Static quenching of tryptophan fluorescence in proteins by a dioxomolybdenum(VI) thiolate complex

Journal of Photochemistry and Photobiology A: Chemistry 293 (2014) 81–87

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Static quenching of tryptophan fluorescence in proteins by a dioxomolybdenum(VI) thiolate complex Alexander A. Rhodes a , Brandi L. Swartz a , Erik R. Hosler a , Deanna L. Snyder a , Kristen M. Benitez a , Balwant S. Chohan b , Swarna Basu a,∗ a b

Department of Chemistry, Susquehanna University, 514 University Avenue Selinsgrove, PA 17870, United States Math & Natural Sciences Department, Centenary College, Hackettstown, NJ 07840, United States

a r t i c l e

i n f o

Article history: Received 26 June 2014 Received in revised form 25 July 2014 Accepted 29 July 2014 Available online 7 August 2014 Keywords: Molybdenum complex Serum albumins Tryptophan fluorescence Static quenching

a b s t r a c t The binding of cis-dioxobis(dithiocarbamato) molybdenum(VI) with the proteins bovine serum albumin, human serum albumin, lysozyme, and free tryptophan was studied using fluorescence spectroscopy and Stern–Volmer kinetics. The quenching of tryptophan fluorescence was determined to be static with binding constants on the order of 104 –105 M−1 , and with a binding site number of one. The interaction was studied over a range of temperatures, and the binding was found to be exothermic with a negative change in entropy. Quantum chemical calculations were also conducted to identify optimal spatial contacts and the resulting energetic contributions between the complex and free tryptophan. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Transition metal ions are essential for a great variety of biochemical functions. These ions can be incorporated into or associate closely with proteins, giving rise to functions ranging from cell signaling, gene expression, antioxidant activity to electron transport and catalysis, and in the case of molybdenum, amino acid metabolism. Earlier studies focused on the interaction of transition metal ions with biologically important proteins, particularly with bovine serum albumin (BSA) [1,2]. More recent studies have shifted focus to interaction of proteins with transition metal complexes, including nickel, chromium and molybdenum complexes [3–7]. Based on whether the ligand is hydrophobic, hydrophilic, or possess certain chemical features, the study of a wide number of complexes can give insight into how proteins behave in different chemical environments. Serum albumins are widely studied and well-characterized proteins [3,4,8–16]. The essential difference between human serum albumin (HSA) and BSA is the number of tryptophan residues (Trp). BSA contains two tryptophans, one on the surface of the molecule (Trp134) and one closer to the interior of the protein in a hydrophobic fold (Trp212). HSA contains only one tryptophan (Trp214), in a similar chemical environment as Trp212 in BSA [17]. The binding

∗ Corresponding author. Tel.: +1 570 372 4223; fax: +1 570 372 2774. E-mail address: [email protected] (S. Basu). http://dx.doi.org/10.1016/j.jphotochem.2014.07.023 1010-6030/© 2014 Elsevier B.V. All rights reserved.

capabilities of serum albumins are largely due to the presence of these tryptophan binding sites. Lysozyme is a single polypeptide chain that consists of 129 amino acid residues with six tryptophans which are located in a hydrophobic fold. The tryptophan is responsible for a large portion of the reactivity in the active site of the lysozyme enzyme [18–20]. The interaction of lysozyme with various quenchers has been reported [3,21,22]. The underlying motivation for this work is our interest in identifying and synthesizing water-soluble transition metal complexes that can be used as probes for protein activity. Fluorescence quenching is a useful tool for measuring the extent and accessibility of protein binding sites to small molecules. Using Stern–Volmer kinetics and Forster’s theory, the number of binding sites, the binding constant Ka , and the distance between donor and acceptor can be determined [23–25]. The fluorescence signal of a protein is derived from its aromatic residues, primarily tryptophan [3]. Tryptophan fluorescence is sensitive to its environment, and is a convenient spectroscopic probe for the structure and rotational dynamics surrounding the residue [24]. Fluorescence quenching studies are not limited to just proteins: Fluorescence quenching of non-biological systems by nanoparticles, C60 fullerenes and carbon tetrachloride have been reported in recent years [26–28]. In this study we have explored the binding interaction of a water soluble dioxo-molybdenum(VI) complex containing a nonaromatic diethyldithiocarbamate ligand [MoO2 (S2 CNEt)2 ], with BSA, HSA, lysozyme and free tryptophan (free-Trp). The structure of this dioxo-Mo(VI) complex is shown in Fig. 1. The results

