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Site-Selective Interactions: Squaraine Dye-Serum Albumin Complexes with Enhanced Fluorescence and Triplet Yields Vadakkancheril S. Jisha, Kalliat T. Arun,† Mahesh Hariharan,‡ and Danaboyina Ramaiah* Photosciences and Photonics, Chemical Sciences and Technology DiVision, National Institute for Interdisciplinary Science and Technology (NIIST), CSIR, TriVandrum 695019, India ReceiVed: January 14, 2010; ReVised Manuscript ReceiVed: March 05, 2010
We report the effect of steric factors of a few squaraine dyes, bis(2,4,6-trihydroxyphenyl)squaraine (1), bis(3,5dibromo-2,4,6-trihydroxyphenyl)squaraine (2), and bis(3,5-diiodo-2,4,6-trihydroxyphenyl) squaraine (3), on their binding with human (HSA) and bovine (BSA) serum albumins employing photophysical, chiroptical, biophysical, and microscopic techniques. These dyes interact with serum albumins very efficiently and exhibit site selectivity, involving synergistic effects of hydrophobic, hydrogen bonding, and electrostatic interactions. The association constants of these complexes have been determined and are found to be 4.9 × 106 and 4.1 × 105 M-1, respectively, for the dyes 2 and 3 with BSA, while HSA showed relatively higher association constants of 6.0 × 106 and 9.9 × 105 M-1. Highly clear distinction in site-selective binding can be ascertained from time-resolved fluorescence, displacement cum fluorimetry, and circular dichroism (CD) studies. The increased affinity toward the major binding site (site II, domain III) over the relatively smaller binding site (site I, domain II) in the serum albumin with the increasing size of the heavy atoms present in 2 and 3 as compared to 1 indicates the importance of steric factors thereby confirming that the dye structure has a predominant role in deciding site selectivity. The distance between the energy donor and acceptor was calculated using Fo¨rster theory, which agrees well with the reported site 1 binding agent dansylamine. In contrast, no energy transfer was observed between tryptophan (Trp-214) present in domain II of the albumins and the dyes 2 and 3, indicating that these derivatives bind less efficiently at site I due to steric conatraints but preferentially bind at site II. Laser flash photolysis studies of the dyes 2 and 3 in the presence of HSA exhibited ca. 2.5-fold enhancements in the triplet lifetimes and quantum yields when compared to that obtained in buffer. The uniqueness of these dyes is that they show substituent size-dependent selectivity at site II of serum albumins and signal the event through “turn on” fluorescence intensity as well as enhanced triplet excited state lifetimes and quantum yields, thereby indicating their potential use as NIR noncovalent protein labeling and photodynamic therapeutic agents. Introduction Protein-ligand interactions are important in biological processes such as enzyme-substrate recognition, hormone action, signal transduction, and cell communication.1,2 Of all the proteins, serum albumin is found abundantly in the bloodstream and is principally characterized by its remarkable ability to bind and transport a wide range of endogenous and exogenous ligands like drugs, amino acids, fatty acids, bilirubin, bile acids, and thyroxine.3 Because of the ability of the serum albumins to interact with a wide variety of molecules, it is of current interest to exploit its various favorable properties for the development of novel therapeutic agents, drug delivery pharmacokinetics, and pharmacodynamic modulations.4,5 The specific physiological activity of the ligands upon complexation with serum albumin originates from the presence of two major and structurally selective binding sites, namely, site I and site II, which are located in three homologous domains that form a heart-shaped protein.6 The crystal structure of the * To whom correspondence should be addressed. Tel.: (+) 91 471 2515362. Fax: (+) 91 471 2491712. E-mail:
[email protected] or
[email protected]. † Current address: Department of Chemistry, Northwestern University, Illinois. ‡ Current address: Indian Institute of Science Education and Research, Trivandrum.
binding region has identified that site I is dominated by the presence of 16 hydrophobic residues including tryptophan (Try214), lysine (Lys-199), and histidine (His-242) which play an important role in the protein-ligand interactions. The most important amino acid residues of the IIIA subdomain (site II) binding site are tyrosine (Tyr-411) and arginine (Arg-410). The extensive studies on drug binding have identified that binding site I is dominated by strong hydrophobic interactions. The ligands bound at this site are located in the immediate vicinity of tryptophan (Trp-214), which can serve as an efficient energy donor. In contrast, the interactions at site II involve a combination of hydrophobic, hydrogen bonding, and electrostatic interactions.7,8 Of all the amino acid residues, tyrosine (Tyr411) is the most probable candidate for the complex formation at this site since its phenolic hydroxyl group can undergo effective hydrogen bonding with the bound ligands. Moreover, it has been reported that photosensitizers possessing higher affinity for serum albumin and showing preferential binding at site II were found to exhibit efficient photodynamic therapeutic (PDT) applications.9 However, only very few reports are available in the literature citing the selectivity of ligand binding at site II through various interactions such as steric, hydrogen bonding, and electrostatic interactions. In this context, the design of novel functional molecules that can undergo selective interactions with serum albumin exclusively at site II
10.1021/jp100369x 2010 American Chemical Society Published on Web 04/12/2010
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Figure 1. (A) Structural models indicating the variation in size of the squaraine dyes 1, 2, and 3 investigated in the present study. (B) Crystal structure of HSA showing the major ligand binding sites (Sudlow site I and Sudlow site II), and the structure was obtained from the Protein Data Bank (ID code 1ha2).
