Interaction of phenazinium-based photosensitizers with the ‘N’ and ‘B’ isoforms of human serum albumin: Effect of methyl substitution

Accepted Manuscript Interaction of phenazinium-based photosensitizers with the ‘N’ and ‘B’ isoforms of human serum albumin: Effect of methyl substitution

Swagata Sen, Riya Sett, Bijan K. Paul, Nikhil Guchhait PII: DOI: Reference:

S1011-1344(17)30665-6 doi: 10.1016/j.jphotobiol.2017.08.002 JPB 10937

To appear in:

Journal of Photochemistry & Photobiology, B: Biology

Received date: Revised date: Accepted date:

17 May 2017 31 July 2017 1 August 2017

Please cite this article as: Swagata Sen, Riya Sett, Bijan K. Paul, Nikhil Guchhait , Interaction of phenazinium-based photosensitizers with the ‘N’ and ‘B’ isoforms of human serum albumin: Effect of methyl substitution, Journal of Photochemistry & Photobiology, B: Biology (2017), doi: 10.1016/j.jphotobiol.2017.08.002

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ACCEPTED MANUSCRIPT Interaction of Phenazinium-Based Photosensitizers with the ‘N’ and ‘B’ Isoforms of Human Serum Albumin: Effect of Methyl Substitution

Swagata Sen†, Riya Sett†, Bijan K. Paul†,*, Nikhil Guchhait* Department of Chemistry, University of Calcutta, Kolkata 700 009, India Equal contribution.

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*Corresponding authors: [email protected] (BKP), [email protected] (NG).

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ACCEPTED MANUSCRIPT Abstract The present work is focused on exploring the interaction of two phenazinium-based biological photosensitizers, phenosafranin (PSF) and safranin-O (SO), with human serum albumin (HSA), with particular emphasis on the physiologically significant N-B conformational transition of the

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protein on the dye:HSA interaction. In addition, the presence of methyl substitution on the planar

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phenazinium ring in SO paves way for looking into the effect of simple chemical manipulation

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(that is, methyl substitution on the dye nucleus) on the dye:protein interaction behavior as a

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function of various (pH-induced) isoforms of HSA. Our results reveal a significantly stronger binding interaction of SO with the B isoform of HSA (at pH 9.0) compared to that with the N

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isoform (at pH 7.4). On the contrary, the PSF:HSA interaction is found to be reasonably insensitive to the aforesaid conformational transition of HSA. However, the probable binding

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location of both the dye molecules (PSF and SO) is found to be within the protein scaffolds

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(domain IB). This is further quantified from the modulation of fluorescence decay behavior of

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the dyes within the protein scaffolds. It is important to note that the rotational relaxation behavior of the protein-bound dyes reveals an unusual ‘dip-rise-dip’, an observation not reported earlier.

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Such unusual anisotropy decay is meticulously analyzed by an associated (or multicomponent)

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exponential decay model which emphasizes on the fractional contributions from differential classes of fluorophore populations characterized by the fast (due to unbound or solvent exposed part of the fluorophore) and slow (due to embedded or bound part) motions, in combination with their different local mobilities. Furthermore, the translational diffusion of the dye molecules in the presence of the protein in different isoforms (N-form or B-form) at a single molecule level is also measured by Fluorescence Correlation Spectroscopy (FCS).

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ACCEPTED MANUSCRIPT Keywords: Phenazinium dyes; Human serum albumin; ‘N’ isoform; ‘B’ isoform; Differential association constants; Dip-rise-dip anisotropy decay; Fluorescence Correlation Spectroscopy. 1. Introduction Human Serum Albumin (HSA) is the major transport protein of circulatory system comprised of 585 amino acids [1]. HSA is asymmetric in structure composed of three domains

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(domain I, II and III) with the principal dye binding sites located in hydrophobic subdomains IIA

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and IIIB [1]. HSA undergoes different pH-dependent conformational transitions, e.g., the N-F

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transition between pH 5.0 and 3.5, the F-E transition (or acid expansion) between pH 3.5 and 1.2,

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and the N-B transition between pH 7.0 and 9.0 [1-4]. The N-B transition is argued to have particular physiological importance with a view to the predominance of the B-isoform in blood

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plasma under enhanced Ca2+ ion concentration [5-7]. Besides, this transition (or analogous mechanism) is believed to play crucial regulatory role underlying the transport function of HSA

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[6-10], which in turn underscores the enormous physiological importance associated with the N-

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B structural transition of HSA [11]. Thus, any modulation of the binding affinity of a given dye

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with various isoforms of HSA may critically influence the dye distribution profile within the body which in consequence may alter the dose-response relationship as well as the rate of

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excretion from the body [12-14]. Naturally, quantitative knowledge of the differential interaction

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behaviors of a dye with various isoforms of HSA (particularly accompanying the N-B transition) may be critical to complement the optimization of ADME (adsorption-distribution-metabolismexcretion) profile of a given dye. Though in general the blood pH is stable, differences in blood pH are known in comparison to cerebral blood flow, and extracellular and intracellular milieus which offer prospective avenues for the occurrence of dye(s):HSA interaction [15,16]. To this end, the present work is designed to focus on deciphering the interaction behaviors of Phenosafranin (PSF, 3,7-diamino-5-phenyl phenazinium chloride) and Safranin-O