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The static quenching constant or binding constant, Ka , and number of binding sites (n) between the protein and metal complex were calculated using the following equation [26,27]: log

F − F  0 F

= log Ka + n log[Q ]

(3)

The changes in enthalpy, H, and entropy, S, were determined using the van’t Hoff equation: Fig. 1. Structure of cis-dioxobis(N,N-diethyldithiocarbamato) molybdenum(VI) [MoO2 (S2 CNEt)2 ].

presented here complement previously published work by our group on the interaction of transition metal complexes with various proteins [3,4]. Wu and co-workers [6,29] have previously described the interaction between a highly aromatic phenylfluorone molybdenum(VI) complex with BSA and HSA. Although the ligand systems are very different, some comparison between the Mo(VI) complexes as they interact with globular proteins can be made. 2. Materials and methods 2.1. Chemicals The proteins BSA, HSA and lysozyme, and free-Trp were purchased from Sigma–Aldrich and used without further purification. Protein solutions were prepared in 50 mM Tris–HCl buffer (pH 7.4). The dioxo-Mo(VI) complex, cis-dioxobis(N,Ndiethyldithiocarbamato) molybdenum(VI) [MoO2 (S2 CNEt)2 ], was synthesized using the procedures described by Moore and Moloy [30,31]. The complex was recrystallized from dichloromethane, and thoroughly dried under vacuum before use. All solutions were prepared using deionized water (reverse osmosis, 18.2 M). 2.2. Instrumentation and quenching protocol Absorption spectra were measured using a Cary 4000 UV–Vis Spectrophotometer. Fluorescence emission spectra were measured using a Perkin-Elmer LS50B Luminescence Spectrometer. For the fluorescence quenching experiments, the protein concentration was fixed at 1.0 × 10−4 M and the concentration of the dioxo-Mo(VI) complex was increased through the preparation of individual samples. The maximum dioxo-Mo(VI) complex concentration ranged from 2.1 × 10−4 M to 2.7 × 10−4 M, depending on the extent of quenching. Excitation wavelengths ranged from 279 nm to 281 nm and slit widths were varied as needed. Fluorescence spectra were obtained over a 25–55 ◦ C range. Emission maxima were at 345 nm for the protein samples and 363 nm for tryptophan. 2.3. Data analysis The dynamic quenching constant (KSV ) was determined using the Stern–Volmer equation [25,32]: F0 = 1 + KSV [Q ] F

(1)

F0 and F correspond to the fluorescence intensities of the protein without quencher and in the presence of quencher, respectively, and [Q] is the concentration of the quencher. KSV is related to the lifetime of the system according to the following equation [25]: KSV = kq 0

(2)

kq is the bimolecular quenching constant and  0 is the lifetime of the protein (order of 10−8 s).

ln Ka = −

H S + RT R

(4)