and exhibit efficient photodynamic activity has been challenging because such molecules can have potential use as drugs and sensitizers in PDT applications.10 Squaraines (SQ) form a class of novel dyes possessing sharp and intense absorption in the red to near-infrared region due to intramolecular charge transfer (ICT) interaction and hence are the object of intense investigations as molecular components of technological applications.11,12 These include electrophotography, optical data storage,11 solar cells,12 ion and molecular sensors,13-16 and nonlinear optics.17 These dyes exhibit ICT and are sensitive to their environment and give significant enhancement in their fluorescence quantum yields in the presence of membrane mimics like micelles, revealing their potential application as fluorescent sensors in biological applications.18 We have proposed earlier squaraine dyes as a possible new class of photosensitizers for PDT applications because of their highly favorable photophysical and photobiological properties.19 For example, the squaraine dyes, bis(2,4,6-trihydroxyphenyl)squaraine (1), bis(3,5-dibromo-2,4,6-trihydroxyphenyl)squaraine (2), and bis(3,5-diiodo-2,4,6-trihydroxy-phenyl)squaraine (3), based on the phloroglucinol moiety (Figure 1), exhibited high solubility in buffer and showed sharp and intense absorption in the region 580-620 nm. As compared to the unsubstituted dye 1, the heavy atom substituted squaraine dyes 2 and 3 exhibited significant phototoxicity in different cell lines, thereby indicating their potential as efficient sensitizers in PDT.20 In this context, it was our objective to evaluate the probable in vivo transportation pathways of the squaraine dyes 2 and 3 and investigate their interactions with human (HSA) and bovine serum albumins (BSA) when compared to the unhalogenated squaraine dye 1.21 Our results demonstrate that the halogenated squaraine dyes 2 and 3 exhibit high selectivity (>90%) toward site II of serum albumins with a high association constant (∼106 M-1) involving hydrogen bonding and electrostatic interactions in contrast to the marginal selectivity observed with the dye 1. Further, the microencapsulation of these dyes with serum albumins resulted in significant enhancement in fluorescence intensity and excited state triplet lifetimes and quantum yields, and hence these dyes can have potential PDT and immunological applications. Results Interactions of Squaraines with Serum Albumins. The absorption of the squaraine chromophore is very sensitive to the environment it experiences22,23 and can undergo large changes upon binding to protein due to the hydrophobic environment. The absorption spectra of squaraine dyes in the presence of both BSA and HSA showed large red shifts as well
Figure 2. ( A) Changes in the absorption spectra of the dye 1 (3.0 µM) with the addition of HSA in phosphate buffer. [HSA] (a) 0, (g) 1.5, (h) 1.8, and (m) 7 µM. Inset shows the visual detection of protein. (B) Changes in the absorption spectra of 2 (3.0 µM) with the addition of HSA in phosphate buffer. [HSA] (a) 0, (g) 0.19, (h) 0.28, and (m) 7 µM. Inset shows the half-reciprocal plot.
as hypochromism. For example, in the case of 1 the initial addition of HSA up to 1.5 µM led to the decrease in absorbance at 584 nm, corresponding to the squaraine chromophore (Figure 2A). However, the subsequent additions resulted in a gradual increase in absorbance but with the formation of a new band at 610 nm. The formation of the bathochromic-shifted band interestingly resulted in naked eye visualization of color change from pinkish-red (1 alone) to blue in the presence of HSA (inset of Figure 2A). Figure 2B shows the absorption changes of 2 with the addition of HSA. The initial addition of HSA (up to 0.19 µM) led to a decrease in absorbance at 610 nm, corresponding to the squaraine chromophore. However, the subsequent additions (0.19-7 µM) resulted in a gradual increase in absorbance and formation of a new band at 624 nm. Similar observations have been made for the dye 3 in the presence of HSA (Figure S1. Supporting Information). The observed red shift and hypochromicity indicate the hydrophobic environment surrounding the squaraine chromophore. Half-reciprocal analysis of the absorption data (inset of Figure 2B) gave a 1:1 stoichiometry for the complex between 2 and HSA, with an association constant of 6.0 × 106 M-1 and change in free energy of -38 kJ/mol. Table 1 summarizes the photophysical, binding constants, and changes in free energy of the dyes 1, 2, and 3 in the presence of HSA and BSA. Figure 3A shows the corresponding changes in fluorescence spectra of 1 with increasing concentration of HSA. Addition of HSA gave initially a gradual enhancement in fluorescence intensity, with a bathochromic shift in emission from 600 to 623 nm. The inset of Figure 3A shows the half-reciprocal analysis of the HSA-1 complex. Figure 3B shows the changes in the
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TABLE 1: Photophysical Properties of Squaraine Dyes 1-3 in the Presence and Absence of Serum Albumins HSA and BSA in Phosphate Buffera dye 1 1 1 2 2 2 3 3 3
+ HSA + BSA + HSA + BSA + HSA + BSA
λab, nm
λem, nm
ΦFb (10-2)
τ (ns)
KSA, M-1
-(∆G), kJ mol-1
584 610 610 610 624 624 613 628 628
600 623 620 625 640 643 634 646 645
0. 14 12 11 0. 018 2.5 2 0. 023 0. 65 0. 7
121 0.6 (60%), 1 (40%) 0.5 (60%), 1.5 (40%)
