 and 500 nm, the initial excitation wavelength was set to 250 nm with increments of 5 nm, the number of scanning curves was 14, emission and excitation

G. Vignesh et al. / Journal of Luminescence 145 (2014) 269–277

slit widths were fixed at 5 nm and 3 nm respectively and the synchronous fluorescence spectra were recorded with Δλ ¼15 nm as well as Δλ¼ 60 nm between excitation and emission wavelengths. The nature of interaction of complexes with HSA/BSA was studied only below the cmc values (lower concentration) so that the absorbance values were low (slightly greater than 0.1) so as to eliminate the inner filter effect. Wherever applicable, if any inner filter effect, Eq. (1) was used for all of the fluorescence results to obtain accurate data [27]. F cor ¼ F obs  eðAex þAem Þ=2

ð1Þ

where Fcor and Fobs are the fluorescence intensities corrected and observed, respectively, and Aex and Aem are the sum of the absorbance of protein and ligand at the excitation and emission wavelengths, respectively. For complexes 3 and 4 the absorbance of the complexes found to be below 0.1 in the studied concentration range, so no inner filter correction was made.

271

3.2. Critical micelle concentration values (CMC) The specific conductivity of the complexes 1–4 increases with the complex concentration and temperature. When plots are made of [complex] vs specific conductivity, the slope is reduced after a particular value of concentration. This particular concentration value at which slope of the plot changes shows micellization and this concentration is considered as CMC. As seen from Supplementary Table 2 it is found that CMC value of all the complexes increase with increase in the temperature. The increase in temperature causes a decrease in hydration in the hydrophilic group which facilitates micellisation and also temperature disrupts the water surrounding the hydrophobic groups and this retards micellisation. The relative magnitude of these two opposing effects will determine CMC behavior. Further with the increase in the alkyl chain on polar head group, the CMC value decreases. This may be due to an increase in hydrophobic character of the molecule in the coordination sphere. 3.3. Analysis of fluorescence quenching mechanism

3.1. Spectroscopic characterization of surfactant-cobalt(III) complexes Several workers have employed the NH2 deformation mode (1700–1500 cm  1 region), the CH2 rocking (950–850 cm  1 region) and Co–N stretching mode in the 600–500 cm  1 region to distinguish between cis and trans isomers [28–31]. The cis isomers always show two peaks, whereas the trans isomers usually have only one peak in the CH2 rocking region. In the present work, the NH2 deformation mode shows two bands in the region 1630– 1530 cm  1[32], two bands for the CH2 or NH2 twist in the region 1060–980 cm  1[33] and two bands for the CH2 rock mode in the region 943–850 cm  1. Schilt and Talyor [34] studied the infra red spectra of phenanthroline complex, the IR bands, δ (C–H) 853, 737 cm  1, observed for phenanthroline are red shifted to 847 and 715 cm  1 in the complex 2. This shift can be explained on the basis of the fact that the nitrogen atoms of phenanthroline ligands donate a pair of electron each to the central cobalt metal forming a coordinate covalent bond [35]. Besides, it is also confirmed by the shift of υ(C–N) of phenanthroline from about 1670 cm  1 in free ligand to 1634 cm  1 after coordination [36]. In both the complexes, the bands that exhibited around 2917 and 2848 cm  1 can be assigned to C–H asymmetric and symmetric strectching vibration of aliphatic CH2 of undecylamine. Perchlorate bands at ca. 1090 and 626 cm  1 belong to an ionic species; this means that this counter ion is not involved in the cobalt ligand coordination [37]. In the 1H NMR spectrum of the surfactant-cobalt(III) complexes, the methylene protons of the long chain moiety (undecylamine) gave rise to a multiplet at 1.2 to 1.8 ppm whereas terminal methyl group of hydrocarbon chain substituent gave a triplet around 0.85 ppm. The 1H-NMR spetra of complex 4 exhibits signals in the region 2.4–3 ppm attributable to the –CH2 group of the ethylenediamine chelate ring. The aromatic proton of phenanthroline ligands appeared in the region 7.4–9.5 ppm and assigned in the similar manner to the case of parent complex. The 13C-NMR spectrum of surfactant-cobalt(III) complexes exhibit only one singal around 37–40 ppm because of merging of undecylamine and chelating ligand singnals. For long chain undecylamine, the aliphatic methylene carbons of surfactant-cobalt(III) complexes appeared around 22–40 ppm and the terminal carbon atom appeared around 13.9 ppm. The signal around 124–157 ppm corresponding to phenanthroline rings.