2.4. Computational chemistry Quantum chemical calculations were carried out using the Gaussian09 program (Gaussian, Inc., Wallingford, CT, USA). The structures of tryptophan, the dioxo-Mo(VI) complex and various orientations of the complex were first optimized using Hartree–Fock theory and the LanL2DZ basis set. Excited states were modeled using the time-dependent self-consistent-field (TD-SCF) method following computational methodologies described in the literature [33–35]. Structures were first optimized in the gas-phase and then in water, where the Polarized Continuum Model (PCM) was used. In this model, the solvent cavity is defined as a series of interlocking spheres. 3. Results and discussion 3.1. Fluorescence quenching The quenching of the intrinsic fluorescence of tryptophan was monitored in order to study the interaction between the dioxo-Mo(VI) complex and proteins, and free-Trp. Fluorescence quenching plots are shown in Fig. 2. The onset and extent of fluorescence quenching varied. The fluorescence of BSA was quenched 83% at a complex concentration of 2.1 × 10−4 M, with negligible change when the concentration was raised to 2.5 × 10−4 M. At the same two concentrations, the fluorescence of HSA was quenched 55%. The fluorescence of lysozyme was quenched 69% at a complex concentration of 2.7 × 10−4 M, while the fluorescence of free-Trp was quenched 59% at a complex concentration of 2.1 × 10−4 M. At lower dioxo-Mo(VI) complex concentration (1.2 × 10−5 M) the fluorescence of BSA was quenched 14%, whereas the fluorescence of HSA and free-Trp was quenched by 3% and 2%, respectively. The fluorescence of lysozyme did not measurably quench until the complex concentration reached 3.8 × 10−5 M. The fluorescence quenching is due to the interaction of the dioxo-Mo(VI) complex with the Trp residue(s) of the protein and amino acid residues in the immediate vicinity in the protein. There are three factors that influence the quenching: (i) The hydrophobicity of the ligand, (ii) the charge of the complex, and (iii) the immediate environment of the Trp in the protein [3,4]. In all three proteins, the tryptophans are surrounded by both hydrophobic and partially hydrophobic residues such as valine (BSA, HSA and lysozyme), partially hydrophobic tyrosine (BSA and HSA), leucine and phenylalanine (BSA) and isoleucine (lysozyme) [3]. The Trp residues in BSA and lysozyme are also surrounded by negatively charged glutamine and polar uncharged amino acids such as serine (BSA and lysozyme) as well as glutamine and threonine (lysozyme). Given the presence of positively charged amines in the dioxoMo(VI) complex, the polar Mo O bonds and non-polar N-diethyl groups, a strong interaction between the Trp residues and its immediate environment is expected. It is worth noting that Wu and co-workers [6,29] reported a dramatic blue shift in the emission maxima in BSA and HSA as the concentration of their phenylfluorone-Mo(VI) complex was

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Fig. 2. Fluorescence spectra of 1.0 × 10−4 M of (a) bovine serum albumin (BSA), (b) human serum albumin (HSA), (c) lysozyme and (d) free tryptophan (Trp) in the presence of various concentrations of the dioxo-Mo(VI) complex at 25 ◦ C.

increased. They attributed this shift to the possible changes in protein conformation being induced by the complex. In this work, we did not see any shift in the emission maxima of BSA or HSA, though a very slight (∼5 nm) blue shift in lysozyme and free-Trp was observed.

3.2. Static and dynamic quenching Fluorescence quenching of a protein can be either dynamic or static. The linearity of the Stern–Volmer plot and the value of the bimolecular quenching constant kq are both used to determine the type of quenching [3,4,6,29]. kq is related to  0 (Eq. (2)) and  0 is relatively small, 10−8 –10−9 in the case of proteins. This leads to Stern–Volmer plots that curve upwards, indicative of static quenching (Fig. 3). Static quenching occurs as a result of the formation of a non-fluorescent ground state complex between fluorophore and quencher. The dynamic quenching mechanism results from diffusive encounters between fluorophore and quencher during the lifetime of the excited state. For dynamic quenching, the maximum value of the quenching constant is 2.0 × 1010 M−1 s−1 [6,32]. In this work, values of kq were on the order of 1011 –1012 M−1 s−1 suggesting that the nature of the quenching is static in all cases. The kq values (Table 1) are a little lower than the values reported in the literature (1012 –1013 M−1 s−1 ) [5,17,32]. We have previously reported values on the order of 1013 M−1 s−1 for a Cr(III) complex and values on the order of 1012 –1013 M−1 s−1 for various Ni(II) complexes [3,4]. Wu and co-workers [6,7,29] have reported kq values on the order of 1013 for BSA and HSA with their Mo(VI) complexes.

Fig. 3. Dynamic quenching plots for lysozyme (), free tryptophan (♦), bovine () and human (䊉) serum albumin at 25 ◦ C. F0 and F are the fluorescence of the protein or tryptophan in the absence and presence of the dioxo-Mo(VI) complex, respectively.

Fluorescence quenching plots based on Eq. (3) are shown in Fig. 4. These plots are linear with high R2 values. The binding constants, Ka , binding site numbers, n, and the calculated Gibbs energy, G, are shown in Table 1. BSA, HSA and free-Trp have

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Table 1 Summary of results for BSA (bovine serum albumin), HSA (human serum albumin), lysozyme and free tryptophan at 25 ◦ C. G is the change in Gibb’s free energy for the complexation process. Protein

Stern–Volmer constant, Ksv (M−1 )

Quenching constant, kq (M−1 s−1 )

Binding constant, Ka (M−1 )

Binding site, n

G (kJ/mol)