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ACCEPTED MANUSCRIPT (SO, 3,7-diamino-2,8-dimethyl-5-phenyl phenazinium chloride) with the two isoforms of HSA, namely, the N-form (at pH 7.4) and B-form (at pH 9.0). These phenazinium-based dye molecules (PSF and SO) find extensive applications as photosensitizers [17,18], biological probes for estimation of hyaluronic acid, study of DNA intercalation, protein immobilization, membrane organization, inhibition of human ribonuclease reductase and so on [19-23]. The dye molecules

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(PSF and SO) are structurally analogous barring the degree of methyl substitution on the planar

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phenazinium nucleus (Scheme 1), which in turn paves way for looking into the effect of simple

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chemical manipulation (that is, methyl substitution on the dye nucleus) on the dye:protein

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interaction behavior as a function of various (pH-induced) isoforms of the transport protein, HSA.

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Although the interaction of PSF with HSA has been reported in the literature [24,25], the present work emphasizes on various aspects of the overall interaction scenario which remain

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hitherto unexplored, e.g., the effect of protein conformational transition (N-B transition) on the

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binding affinity of the dyes with HSA. Concurrently, the study also reveals the effect of methyl

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substitution (PSF and SO differs in the degree of methyl substitution on the planar phenazinium ring, Scheme 1) on the binding affinity of the dyes leading to differential association constants of

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PSF and SO with various (N-form and B-form) isoforms of HSA. This is further corroborated

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from the differential fluorescence relaxation behaviors of the dyes with various isoforms of the protein and translational diffusional behaviors at a single molecule level as studied by Fluorescence Correlation Spectroscopy (FCS).

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Scheme 1: (a) Schematic and (b) Optimized (B3LYP/6-31++G(d,p)) Structures of PSF and

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SO.

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2. Experimental 2.1. Materials

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The dyes (PSF and SO, Scheme 1), and HSA (fatty acid free) were used as procured from

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Sigma-Aldrich Chemical Co., USA. The solvent 1,4-dioxane was obtained from Spectrochem, India (UV spectroscopy grade) and stored in the dark over molecular sieves (5.0 Å, E-Merck Ltd.). 10.0 mM phosphate buffer solution of desired pH (that is, pH 7.4 and 9.0) was prepared from the stock solution of phosphate buffer obtained from Sigma-Aldrich Chemical Co., USA by appropriate dilution in triply distilled deionized Milli pore water. 2.2. Instrumentation and Methods

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ACCEPTED MANUSCRIPT The absorption and fluorescence spectra were acquired on a Hitachi U-3501 UV-Vis spectrophotometer and Jasco FP-8500 fluorometer, respectively. All the spectroscopic measurements were performed using a low dye concentration (ca. 2.0 µM) in order to minimize inner-filter effects [26-28]. The fluorescence lifetime and depolarization decay profiles were obtained by Time-

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Correlated Single Photon Counting technique following excitation of the samples at λex = 450 nm. The circular dichroic (CD) spectra were obtained on a Jasco J-815 spectropolarimeter using

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a cylindrical cuvette (path-length = 0.1 cm) at 25 °C. The dynamic light scattering (DLS)

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measurements were carried out on a Malvern Nano-ZS instrument equipped with a 4 mW He-Ne laser having λ = 632.8 nm. The FCS measurements were carried out on a Confocal Laser

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Scanning Microscope system from Becker & Hickl DCS-120 equipped with an inverted optical

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microscope of Zeiss (Carl Zeiss, Germany) [29,30]. For docking simulation the native structure of HSA was obtained from the Protein Data Bank, PDB ID: 1AO6 [31]. The docking simulation

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was performed on the AutoDock 4.2 software package [32,33]. The three-dimensional structures

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of the dye molecules (PSF and SO) were prepared on AutoDock 4.2 [32] software utilizing the optimized geometries of PSF and SO (DFT//B3LYP/6-31++G(d,p)) as obtained from calculation

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on Gaussian 03W [34] suite of programs (Scheme 1). The PyMOL software package [35] was

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used for visualization of the docked conformations. A detailed description of the experimental methods and protocols is given in the Supporting Information. 3. Results and Discussion 3.1. Characterization of Various Conformational States of HSA The characterization of various conformational states of HSA is studied by (i) circular dichroism (CD) spectroscopy [1-4,16,24,27,28,36], (ii) intrinsic fluorescence profile of native

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ACCEPTED MANUSCRIPT HSA (at pH 7.4) [2-4,36], and (iii) dynamic light scattering (DLS) measurements. The results are presented in the Supporting Information. 3.2. Interaction of PSF and SO with HSA 3.2.1. Absorption Spectroscopic Studies. The absorption profile of the dyes (PSF and SO) is characterized by a broad unstructured band at ~ 520 nm in aqueous buffer (pH 7.4 and 9.0),

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which undergoes a subsequent decrease of absorbance coupled with a red-shift following

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interaction with the protein, Figure 1.

Figure 1: Modulation of absorption profile of (a) PSF and (b) SO following incremental addition of HSA in aqueous buffer of varying pH values as specified in the figure legends. Curves i ix correspond to [HSA] = 0, 12, 24, 36, 61, 121, 242, 364, 485 µM.