The intrinsic fluorescence of HSA/BSA is due to three fluorophores (Trp, Try and Phe). Due to low quantum yield of phenylalanine and fluorescence of tyrosine is completely quenched if it ionized (or) near a tryptophan residues. Thus intrinsic fluorescence of HSA/BSA is mainly due to Trp residues (Trp-214) for HSA and Trp-134 and 212 for BSA. The characteristic of the intrinsic fluorescence of HSA/BSA is very sensitive to its microenvironment. Intrinsic fluorescence of HSA/BSA gets quenched if there is a slight change in the microenvironment of HSA/BSA. This intrinsic fluorescence can be quenched by a variety of molecules and the mechanism of the quenching may be either through static or dynamic quenching [38,39]. Static quenching refers to quenching through fluorophore-quencher complex formation and dynamic quenching refers to a process that the fluorophore and the quencher come into contact during the transient existence of the excited state. Static and dynamic quenching can be distinguished by the effect of temperature on the quenching behavior. The Fig. 2 and Supplementary Figs. 1 and 2 show that intrinsic fluorescence of HSA/BSA in the presence of surfactant-cobalt(III) complexes of the present study. It is observed from the figure that in the presence of both the complexes fluorescence intensity of HSA/ BSA decreases regularly with the increase in concentration of complexes at 291 K, by comparing the quenching effect of same

a

3+

[Co(phen)2(UA)Cl] - HSA

200 175

T = 291 K

150

Intensity

3. Results and discussion

i 125 100 75 50 25 300

320

340

360

380

400

420

440

Wavelength (nm) Fig. 2. Emission spectra of HSA in the presence of various concentration of [Co(phen)2(UA)Cl]2+, c(HSA)¼1  10  5 mol L  1, c([Co(phen)2(UA)Cl]2+)¼0–45.8 mM.

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2.2

0.18

2+

[Co(phen)2(UA)Cl] - HSA

0.16

291 K R = 0.9933 298 K R = 0.9945 301 K R = 0.9875

2.0

0.14

Absorbance

F0/F

1.8 1.6 1.4 1.2

C

0.12 0.10 0.08

B

0.06 0.04

A

1.0

0.02 0

1

2

3

4

5

[Q]

240

Fig. 3. Stern-Volmer plot for the quenching of HSA by [Co (phen)2(UA)Cl]2+, [HSA]¼ 1  10  5 mol L  1.

Table 1 Stern-Volmer constant for the interaction of HSA-surfactant-cobalt(III) complexes. kq  1012 (L mol  1 s  1)

Ra

291 2.511 296 2.409 301 2.024

2.511 2.409 2.024

0.9933 0.0486 0.9945 0.0425 0.9875 0.0542

[Co(phen)2(UA)2]3+

291 1.912 296 1.742 301 1.540

1.912 1.742 1.540

0.9978 0.0212 0.9956 0.0274 0.9961 0.0228

[Co(en)2(UA)Cl]2+

291 0.655 296 0.705 301 0.823

0.655 0.705 0.823

0.9949 0.0110 0.9974 0.0084 0.9967 0.0112

[Co(en)2(UA)2]3+

291 0.888 296 0.822 301 0.683

0.888 0.822 0.683

0.9964 0.0125 0.9985 0.0061 0.9985 0.0062

Complexes

T (K)

[Co(phen)2 (UA)Cl]2+

a b

Ksv  104 (L mol  1)

SDb

R is the correlation coefficient. SD is the standard deviation for Ksv.