BSA HSA Lysozyme Tryptophan

2.8 × 10 5.2 × 103 8.4 × 103 6.6 × 103

2.9 × 10 5.3 × 1011 8.6 × 1011 6.7 × 1011

1.8 × 105 3.0 × 104 3.6 × 105 9.9 × 104

1.0 1.0 1.5 1.0

−30.0 −25.8 −33.6 −28.5

4

12

to the protein or free-Trp and forming a complex in the ground state. This complex is less fluorescent than the free proteins (or free-Trp) and the fluorescence intensity decreases as a function of complex concentration. This can be represented by the following equation: Fluorophore + Mo(VI)complex ↔ Non-fluorescent complex

(5)

As the concentration of the Mo(VI) complex is increased, the reaction is driven to the right and this results in high Ka values and negative G values, consistent with a product-favored reaction. Also, the stability of the non-fluorescence complex is temperaturedependent, as discussed in the next section. 3.3. van’t Hoff analysis

Fig. 4. Static quenching plots for lysozyme (), free tryptophan (♦), bovine () and human (䊉) serum albumin at 25 ◦ C. Best fits: bovine serum albumin (solid line, R2 = 0.9882), human serum albumin (dotted line, R2 = 0.9963), lysozyme (solid line, R2 = 0.9988) and free tryptophan (dashed line, R2 = 0.9792). F0 and F are the fluorescence of the protein or tryptophan in the absence and presence of the complex, respectively, and [Q] is the concentration of the dioxo-Mo(VI) complex.

one binding site for the complex, while lysozyme has a binding site number closer to 1.5, indicative of multiple binding sites. The presence of multiple binding sites is a possibility given that lysozyme has six tryptophans, with Trp-62, Trp-63, and Trp-108 located in the active site [19,20]. The binding constants are on the order of 104 –105 M−1 , with lysozyme showing the strongest binding (3.6 × 105 M−1 ) even though the onset of quenching occurred at a higher complex concentration when compared to the other protein systems. The corresponding negative G values indicate a highly spontaneous process under standard conditions. Wu and coworkers [6,7,29] have reported Ka values of 3.6 × 104 M−1 for HSA and 8.8 × 104 M−1 for BSA with their Mo(VI) complexes at 20 ◦ C. Surprisingly, free-Trp showed a slightly stronger affinity towards the dioxo-Mo complex than HSA. The oxo atoms on the dioxoMo(VI) complex are capable of H-bonding interactions with the indole N–H group, and the amine and carboxylic acid groups in tryptophan, thereby offering additional close contacts that are not available in the hydrophobic pocket in HSA. The possibility that H-bonding is one of the main contributors to complex formation is explored via a temperature-dependent (van’t Hoff) analysis that is discussed in the next section. Studies have shown that H-bonding can also result in fluorescence quenching [36,37]. The quenching of tryptophan fluorescence by a variety of protic solvents has been reported [37], where quenching is attributed to intermolecular photoinduced electron transfer [36]. The high binding constants (Ka , 104 –105 M−1 ), and the data from the dynamic quenching experiments confirms that static quenching is the predominant form of quenching in all of the protein-Mo(VI) complex systems. The Mo(VI) complex is binding

The binding constants for the three proteins and free-Trp with the dioxo-Mo(VI) complex were measured at different temperatures (Table 2). The binding constant, Ka , decreased appreciably for all four systems between 25 ◦ C and 55 ◦ C, with slight increases for HSA and free-Trp at 55 ◦ C. The decrease in the binding constant indicates that the protein–complex interactions are weakening at higher temperatures, resulting in a decrease in stability. This observation eliminates the dynamic quenching mechanism, since dynamic quenchers do not physically bind to the target. Thus the most probable quenching mechanism is initiated by protein–complex formation rather than by dynamic collision. The slight increase observed with HSA could be attributed to the onset of protein denaturation, which opens up the protein and makes other binding modes accessible to the complex. Protein denaturation typically occurs around 60 ◦ C with values in the 62–64 ◦ C range for albumins having been reported in the past [38]. In the case of free-Trp, many binding modes are available, and the complex may be reorienting itself due to the increase in thermal energy. It is interesting to note that at higher temperatures, the KSV values remains in the order of 104 for BSA and 103 for the other systems, whilst the quenching constant, kq , is on the order of 1011 –1012 . The latter value continues to be higher than the maximum range for dynamic quenching to occur, therefore fluorescence quenching can still be considered to follow the static mechanism at these higher temperatures. van’t Hoff plots were generated using Eq. (4), from which changes in enthalpy (H) and entropy (S) were calculated. These plots are given in Fig. 5, and pertinent data is collated in Table 2. The combination of high Ka values (104 –105 M−1 ), and negative H values clearly shows that the binding process is highly spontaneous and exothermic. The exothermic interaction is consistent with the idea that “bond formation” or binding results in the product being lower in energy. It also validates the previously stated observation that the binding weakens with increasing temperature. According to Le Chatelier’s principle, exothermic reactions favor reactants as the temperature increases. Negative values for both H and S mean that the interaction between the Mo(VI) complex and the proteins was a result of van der Waal’s forces and H-bonding [29]. Strong binding also decreases the disorder of a system, leading to very negative S values. Wu and co-workers [6,22] reported negative entropy values for HSA and a positive entropy value for BSA,