The red-shift in the absorption profile of the dyes with added HSA is suggestive of the lowering of polarity at the interaction site of the dyes within the protein scaffolds in comparison to that in bulk aqueous buffer [22-24,37-39]. Here, it could be interesting to note that the extent 7

ACCEPTED MANUSCRIPT of the red-shift for PSF (from ~ 520 nm in aqueous buffer to ~ 524 nm in the presence of HSA) is comparable in both the cases under investigation, that is, in aqueous buffer at pH 7.4 (N-form of HSA) as well as at pH 9.0 (B-form of HSA); whereas for SO the extent of red-shift for interaction with the B-form of HSA (from ~ 520 nm in aqueous buffer to ~ 530 nm in the presence of HSA) is relatively greater in comparison to that with the N-form of HSA (from ~

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520 nm in aqueous buffer to ~ 524 nm in the presence of HSA). This probably reflects a

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relatively stronger interaction of SO with the B-form of HSA compared to the N-form of the

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protein, while on the contrary the N-B conformational transition of the protein appears to have

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only nominal influence on the PSF:HSA binding interaction. This argument is further substantiated in the forthcoming sections.

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3.2.2. Fluorescence Spectroscopic Studies. The fluorescence profile of the dyes (PSF and SO) displays a broad unstructured band with maximum at ~ 585 nm in bulk aqueous buffer of pH 7.4

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and pH 9.0 [22-24,37-39]. The occurrence of the dye:HSA interaction is manifested through the

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marked modulation of the fluorescence properties of the dyes in terms of a significant blue-shift

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of the characteristic fluorescence maximum of the dyes to ~ 561 nm within the protein-bound state coupled with a remarkable fluorescence intensity enhancement as depicted in Figure 2. In

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parity with reported literature [22-24,37-39], the observed modulation of the fluorescence

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properties of the dyes following interaction with HSA can be ascribed to the lowering of polarity in the microenvironment surrounding the dye binding site within the protein. Furthermore, a significant increase of fluorescence intensity of the dye molecules may be argued to accompany reduction of nonradiative decay channels usually available in bulk aqueous buffer phase [24,25,39,40]. In this context, the differential extents of fluorescence intensity enhancement of the dye molecules (PSF and SO) following interaction with various isoforms of HSA is imperative note.

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ACCEPTED MANUSCRIPT For PSF:HSA interaction the extents of increase of fluorescence intensity of the dye (PSF) with added HSA are found to be comparable with the N and B-forms of the protein, Figure 2a. On the contrary, for SO:HSA interaction the extent of fluorescence intensity enhancement of the dye (SO) upon interaction with the B-form of HSA (at pH 9.0) is found to be significantly greater in comparison to that with the N-form of HSA (at pH 7.4) , Figure 2b. This is consistent with the

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aforesaid argument of a stronger interaction of SO with the B-form of HSA compared to the N-

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form, while the N-B conformational transition of the protein having only nominal influence on

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the PSF:HSA binding interaction.

Figure 2: Modulation of fluorescence profile (λex = 520 nm) of (a) PSF and (b) SO following incremental addition of HSA in aqueous buffer of varying pH values as specified in the figure legends. Curves i xv correspond to [HSA] = 0, 12, 20, 24, 36, 60, 120, 182, 242, 363, 485, 606, 727, 849, 970 µM.

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ACCEPTED MANUSCRIPT 3.2.3. Dye:Protein Interaction. The modulation of fluorescence intensity of the dye molecules (PSF and SO) following interaction with various isoforms of HSA is processed according to the Benesi-Hildebrand equation [24,39,41] in order to quantitatively assess the dye:HSA binding: (1) where, I0, I and I respectively represent the fluorescence intensities of the dye in the absence of

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HSA, at an intermediate concentration of HSA, and at saturation of interaction. The K term

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denotes the dye:HSA association constant. The plots of 1/(I – I0) versus 1/[HSA] represent a straight line in all the cases under investigation (Figure 3) conforming to a 1:1 stoichiometry for

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the dye:HSA interactions studied here.

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Figure 3: Double reciprocal plot of 1/[I - I0] versus 1/[HSA] (in mM-1) for estimation of dye:HSA association constant for interaction of (a) PSF with HSA in aqueous buffer of pH 7.4 (□) and pH 9.0 (■), and (b) SO with HSA in aqueous buffer of pH 7.4 (○) and pH 9.0 (●).