surfactant-cobalt(III) complex on the HSA and BSA spectra no significant change is observed between the two proteins (Fig. 2 and Supplementary Fig. 2) this may conclude that the Trp-134 is a not target in the BSA thus indicates that changes in the microenvironment have occurred around the single tryptophan residue Trp 214 and Trp 212 within the hydrophobic pocket of sub-domain IIA of HSA and BSA. In order to study the fluorescence quenching mechanism, the fluorescence quenching data at different temperatures (291, 296 and 301 K) were analyzed using classical Stern–Volmer equation [38] F 0 =F ¼ 1 þ K sv ½Q  ¼ 1 þ kq τ0 ½Q 

0.00

ð2Þ

where F0 and F are the fluorescence intensities in the absence and presence of the quencher, respectively. Ksv is the Stern–Volmer constant, [Q] is the concentration of the quencher, kq the quenching rate constant and τ0 the fluorescence lifetime in the absence of quencher. Fig. 3 and Supplementary Figs. 3 and 4 show the Stern Volmer plots. As it is known that linear Stern Volmer plot represent a single quenching mechanism, either static or dynamic. As seen from the Table 1 and Supplementary Table 3, it is found that for the surfactant-cobalt(III) complex–HSA system containing complex 1, 2, 4 and in the case of the surfactant-cobalt(III) complex–BSA system containing complex 2, the Ksv value decreases with increasing temperature indicating that static quenching prevails. In the case of surfactant-cobalt(III) complex–HSA system containing complex

270

300

330

360

390

Wavelength (nm) Fig. 4. The UV–vis absorption spectra of HSA in the absence and presence of surfactant. cobalt(III) complexes. (A) Absorption spectrum of [Co(phen)2(UA)Cl]2+, (B) the absorption spectrum of HSA only, and (C) the difference absorption spectrum between {[Co(phen)2(UA)Cl]2++HSA} and [Co(phen)2(UA)Cl]2+ at the same concentration, c(HSA)¼([Co(phen)2(UA)Cl]2+)¼ 2.0  10  6 mol L  1.

3 and in surfactant-cobalt(III) complex–BSA system containing complex 1, 3 and 4, Ksv value increases with temperature which indicates that quenching has occurred via a dynamic quenching mechanism. But the obtained bimolecular quenching constants for all the above systems are in the order of 1011–1012 L mol  1 s  1. which is 10–100 fold higher than the maximum value possible for diffusion controlled quenching (2  1010 L mol  1 s  1) [40]. These large values of kq for all the systems show that the dominating quenching mechanism is static rather than dynamic mechanism. In order to confirm the ground state complex formation between HSA/BSA and surfactant-cobalt(III) complexes UV–vis absorption spectra of HSA/BSA in the presence and absence of complexes were taken. Collisional quenching can only affect the excited state of the fluorophores with no change in the absorption spectrum. In contrast ground state complex formations will frequently perturb the absorption spectrum of the fluorophores. The UV–vis absorption spectra of HSA/BSA in the presence of complexes (1, 2, 3 and 4) are shown in the Fig. 4 and Supplementary Figs. 5 and 6. The UV–vis absorption spectra of HSA/BSA in the absence and presence of complexes indicate that the probable quenching of HSA/BSA by surfactant-cobalt(III) complexes is static in nature [41]. 3.4. Analysis of binding equilibria For the static quenching, when molecules bind independently to a set of equivalent sites on a macromolecule, the binding constant (Kb) and the number of binding sites (n) can be determined by the following equation [42]:
 log ðF 0 FÞ=F ¼ log K b þ n log ½Q  ð3Þ where F0 and F are the fluorescence intensities in the absence and presence of the quencher, [Q] the concentration of the quencher, Kb is the binding constant and n is the number of binding sites. Using this equation for all the surfactant-cobalt(III) complex-HSA/BSA systems of the present work Kb and n values at three different temperatures were obtained from the intercept and slope of the plots of log[(F0  F)/F] vs log[Q] (Fig. 5 and Supplementary Figs. 7 and 8). From the figures it is found that all the correlation coefficients of samples almost equal to 0.99, indicating that the binding of surfactant-cobalt(III) complex to BSA/HSA agreed well with the site-binding model as described by Eq. (3). From the Table 2 and Supplementary Table 4, it is noticed that binding