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Table 2 Binding constants, Ka , for bovine serum albumin (BSA), human serum albumin (HSA), lysozyme and free tryptophan (free-Trp) with the dioxo-Mo(VI) complex at different temperatures, along with experimentally determined values for changes in enthalpy (H) and entropy (S). Temperature (◦ C)

BSA

HSA

Lysozyme

Free-Trp

25 35 50 55 H (kJ/mol) S (J/mol K)

1.8 × 105 1.3 × 105 1.7 × 104 1.5 × 104 −75 −150

3.0 × 104 1.7 × 104 8.2 × 102 8.8 × 102 −108 −275

3.6 × 105 1.6 × 105 8.3 × 102 2.8 × 102 −209 −588

9.9 × 104 1.6 × 103 6.4 × 101 1.1 × 102 −190 −548

Fig. 5. Plots of the natural log of the binding constant (Ka ) versus reciprocal temperature (1/T) (van’t Hoff plots) for lysozyme (), free tryptophan (♦), bovine () and human (䊉) serum albumin. Best fits: bovine serum albumin (solid line, R2 = 0.9421), human serum albumin (dotted line, R2 = 0.9412), lysozyme (solid line, R2 = 0.9475) and free tryptophan (dashed line, R2 = 0.9201).

which they attributed to electrostatic gravitational contributions. Given the complex structure of proteins, many forces exist between the protein and the metal complex, and it is a combination of these attractive forces that drive the reaction forward. 3.4. Fluorescence resonance energy transfer (FRET) analysis The possibility that the fluorescence quenching is also a consequence of long-range energy transfer was explored. Forster resonance energy transfer (FRET) provides a high degree of specificity through the requirement of a spectral overlap between the donor emission and the acceptor absorption spectrum. The dioxoMo(VI) complex shows a weak absorption spectrum with poor overlap with the emission spectrum of each of the proteins and free-Trp (Fig. 6). Therefore, any Forster type long-range energy transfer (FRET) from the tryptophan residues to the dioxo-Mo(VI) complex is absent. This aspect is further confirmed by calculation of the Forster distances, R0 , which are found to be at or below the lower limit typically associated with FRET (Table 3) [4,6,29,32]. Table 3 Summary of the FRET analysis for the overlap between the absorption spectrum of the dioxo-Mo(VI) complex and the emission spectra of bovine serum albumin (BSA), human serum albumin (HSA), lysozyme and free tryptophan (free-Trp). Protein

Spectral overlap (J) L cm3 /mol

R0 (nm)

BSA HSA Lysozyme Free-Trp

5.41 × 10−15 5.24 × 10−15 3.14 × 10−16 2.27 × 10−15

2.15 2.14 1.17 1.84

Fig. 6. Overlap of the absorption spectrum of dioxo-Mo(VI) complex (dotted line) with the emission spectrum of bovine serum albumin or BSA (solid line, Excitation wavelength = 280 nm). Concentrations of both samples were 1.0 × 10−5 M. The absorption spectrum of the complex was calculated using the extinction coefficients obtained from a higher concentration.