However, it is intriguing to note that the interaction of the dye PSF with various isoforms of HSA yields comparable magnitudes of the PSF:HSA association constant: KPSF:HSA = (1.24 ± 0.12) × 103 M-1 at pH 7.4 (N-form of HSA) and KPSF:HSA = (1.17 ± 0.12) × 103 M-1 at pH 9.0 (Bform of HSA). On the contrary, the SO:HSA interaction is found to yield a significantly higher magnitude of the association constant (~ 5.13 times higher) for interaction with the B-form of HSA compared to the N-form, KSO:HSA = (1.15 ± 0.02) × 103 M-1 at pH 7.4 (N-form of HSA) and

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ACCEPTED MANUSCRIPT KSO:HSA = (5.9 ± 0.5) × 103 M-1 at pH 9.0 (B-form of HSA). This indicates a stronger interaction of SO with the B-form of HSA compared to the N-form, while the PSF:HSA interaction is reasonably insensitive to the N-B conformational transition of the protein. The corresponding free energy changes for the studied interaction processes are evaluated using the standard , and the as-calculated values are: ΔGPSF:HSA = -

thermodynamic relationship,

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17.65 kJ mol-1 at pH 7.4 (N-form of HSA) and ΔGPSF:HSA = - 17.5 kJ mol-1 at pH 9.0 (B-form of

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HSA), and ΔGSO:HSA = - 17.46 kJ mol-1 at pH 7.4 (N-form of HSA) and ΔGSO:HSA = - 21.51 kJ

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mol-1 at pH 9.0 (B-form of HSA). A negative free energy change (ΔG < 0) indicates the

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spontaneity of the dye:HSA interaction processes under investigation. However, it is important to realize at this stage that differential binding forces underlying the interaction of the dyes (PSF

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and SO) with various conformational states of the protein (N-form or B-form) could be playing

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an important role here [42,43]. A conclusive statement to this effect would require a precise evaluation of the thermodynamic parameters (such as, enthalpy change and entropy change)

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governing the interaction of the dyes (PSF and SO) with HSA (N-form and B-form) and thus we

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refrain from making any comment on the matter at this stage as the literature reports diverging results regarding the evaluation of the concerned thermodynamic parameters depending on the

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experimental method used. We believe that this important issue should be extensively addressed

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in order to be able to make a unanimous conclusion and hence work along this direction is still underway in our laboratory. 3.2.4. Polarity at the Dye Binding Site. The polarity sensitive fluorescence behavior of the dyes (PSF and SO) is exploited here to implement a quantitative assessment of the micropolarity in the vicinity of the dye binding site within the protein. The characteristic fluorescence wavelength of the dyes (PSF and SO) at λfl. ~ 585 nm in aqueous medium undergoes a significant blue-shift with decreasing solvent polarity (e.g., λfl. ~ 564 nm in 1,4-dioxane), Figure 4. To this effect, the

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ACCEPTED MANUSCRIPT fluorescence behavior of the dyes (PSF and SO) is meticulously monitored in a reference solvent mixture of water and 1,4-dioxane of known polarity (on ET(30) scale [44]) and the characteristic fluorescence wavelengths are plotted as function of the polarity index, ET(30) to construct a calibration curve (Figure 4). The polarity of the dye microenvironment within the protein scaffolds is then evaluated by extrapolation of the characteristic fluorescence wavelength of the

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HSA-bound dye on the calibration curve (Figure 4). A marked reduction of polarity of the dye

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microenvironment within the protein-bound state (ET(30) = 46.3 kcal mol-1) in comparison to

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that in bulk aqueous buffer phase (ET(30) = 63.1 kcal mol-1 [44]) comprehensively suggests

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binding of the dyes in considerably hydrophobic region within the protein scaffolds [39]. It is intriguing to note that neither of the dye molecules (PSF or SO) displays a

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discernible difference in the micropolarity at the binding site as a function of the conformational state (N or B isoform of HSA) of the protein. This indicates equivalent binding location of both

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the dye molecules in the N as well as B-form of HSA. This is further corroborated from the

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elucidation of the dye binding location from docking simulation which indicates binding of the

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dye molecules (PSF and SO) in domain IB of the native protein (N-form of HSA). This is further

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elaborated in a forthcoming section.

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Figure 4: Modulation of fluorescence profile (λex = 520 nm) of (a) PSF and (b) SO in reference solvent mixture of water and 1,4-dioxane. Curves i x correspond to increasing volume proportion of 1,4dioxane, that is, 0, 10, 20, 30, 40, 50, 60, 70, 80 and 90% of 1,4-dioxane (v/v). The right panels display the variation of fluorescence wavelength (λem in nm) of the respective dye molecules as a function of solvent polarity equivalent parameter (ET(30) in kcal mol-1). The estimated polarity values of the proteinbound dyes are also indicated on the respective calibration curves (PSF in HSA: □ and SO in HSA: ○).

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3.2.5. Time-Resolved Fluorescence Decay. Figure 5 represents the time-resolved fluorescence

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decay traces of the dyes (PSF and SO) in bulk aqueous buffer and their subsequent modulation

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Table S1 of the Supporting Information.

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following gradual addition of the protein. The deconvoluted decay parameters are comprised in

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Figure 5: Modulation of fluorescence decay traces (λex = 450 nm) of (a) PSF and (b) SO following incremental addition of HSA in aqueous buffer of varying pH values as specified in the figure legends. Curves i ix correspond to [HSA] = 0, 12, 36, 60, 178, 347, 456, 664, 860 µM. The solid lines are the best fit lines to the experimental data points. The sharp black profile on the extreme left in each panel designates the instrument response functions (IRF).