G. Vignesh et al. / Journal of Luminescence 145 (2014) 269–277

0.2

2+

[Co(phen)2(UA)Cl] - HSA

9.0 8.8

-0.2

Complex1 Complex2 Complex3 Complex4

8.6

lnK

log[(F0-F)/F]

9.2

291 K R = 0.9952 296 K R = 0.9980 301 K R = 0.9908

0.0

273

-0.4

8.4 8.2

-0.6

8.0

-0.8

7.8

-5.2

-5.0

-4.8

-4.6

-4.4

-4.2

0.00332

0.00334

log[Q]

0.00336

0.00338

0.00340

0.00342

0.00344

(1/T) K-1

Fig. 5. Plot of log[(F0–F)/F] vs log[Q] for HSA-[Co(phen)2(UA)Cl]2+. 12 11

Table 2 Binding constant and the number of binding sites of the HSA-surfactant-cobalt(III) complexes.

10

T (K)

Kb (  103 M−1)

n

Ra

SDb

[Co(phen)2(UA)Cl]2+

291 296 301 291 296 301 291 296 301 291 296 301

8.222 6.638 5.448 6.932 8.247 9.954 3.034 2.675 2.307 2.850 4.228 6.117

0.88 0.87 0.86 0.89 0.92 0.95 0.93 0.90 0.87 0.89 0.94 0.99

0.9952 0.9980 0.9908 0.9994 0.9987 0.9977 0.9925 0.9990 0.9951 0.9920 0.9945 0.9964

0.027 0.0174 0.0380 0.0096 0.0149 0.0216 0.0368 0.0128 0.0278 0.0364 0.0319 0.0272

[Co(phen)2(UA)2]3+

[Co(en)2(UA)Cl]2+

[Co(en)2(UA)2]3+

a b

lnK

9

Complexes

8 7

Complex1 Complex2 Complex3 Complex4

6 5 4 0.00332

0.00334

0.00336

0.00338

0.00340

0.00342

0.00344

(1/T) K-1 Fig. 6. van′t Hoff plot for the interaction of HSA (A)/BSA (B) with surfactant-cobalt (III) complexes.

R is the correlation coefficient. SD is the standard deviation for Kb.

constant values of double chain surfactant-cobalt(III) complexes is found to be greater than the respective single chain complex. This illustrates that there is a strong binding force between double chain surfactant complex and HSA/BSA. This shows that double chain surfactant-cobalt(III) complexes that confer it with unique characteristic such as much lower CMC (Supplementary Table 1) and its higher hydrophobicity. It also observed the values of Kb is significant to understand that the surfactant-cobalt(III) complex containing phenanthroline ligand binds more strongly than the ethylenediamine ligand due to the higher hydrophobic nature of the phenanthroline complex than the ethylenediamine complex. This result shows that the head groups of the surfactant metal complex plays an important role in the binding process. The values of n approximately equal to 1 which indicates the existence of a single binding sites for both type of complexes with HSA/BSA. It is well know that the binding sites of long chain fatty acids (C12, C14 and C16) are mostly occupied in the sub-domains IIA and IIIA of HSA/BSA and also simple cobalt complexes bind to domain IIA of HSA/BSA. Based on these facts, it is concluded that in the present work, the binding of complex 1 and 2 with HSA/BSA is through sub-domain IIA [43–45]. 3.5. Thermodynamic parameters and binding forces The interaction forces between drug and biomolecules includes hydrogen bonds, van der Waals forces, electrostatic and hydrophobic

attraction [46]. The sign and magnitude of ΔS0 and ΔH0 for protein binding can account for the main force contributing to protein stability. When the temperature change is not very enormous, the ΔH1 of a system can be regarded as a constant and its value and that of entropy change (ΔS1) can be calculated from the van′t Hoff equation: log K ¼ ΔH1=2:303RTΔS1=2:303RT