Therefore, the dioxo-Mo(VI) complex is clearly quenching tryptophan fluorescence via a static quenching mechanism, with short (<2.0 nm) binding distances. Lab-based experiments alone cannot provide a complete description of the interactions observed, therefore theoretical quantum chemical calculations were performed with two specific goals in mind: To determine the most energetically favorable orientation of the dioxo-Mo(VI) complex with respect to free-Trp, and to determine the binding energy and approximate distance between tryptophan and the complex. 3.5. Quantum chemical calculations The gas phase binding energy for the dioxo-Mo(VI) complex with tryptophan when the indole N H group hydrogen bonds with ˚ the Mo O group in the complex is −35 kJ/mol, at a distance of 2.3 A. This shows very good agreement with the G values obtained using the Ka value of tryptophan at 25 ◦ C (G = −28 kJ/mol) and also from the van’t Hoff plot at 25 ◦ C (G = −27 kJ/mol). When the orientation of the dioxo-Mo(VI) complex is changed such that the Mo O group is interacting with the amine group in the amino acid, the binding energy increases significantly to −19 kJ/mol, but the dis˚ These two orientations with the tance remains unchanged at 2.3 A. indole N H and the amide group are shown in Fig. 7. The binding energy increases further when the solvation model is used (E = −12 kJ/mol), but the product is still lower in energy than the individual species. The addition of solvent (water) introduces numerous interactions that cannot be individually quantified but overall the calculations are in good agreement with experimental results in terms of predicting the energetics of binding and the spontaneity of complex formation. The only difference between

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Fig. 7. Optimized gas-phase calculations showing the orientations of the dioxo-Mo(VI) complex with tryptophan. Left: (a) interaction with the indole N H, right: (b) and the amide group. The dashed line in each illustration indicates the H-bond.

the gas-phase geometry and the solution-phase geometry is the distance between the Mo O group and the indole N H group. ˚ Calculations on the excited state This distance decreased to 1.99 A. showed no change in orientation but the distance decreased to ˚ indicating a strengthening of the interaction upon excita1.68 A, tion. A decrease in the length of hydrogen bonds has recently been reported for tryptophan-water clusters [39]. To further characterize the interactions and energetics of the binding between transition metal complexes and these globular proteins, theoretical molecular docking studies are currently underway. Recent studies have shown that the Rose Bengal molecule, which shows similar binding characteristics to transition metal complexes, can interact (dock) at a site that is less than 3 A˚ from Trp-212 in BSA with a binding energy of −26 kJ/mol [40]. Unpublished docking data from our own group also show similar binding energies between various small nickel complexes and the BSA protein. 4. Conclusions Fluorescence quenching studies show that a non-aromatic dioxo-Mo(VI) complex exhibits strong and favorable interactions with BSA, HSA, lysozyme, and free-Trp. The quenching mechanism is static with binding constants on the order of 104 –105 M−1 and the interactions are highly spontaneous and exothermic, with van der Waal and H-bonding forces predominating. Acknowledgement We thank Susquehanna University for financial support for this project. References [1] S.H. Laurie, D.E. Pratt, A spectroscopic study of nickel (II)-bovine serum albumin binding and reactivity, J. Inorg. Biochem. 28 (1986) 431–439. [2] S.H. Laurie, D.E. Pratt, Copper–albumin: what is its functional role? Biochem. Biophys. Res. Commun. 135 (1986) 1064–1068. [3] H.F. Crouse, J. Potoma, F. Nejrabi, D.L. Snyder, B.S. Chohan, S. Basu, Quenching of tryptophan fluorescence in various proteins by a series of small nickel complexes, Dalton Trans. 41 (2012) 2720–2731. [4] H.F. Crouse, E.M. Petrunak, A.M. Donovan, A.C. Merkle, B.L. Swartz, S. Basu, Static and dynamic quenching of tryptophan fluorescence in various proteins by a chromium (III) complex, Spectrosc. Lett. 44 (2011) 369–374. [5] H.Y. Shrivastava, B.U. Nair, Fluorescence resonance energy transfer from tryptophan to a chromium (III) complex accompanied by non-specific cleavage of albumin: a step forward towards the development of a novel photoprotease, J. Inorg. Biochem. 98 (2004) 991–994. [6] W. Dan, W. Qin, L. Yan, D. Bin, X. Guiying, Quenching of the intrinsic fluorescence of bovine serum albumin by phenylfluorone–Mo(VI) complex as a probe, Int. J. Biol. Macromol. 37 (2005) 69–72. [7] W. Qin, W. Dan, D. Bin, L. Yan, D. Caihong, Interaction of mnitrophenylfluorone–Mo(VI) complex as a probe with human serum albumin: a fluorescence quenching study, Spectrochim. Acta A: Mol. Biomol. Spectrosc. 63 (2006) 532–535.

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