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The fluorescence decay profile of the dyes in bulk aqueous buffer is characterized by a

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monoexponential decay pattern (the characteristic lifetimes are: for PSF τ1 = 868 ps (at pH 7.4)

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and τ1 = 820 ps (at pH 9.0) and for SO τ1 = 1.02 ns (at pH 7.4) and τ1 = 1.29 ns (at pH 9.0)). This

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is in good agreement with earlier reports on the decay behavior of the dye molecules in bulk

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ACCEPTED MANUSCRIPT medium [24,38]. The data presented in Table S1 show that the fluorescence decay traces of the dyes in the presence of the protein require a complex triexponential decay function for an adequate deconvolution, in which the appearance of a slow (τ2) and an ultrafast (τ3) decay component reveals the occurrence of the dye:HSA binding interaction, while the decay component τ1 is reasonably attributed to the contribution from the free (or unbound) fraction of

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the dye molecules on the basis of its resemblance to the characteristic lifetimes of the respective

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dye molecules in bulk aqueous buffer. Thus, a careful perusal of the fluorescence decay

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parameters (Table S1) enabled a legitimate estimation of the variation of the fraction of the dye

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molecules bound to the protein. A gradual decrease of the relative amplitude (α1) corresponding to the free (or unbound) fraction of the dye molecules with incremental addition of the protein

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can be correlated with the occurrence of progressive binding of the dyes with the protein, that is, progressive increase of the population of the bound dyes. The variation of relative amplitudes

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molecules as a function of HSA concentration under various experimental conditions is displayed in Figure 6. 19

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Figure 6: Variation of relative amplitudes (α1: ○, α2: ●, α3: ▲) corresponding to the fluorescence decay components of (a) PSF and (b) SO as a function of HSA concentration in aqueous buffer of varying pH values as specified in the figure legends. The solid lines provide only visual guide.

In this context, it is imperative to note that for interaction of PSF with HSA the relative

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populations (that is, α1) of the free (or unbound) dye at the maximum concentration of the protein used in the study are strikingly similar at pH 7.4 (N-form of HSA, α1 = 0.45) and pH 9.0 (B-form

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of HSA, α1 = 0.46). On the contrary, the variation of relative amplitudes (αi) for interaction of

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SO with HSA indicate a noticeably grater population of the free (or unbound) dye at pH 7.4 (Nform of HSA, α1 = 0.46) compared to that at pH 9.0 (B-form of HSA, α1 = 0.18), Table S1. This is found to be in good harmony with the steady-state spectroscopic results stated earlier in the sense that the interaction of SO with the B-form of HSA is relatively stronger in comparison to the N-form, while the PSF:HSA interaction is reasonably insensitive to the N-B conformational transition of HSA. In general, a steady increase of the fluorescence lifetime values (Table S1)

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ACCEPTED MANUSCRIPT with gradual addition of HSA suggests the impartation of increasing degree of motional restrictions on the dye molecules [23,24,26-28,39,40,45]. 3.2.6. Rotational Relaxation Dynamics. The fluorescence depolarization traces of the dyes (PSF and SO) in bulk aqueous buffer and their modulations with subsequent addition of HSA are depicted in Figure 7. In bulk aqueous buffer, the fluorescence depolarization of the dyes

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corresponds to a fast rotational relaxation dynamics having the following rotational correlation

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time constants: τr = 147 ps for PSF in aqueous buffer of pH 7.4, τr = 160 ps for PSF in aqueous

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buffer of pH 9.0, and τr = 179 ps for SO in aqueous buffer of pH 7.4, τr = 190 ps for SO in

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aqueous buffer of pH 9.0. This is consistent with the notion of a homogeneous environment surrounding the dye molecules in bulk aqueous buffer phase. Moreover, an ultrafast nature of the

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fluorescence anisotropy decay behavior of the dyes (faster rotational correlation time (τr) than the fluorescence lifetime (τ1, Table S1)) corresponds to an essentially completed fluorescence

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depolarization within the excited-state lifetime [26,27,40,45,46]. This is further corroborated

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from the lack of residual anisotropy in the fluorescence anisotropy decay traces of the dye

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molecules in bulk aqueous buffer (Figure 7). At this stage, it is interesting to note that the fluorescence depolarization traces of the

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dyes display remarkable modification with gradual addition of the protein, as illustrated by the

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“dip-rise-dip” anisotropy decay profiles (Figure 7). Such unusual dip-rise-dip fluorescence depolarization behavior is argued to manifest the signature of coexistence of differential fluorophore populations typically varying in their excited-state lifetimes and rotational correlation times [26,27,40,46-48]. In general, a dip-rise-dip anisotropy decay profile is connected to the presence of a fluorophore population having a fast lifetime as well as a fast rotational correlation time together with another class of fluorophore population characterized by significantly slow fluorescence lifetime/rotational correlation time [26,27,40,46-48].

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Figure 7: Time-resolved fluorescence anisotropy decay traces (λex = 450 nm) of (a) PSF and (b) SO as a function of HSA concentration in aqueous buffer of varying pH values as specified in the figure legends. The HSA concentrations are indicated in the figure legends ([HSA] = 0, ——; 36 µM, ——; 180 µM, ——; 363 µM, ——; 485 µM, ——).