ð4Þ

where Kb is the binding constant at the corresponding temperature and R is the gas constant. ΔH1 and ΔS1 values were calculated from slope and ordinate of the plot of log Kb vs 1/T (Fig. 6). The thermodynamic studies reveal that free energy changes are negative for interaction between all of our surfactant complexes and HSA/BSA which show that binding processes are spontaneous. The thermodynamic parameters thus obtained for the interaction of surfactant-cobalt(III) complexes with HSA/ BSA at different temperatures are shown in Supplementary Tables 5 and 6. As shown in the table ΔH1 and ΔS1 for the binding reaction between double chain surfactant-cobalt(III) complexes and HSA/BSA and single chain complex 1 with BSA are found to be positive, which indicate that hydrophobic force plays a vital role in the binding processes. But in the case of binding between single chain surfactantcobal(III) complexes and HSA/BSA, mostly the value of ΔH1 and ΔS1 are found to be negative, which show that hydrogen bonding and van der walls forces involved in the binding [47].

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3.6. Energy transfer from HSA/BSA to surfactant-cobalt(III) complexes

The efficiency (E) of energy transfer between the donor and acceptor could be calculated by the following equation:

In order to get more information on the surfactant-cobalt(III) complex-HSA/BSA system, distance r between the surfactant-cobalt (III) complex (donor) and the HSA/BSA (acceptor) has been calculated by using the fluorescence resonance energy transfer method (FRET) [48]. According to Forster′s non-radiative energy transfer theory, the energy transfer will happen under the following conditions: (i) the donor can produce fluorescence light, (ii) the extent of overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor and (iii) the distance between the donor and the acceptor is lower than 7 nm. The overlap pattern of the UV-absorption spectrum of surfactant-cobalt(III) complex with the emission spectrum of HSA/BSA was shown in the Fig. 7 and Supplementary Figs. 9 and 10.

E¼

250

R60 ¼ 8:8  1025 K 2 N 4 ΦJ

0.1 0.08

100

0.06

where F(λ) is the fluorescence intensity of the donor in the wavelength range λ to λ+Δλ and ε(λ) is the extinction coefficient of the acceptor at wavelength λ. J can be evaluated by integrating the overlap spectra. According to these equations and using K2 ¼ 2/3, N ¼ 1.36 and Ψ ¼0.15 [49], the J, R0 and r values were calculated from the pattern of the spectral overlap obtained for our surfactant-cobalt(III) complex –HSA/ BSA system which are shown in the Supplementary Tables 7 and 8. As seen from this table it is observed that the r values between surfactant-cobalt(III) complex and HSA/BSA is less than 7 nm which indicates that the energy transfer from HSA/BSA to surfactant-cobalt(III) complexes occurred with high probability.

Absorbance

Intensity

0.14 0.12

0.04

50

0.02 0 300

0 320

340

360

380

400

Wavelength (nm) Fig. 7. Spectral overlap of [Co(phen)2(UA)Cl]2+ absorption (A) and HSA Emission (E), c([Co(phen)2(UA)Cl]2+) ¼ c(HSA)¼1  10  5 mol L  1.

250

ð6Þ

In above equation K2 is the orientation factor of the dipoles, N is the refractive index of the medium, Φ the fluorescence quantum yield of the donor and J is the effect of the spectral overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor which could be calculated by the equation below: Z FðλÞεðλÞλ4 dλ J¼ ð7Þ FðλÞdλ

0.16

150

ð5Þ

R60 þ r 60

where E is the efficiency of energy transfer between the donor and the acceptor, R0 is the critical distance between the acceptor and donor when the efficiency of energy transfer is 50%.

0.18

200

R60

90

2+

[Co(phen)2(UA)Cl] -HSA

2+

[Co(phen)2(UA)Cl] -HSA

80 200

Intensity

150

100

60 50 40

50

30 20

0 300

315

330

345

360

375

390

290

300

Wavelength (nm)

310

320

330

340

Wavelength (nm)

1.0 2+

HSA- [Co(phen)2(UA)Cl]

0.9 0.8 0.7

F/F0

Intensity

70

0.6 0.5

Try Trp

0.4 0.3 0

1

2

3

4

5

[Q] Fig. 8. (a) Synchronous fluorescence spectrum of HSA in presence of [Co (phen)2(UA)Cl]2+, (A) Δλ ¼60 nm, (B) Δλ¼ 15 nm.