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This type of fluorescence depolarization profiles are usually described in terms of an associated (or multicomponent) anisotropy decay model suggested by Ludescher et al. [49] in

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which the total fluorescence decay parameters (excited-state lifetime and amplitude) are

where,

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explicitly connected with individual anisotropy decay parameters as follows: (2)

(3) (4) where, IT(t) represents the total fluorescence intensity decay in which αi is the normalized preexponential factor (amplitude) of the ith lifetime component, that is, τi. Similarly, τir denotes the 22

ACCEPTED MANUSCRIPT ith rotational correlation time, and r(0) is the pre-rotational anisotropy. However, fi(t) probably represents the most crucial parameter in the above description of dip-rise-dip fluorescence depolarization, that is, the time-dependent weighting factor accounting for the coexistence of differential fluorophore populations significantly differing in their characteristic time constants, that is, the rotational correlation time and lifetime [26,27,40,46-49]. The simulated anisotropy

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decay profiles based on the aforementioned equations are presented in Figure 8. Essentially,

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according to the aforesaid description of dip-rise-dip fluorescence depolarization the origin of the

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fast and slow time constants is ascribed to the unbound or solvent exposed moieties of the

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fluorophore and the bound part of the fluorophore, respectively [26,27,40,46-49]. It is imperative to note that the fluorescence decay parameters (Table S1) of the dyes (PSF and SO) within the

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protein-bound state fruitfully meet the important prerequisites in this effect, conforming to the coexistence of slow and ultrafast decay components having varying amplitudes. The origin of the

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dip-rise-dip fluorescence anisotropy decay is believed to emanate from the varying contributions

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to the total fluorescence intensity decay (IT(t)) from the differential classes of fluorophore

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populations characterized by the fast (due to unbound or solvent exposed part of the fluorophore) and slow (due to bound part) motions, in combination with the distinct local mobilities of the

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differential classes of fluorophore populations [26,27,40,46-49].

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The simulated rotational relaxation parameters based on equations (2) - (4) are collected in Table 1. The simulated anisotropy decay parameters reveal that the relative weighting factor (f3) corresponding to the ultrafast rotational relaxation time (τ3r), that is, the weighting factor (f3) conforming to the solvent exposed part of the fluorophore progressively decreases with incremental addition of HSA. This in turn reflects that the relative fraction of the HSA-bound (or solvent exposed) part of the fluorophore gradually increases (or decreases) with increasing HSA concentration, and thus explicitly demonstrating an enhanced degree of binding of the dye(s). A

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ACCEPTED MANUSCRIPT steady increase in the average rotational correlation time (τr, Table 1) of the dye molecules with increasing protein concentration further substantiates this argument in terms increasing degree of rotational constraints imposed on the dye molecules [26,46]. This is also consistent with the observation that the prominence of the dip-rise-dip decay gradually diminishes with increasing HSA concentration (Figure 7). Again it is seen that the decrease of the weighting factor (f3)

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corresponding to the solvent exposed part of the fluorophore with increasing HSA concentration

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(Table 1) occurs relatively smoothly for PSF (at pH 7.4 (N-form of HSA) as well as pH 9.0 (B-

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form of HSA); ~ 90% drop in the magnitude of f3 within the studied range of concentration of

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HSA) in comparison to SO, in which the changes are comparatively sharp in nature (~ 94% and ~ 96% drop in the magnitude of f3 within the studied range of concentration of HSA at pH 7.4

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and pH 9.0, respectively). This appears consistent with the earlier findings that the dye PSF

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exhibits no significant difference in binding affinity with the protein in various conformational states, whereas the binding affinity of SO with the B-form of HSA is greater than with the N-

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form. However, it is pertinent to state here that the simulated rotational relaxation data cannot be

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precisely compared with the binding affinity data as the simulation model does not exactly

be

attempted

here.

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can

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correspond to a binding model of the dyes with the protein, thus only a qualitative comparison

24

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Figure 8: Simulated anisotropy decay curves of (a) PSF and (b) SO as a function of HSA concentration in aqueous buffer of varying pH values as specified in the figure legends as constructed using equations 2 - 4. The HSA concentrations are indicated in the figure legends ([HSA] = 0, ——; 36 µM, ——; 180 µM, ——; 363 µM, ——; 485 µM, ——).

Table 1: Simulated Rotational Relaxation Decay Parameters of HSA-Bound PSF and SO

(µM)

τ1r (ns)

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[HSA]

τ2r (ns)

τ3r (ps)

f1

f2

f3

τr (ns)

PSF:HSA Interaction at pH 7.4 (N-form of HSA) 36

1.47

1.58

110

0.47

0.22

0.31

1.07

180

2.47

2.58

153

0.48

0.39

0.13

2.21

363

3.39

4.10

206

0.49

0.45

0.06

3.52

485

3.71

4.20

222

0.50

0.47

0.03

3.84

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ACCEPTED MANUSCRIPT PSF:HSA Interaction at pH 9.0 (B-form of HSA) 36

1.77

1.16

109

0.36

0.33

0.31

1.05

180

2.12

1.73

124

0.37

0.51

0.12

1.68

363

3.52

3.48

152

0.40

0.56

0.04

3.36

485

3.95

3.95

190

0.41

0.56

0.03

3.84

0.34

1.09

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1.59

1.59

110

0.39

0.27

180

2.12

2.14

134

0.24

0.39

0.37

1.39

363

3.38

3.62

154

0.45

0.50

0.05

3.34

485

3.98

4.21

195

0.47

0.51

0.02

4.02

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36

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SO:HSA Interaction at pH 7.4 (N-form of HSA)

36

1.83

2.12

106

0.29

0.52

1.02

180

2.60

3.75

145

0.26

0.35

0.39

2.04

363

3.65

4.22

190

0.46

0.50

0.04

3.79

485

4.72

5.12

250

0.48

0.51

0.01

4.88

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0.19

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SO:HSA Interaction at pH 9.0 (B-form of HSA)

3.2.7. Circular Dichroism (CD) Studies. The effect of dye binding on the protein

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conformation is investigated by far-UV circular dichroism (CD) spectroscopy. The far-UV CD profile of the native protein (N-form of HSA) is characterized by two minima at ~ 208 nm and ~

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222 nm showing the hallmark of a α-helix rich secondary structure, Figure 9a [3,4,24, 27,28,33,45,46]. The CD profile of native HSA is found to show a regular decrease of mean residue ellipticity (MRE) with no apparent shift of the characteristic minima following progressive addition of the dyes, PSF and SO (Figure 9a). The mean residue ellipticity (MRE) and the percentage of α-helicity of the protein are calculated from the observed ellipticity (θ) values using equations S3 and S4, as discussed in Section 3.1. This result indicates a slight

26

ACCEPTED MANUSCRIPT perturbation of the secondary structure of HSA upon interaction with the dyes (PSF and SO) as evident from the reduction in α-helicity content of the native protein (N-form of HSA, at pH 7.4) from ~ 66 (± 2) % in the absence of the dyes to ~ 60.3 (± 2) % in the presence of 50 µM PSF and ~57.8 (± 2) % in the presence of 50 µM SO (Figure 9a) [3,4,24,27,28,33,45,46]. The interaction of the dyes with the B-form of HSA (at pH 9.0) reflects a similar pattern

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of observation, that is, slight decrease of α-helicity content of the protein following incremental

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addition of the both the dyes (PSF and SO), Figure 9b.

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(a)

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(b)

Figure 9: Circular dichroism (CD) profiles of HSA with added PSF and SO in aqueous buffer of (a) pH 7.4 (N-form of HSA) and (b) pH 9.0 (B-form of HSA) as specified in the figure legends. MRE222 denotes the mean residue ellipticity at 222 nm. The changes in α-helicity (%) of the protein as a function of concentrations of the dyes are specified in the respective figure legends (typical error in calculation ~ ± 2%).

3.2.8. Fluorescence Correlation Spectroscopic (FCS) Studies. Figure 10 displays the normalized autocorrelation traces of the dye molecules (PSF and SO) in bulk aqueous buffer

27

ACCEPTED MANUSCRIPT phase as well as in the presence of incremental concentrations of the protein at pH 7.4 and pH

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9.0. The fitted results are collected in Table 2.

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Figure 10: Examples of normalized fluorescence autocorrelation traces of (a) PSF and (b) SO following incremental addition of HSA in aqueous buffer of varying pH values as specified in the figure legends. The HSA concentrations are [HSA] = 0 (i), 36 µM (ii) and 180 µM (iii). The concentration of the dyes is maintained at ~ 10 pM. The solid lines indicate the fitted curves.

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Fluorescence Correlation Spectroscopy (FCS) is a sensitive experimental technique to measure the translational diffusion of a fluorophore in a small confocal volume and thereby furnishing quantitative information regarding the dynamic microheterogeneity of the fluorophore environment. The translational diffusion coefficient of the dyes in bulk aqueous buffer at pH 7.4 (Dt = 23.81 ± 1.2 µm2 s-1 for PSF, and 22.43 ± 1.1 µm2 s-1 for SO) is found to progressively decrease with added HSA (at pH 7.4, that is, N-form of HSA), Table 2. This result can be rationalized on the basis of impartation of motional constraints on the dye molecules within the

28

ACCEPTED MANUSCRIPT protein-bound state [26,29,30,47,50,51]. A comparison of the data assembled in Table 2 further shows that the diminution of the translational diffusion coefficient (Dt) of SO at a given HSA concentration occurs to a greater extent for interaction with the B-form of the protein (at pH 9.0) compared to that with the N-form (at pH 7.4). On the contrary, the decrease in the magnitude of Dt with added HSA is strikingly comparable for the interaction of PSF with either conformation

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state (N- or B-form) of the protein. This is consistent with our earlier results of relatively

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stronger binding interaction of SO with the B-form of HSA (at pH 9.0) than the N-form (at pH

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7.4) as against a reasonable insensitivity of the binding interaction of PSF to the N-B

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conformational transition of HSA.