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275

3.7. Synchronous fluorescence 5000

3.8. Circular dichroism studies Circular Dichroism (CD) spectroscopy has been one of the valuable methods for analysis of protein secondary structure. So in order to obtain an insight into the structure of BSA/HSA, the CD spectra of HSA/BSA were recorded in the presence and absence of all the four complexes. As we know, the CD spectra of HSA/BSA exhibited two negative bands in the UV-region at 208 nm and 222 nm. The band is characteristic of an α-helical structure of protein. The peak at 220 nm is attributed by the n-π transition of peptide bonds in the α-helix, and peak at 208 nm is contributed by π-π transitions of the peptide bonds on the α-helix [56]. The CD results are expressed in terms of mean residue elipticity (MRE) in deg cm2 dmol  1 according to following equation [57]: MRE ¼

Observed CDðm degÞ 10C p nl

αHelixð%Þ ¼

MRE208 4000  100 330004000

ð8Þ

ð9Þ

where Cp is the molar concentration of the protein, n is the number of amino acid residues and l is the path length. Fig. 9

MRE (deg.cm2.dmol-1)

0 -5000 -10000 HSA Complex 1 Complex 2 Complex 3 Complex 4

-15000 -20000 -25000 200

210

220

230

240

250

260

Wavelength (nm)

5000 0

MRE (deg.cm2.dmol-1)

Synchronous fluorescence spectroscopy can give information about the molecular environment in the vicinity of a chromophore such as tryptophan and tyrosine and it involves simultaneous scanning of the excitation and emission monochromators while maintaining a constant wavelength interval between them. When Δλ is set at 15 nm and 60 nm the shift of the maximum emission wavelength reveals the alternation of polarity microenvironment around Tyr and Trp residues. The shift in the position of fluorescence emission maximum corresponds to change of the polarity around the chromophore molecule. A blue shift of λmax means that the amino acid residues are located in a more hydrophobic environment, while a red shift of λmax implies that the amino acid residues are in a less hydrophobic environment [50–53]. Synchronous fluorescence spectra of HSA/BSA with various amount of surfactant-cobalt(III) complexes are shown in the Fig. 8 and Supplementary Fig. 11, the shifts in the wavelength of tyrosine and tryptophan are summarized in the Supplementary Table 9. The emission maximum of tryptophan residues of BSA/ HSA shows a red shift in the presence of surfactant-cobalt(III) complex which indicates that tryptophan residues buried in the non polar hydrophobic cavities were moved to hydrophilic environment, besides that the complex 3 which does not show any significant shift over the investigated concentration range indicating that the complex 3 has no effect on microenvironment of tryptophan residue of HSA/BSA. When Δλ¼ 15 nm, the emission maximum of tyrosine residues of HSA/BSA show red shift in the presence of complexes 1 and 2 which indicate the polarity around Tyr residues was changed whereas in the complexes 3 and 4 there is almost no shift in the maximum wavelength of tyrosine. These results indicate that in the presence of complexes 1 and 2 the changes in the polarity of Tyr and Trp residues are more compared to complexes 1 and 2. This observation illustrates that head group of the double and single chain surfactant-cobalt(III) complexes play a significant role in the polarity. However it is observed from the Fig. 8 that in the presence of surfactant-cobalt(III) complexes (1 and 2) same type of quenching profile is noticed for both Tyr and Trp residues Tyr-261, 317, 332, 339 and 351 and Trp-212 are located in domain II of BSA with a hydrophobic environment and Tyr and Trp residues Tyr-263, 319, 332, 334, 341, 353 and 370 and Trp-214 which are located in domain II of HSA with a hydrophobic environment [54,55].