Table 2: Translational Diffusion Coefficient (Dt) Values of PSF and SO under Various Experimental Conditions as Obtained from FCS Measurements Interaction of PSF with HSA Dt (µm2 s-1)

29

ACCEPTED MANUSCRIPT pH 7.4

pH 9.0

[HSA] (µM)

(N-Form)

(B-Form)

0

23.81 ± 1.2

25.23 ± 1.3

36

13.47 ± 0.67

13.89 ± 0.69

180

7.15 ± 0.36

7.31 ± 0.36

360

5.86 ± 0.29

6.03 ± 0.30

Interaction of SO with HSA

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Dt (µm2 s-1) pH 7.4

pH 9.0

(N-Form)

0

22.43 ± 1.1

36

16.86 ± 0.84

180

8.51 ± 0.42

5.54 ± 0.28

360

7.86 ± 0.39

3.78 ± 0.19

(B-Form) 21.36 ± 1.1 11.23 ± 0.56

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[HSA] (µM)

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3.2.9. Molecular Modeling: Docking Simulation. The probable binding location of the

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dye molecules (PSF and SO) under investigation within the protein scaffolds is elucidated by AutoDock-based molecular modeling, which has been used extensively in the literature to

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unravel the probable binding location of dye/drug molecules within biomacromolecular assemblies [27,52-54]. To this effect, the blind docking approach is exercised which is argued to

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yield an unbiased result regarding the location of exogenous dyes/ligands within the biomacromolecule [27,32,33,55]. Figure 11 shows the minimum energy docked conformation of the dye molecules with the native protein (that is, N-form of HSA), which shows the negatively charged domain IB of HSA as the probable binding site of the cationic dye molecules. The protein residues in the vicinity of the dye molecules at the respective interaction sites are also shown in Figure 11. The protein residues surrounding (within 4.0 Å) the location of PSF are:

30

ACCEPTED MANUSCRIPT Tyr30, Leu31, Gln32, Gln33, Tyr84, Gly85, Glu86, Met87, His105, Lys106, Asp107 (of which, the polar residues are Glu86, Lys106, Asp107, Tyr30, Gln32, Gln33, Tyr84, His105); the protein residues surrounding (within 4.0 Å) the location of SO are: Arg114, Leu115, Arg117, Met123, Tyr138, Ile142, Arg145, His146, Phe157, Tyr161, Leu185, Arg186, Gly189, Lys190 (of which, the polar residues are Arg114, Arg117, Arg145, Arg186, Lys190, Tyr138, His146, Tyr161). A

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stabilizing binding interaction between the dye molecules and the protein is indicated by the

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negative binding energies as obtained from AutoDock-based docking simulation (EPSF:HSA = -

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5.89 kcal mol-1 and ESO:HSA = - 6.77 kcal mol-1, Table S2 in the Supporting Information). An

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energy decomposition analysis highlighting the major contributors to the stabilizing docking energy of the dye molecules with HSA (Table S2 of Supporting Information) shows a

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comparatively smaller contribution from electrostatic energy for docking of SO with HSA than PSF. Simultaneously, the contribution from van der Waals and desolvation energy is found to be

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slightly greater for docking of SO with HSA than PSF (Table S2). A slightly greater degree of

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stabilizing influence from van der Waals and desolvation energy component for SO may

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originate from the additional methyl substitutions on the planar phenazinium nucleus in SO

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which is rather absent in PSF.

31

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Figure 11: Minimum energy docked conformation of (a) PSF and (b) SO with HSA. The respective right panels display the protein residues in the vicinity (around 4.0 Ǻ) of the dye binding site.

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4. Conclusions

The key finding of the present investigation can be summarized as the effect of the N-B

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conformational transition of HSA on the dye (PSF and SO):HSA interaction behavior which reveals a significantly stronger (~ 5.13 times) binding of SO with the B-form of HSA (at pH 9.0) compared to that with the N-form (at pH 7.4). On the contrary, the aforesaid conformational transition of the protein is found to exert only nominal influence on the strength of binding of PSF with HSA. However, the binding stoichiometry of both the dyes (PSF and SO) is found to remain unaffected upon structural transition of the protein. With a view to the crucial regulatory role of the N-B conformational transition of HSA underlying the transport functionality of the 32

ACCEPTED MANUSCRIPT protein as well as predominance of the ‘B’ isoform under certain physiological conditions (e.g., enhanced Ca2+ ion concentration), our result of the modulation of the dye:HSA association constant as a function of the conformational isoform of HSA may be useful in providing effective guidelines toward understanding the modified dye distribution behavior within the body as well as its consequence on the dose-response relationship and rate of excretion (of dye) from

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the body. Furthermore, the comparison between two structurally analogous dye molecules, that

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is, PSF and SO, may provide a prospective conduit of looking into the effect of simple chemical

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manipulation on the dye nucleus in controlling important physiological functions, such as dye

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distribution.

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Acknowledgments

Research fellowships for SS and RS respectively from CSIR and UGC are gratefully

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acknowledged.

Abbreviations: HSA: Human serum albumin; PSF: Phenosafranin; SO: Safranin-O; CD:

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Circular dichroism; DLS: Dynamic light scattering; FCS: Fluorescence correlation spectroscopy;

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PDB: Protein data bank.

33

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Graphical abstract

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ACCEPTED MANUSCRIPT Research Highlights

Interaction of two phenazinium-based photosensitizers, PSF and SO with HSA is studied



N-B conformational transition of HSA influences binding of SO



The binding of PSF is insensitive to the N-B conformational transition of HSA



Unusual dip-rise-dip anisotropy profile describes the rotational relaxation



Single molecular investigation is carried out using FCS

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IP

T



41