-5000 -10000 BSA Complex 1 Complex 2 Complex 3 Complex 4

-15000 -20000 -25000 200

210

220

230

240

250

260

Wavelength (nm) Fig. 9. CD spectra of HSA (3 mM) (A) and BSA (2 mM) (B) in the absence and presence of surfactant-cobalt(III) complexes.

shows the CD spectra of HSA/BSA in presence of complexes. From the figure it is observed that in the presence of complexes, the intensity of the band decreased without any shift. This alternation of the positive of bands shows that even after bridging of surfactant complexes, α-helix is still predominant conformation of HSA/BSA. Inorder to quantify the content of α-helix HSA/BSA, the changes in the intensity have been analyzed by the Eqs. (1) and (2) and it is found that α-helix content of free HSA decreases from 62.20% to 57.32% (HSA- complex 1), 55.35% (HSA-complex 2), 60.52% (HSA-complex 3) and 59.49% (HSA-complex 4) and in the case of BSA, α-helix content of free BSA decreases from 65.23% to 61.53% (BSA-complex 1), 59.89% (BSA-complex 2), 65.13% (BSAcomplex 3) and 64.11% (BSA-complex 4). 3.9. Three dimensional fluorescence studies Three dimensional fluorescence spectroscopy is a newly a developed analytical technique which can comprehensively exhibit the fluorescence information and conformational changes of proteins in a more convenient and credible way. The information regarding the fluorescence characteristics can be entirely acquired by simultaneously changing the excitation and emission wavelengths. The maximum emission wavelength and fluorescence intensity of the protein residues have a close relationship to the polarity of their environment. If there is a shift at the excitation or emission wavelength around the fluorescence peak, or a new peak appears or the existing peak disappears, it can be an important hint to suggest the conformational changes of the protein. The conformational and microenvironmental

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Peak 2

Peak 1

The distance between HSA/ BSA and surfactant-cobalt(III) complex was calculated to be less than 7 nm. The results of synchronous, three dimensional and circular dichroism spectroscopy reveal that the microenvironment and conformation of HSA/BSA were changed in the presence of all the four complexes. All these experimental results clarify that the additional alkyl chain and the head group of the surfactant metal complexes have a significant effect on the binding.

Acknowledgments

Peak 2

We are grateful to the UGC-SAP & COSIST and DST-FIST programmes of the Department of Chemistry, Bharathidasan University. One of the authors, SA, thanks the CSIR (Grant no. 01 (2461)/11/EMR-II) and UGC Grant no. 41–223/2012 (SR) for sanction of research schemes. G.V thanks UGC Research Fellowship in Science for Meritorious Students. We are thankful to Prof. A. Ramu, Department of Inorganic Chemistry, Madurai Kamaraj University, Madurai, for CD Studies.

Appendix A. Supporting information

Peak 1

Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jlumin.2013.07.050.

References

Fig. 10. Three dimensional fluorescence spectra of HSA (A) and HSA-[Co(phen)2(UA)Cl]2+(B). The concentrations of HSA and [Co(phen)2(UA)Cl]2+ were c([Co (phen)2(UA)Cl]2+) ¼c(HSA)¼ 1  10  5 mol L  1.

changes of BSA and HSA were investigated by comparing their spectral changes in the absence and presence of our surfactant-cobalt(III) complexes [58,59]. Fig. 10 and Supplementary Fig. 12 display three dimensional fluorescence spectra of HSA/BSA in the absence and presence of surfactant-cobalt(III) complex and corresponding spectral parameters are presented in Supplementary Table 10. Fig. 10 and Supplementary Fig. 12 show two typical peaks, Peak 1 and Peak 2. Peak 1 is Rayleigh scattering peak (λex ¼λem), whereas the strong Peak 2 (λex oλem) mainly reveals the spectral characteristic of Trp and Tyr residues [60]. As seen from the Fig.10 and Supplementary Fig. 12 the fluorescence intensity of Peak 2 decreases in the presence of all the surfactant complexes, thus decrease in peak suggests that all the complexes induce some conformational changes in HSA/BSA which are related to the hydrophobic microenvironment near Trp and Try residues. These results in combination with synchronous fluorescence and circular dichroism studies indicate that the interaction of surfactant-cobalt(III) complexes with BSA/HSA changes the microenvironment and conformational changes in HSA/BSA.

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