Photophysical Behavior and Fluorescence Quenching of L-Tryptophan

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B: Liquids, Chemical and Dynamical Processes in Solution, Spectroscopy in Solution

Photophysical Behavior and Fluorescence Quenching of LTryptophan in Choline Chloride-Based Deep Eutectic Solvents Anu Kadyan, Shreya Juneja, and Siddharth Pandey J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.9b04659 • Publication Date (Web): 11 Aug 2019 Downloaded from pubs.acs.org on August 16, 2019

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The Journal of Physical Chemistry

Photophysical Behavior and Fluorescence Quenching of L-Tryptophan in Choline Chloride-Based Deep Eutectic Solvents

Anu Kadyan, Shreya Juneja, and Siddharth Pandey*

Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, 110016, India *To

whom correspondence should be addressed.

E-mail: [email protected], Phone: +91-11-26596503

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Abstract:

Intrinsic fluorescence from L-tryptophan (L-Trp) is routinely used to obtain

insights to the structural features and dynamics of proteins and enzymes. In contrast to aqueous enzymology, different parameters that control and influence the behavior of proteins and enzymes in non-aqueous media depend heavily on the solvent. Detailed analysis of the intrinsic fluorescence from L-Trp dissolved in two deep eutectic solvents (DESs), Reline and Glyceline, prepared by mixing salt choline chloride with H-bond donors urea and glycerol, respectively, in 1 : 2 molar ratio within 298.15–358.15 K temperature range is presented. Fluorescence emission maxima of L-Trp dissolved in DESs show bathochromic shift with increasing temperature. In comparison to water and several organic solvents, the fluorescence quantum yields of L-Trp in both the DESs are significantly higher. While the rates of non-radiative decay in the two DESs are comparable and increase with increasing temperature, radiative decay rates are independent of temperature and are higher in Glyceline in comparison to Reline resulting in higher fluorescence quantum yield of L-Trp in Glyceline. Excited-state emission intensity decays of L-Trp fit best to a double exponential model irrespective of the identity of the DES and the temperature. Average lifetime decreases with increasing temperature due to increased thermal deactivation, however, this decrease is much slower in DESs as compared to water. Both steady-state fluorescence anisotropy and rotational reorientation times for L-Trp are governed by the inherent complexity of the DESs as solubilizing milieu resulting in noncompliance to simple hydrodynamic treatment. Fluorescence quenching of L-Trp by acrylamide in Reline is purely dynamic in nature. This is in contrast to the aqueous media where the decrease in fluorescence is a combined result of both dynamic and static quenching. The quenching within Reline is fairly efficient considering the high viscosity of the medium. Significantly lower activation energy of the bimolecular quenching process as compared to the activation energy of the viscous flow indicates facilitation of the electron/charge transfer quenching of L-Trp by acrylamide within the ionic environment offered by the Reline. The 2 ACS Paragon Plus Environment

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effect of high viscosity is partly overcome by the strongly ionic environment of Reline during the electron/charge transfer between L-Trp and acrylamide. The results highlight the structural complexity of these DESs especially within the cybotactic region of the probe which is absent in the common molecular solvents of similar high viscosity.



Introduction Photophysical investigations have been carried out routinely to obtain key insights to various biological systems and processes. Fluorescence spectroscopy, as a multidimensional tool, has been widely utilized in several biochemical applications. Among various biopolymers (e.g., proteins, lipids, nucleic acids, carbohydrates), some of the proteins show unique and intrinsic fluorescence.1–3 The amino acid L-Trp along with its large size and being the most abundant intrinsic fluorophore with a concentration of about 1 mole% in proteins, offers significant spectroscopic properties.2 Fluorescence from indole ring in L-Trp is highly sensitive to the surrounding local environment, which in turn, has potential to afford key information regarding a protein.4–9 Interactions inside proteins leading to conformational transitions, substrate binding, subunit association and denaturation, among others, result in changes in the spectral response of L-Trp.2 It shows high anisotropy and sensitivity to collisional quenching that are usually sensitive to protein conformation and the extent of motion during the excited-state lifetime. Researchers in the past decades have been exploiting the photophysics of L-Trp residue(s) in proteins for examining protein conformational dynamics and function.3,10–12 However, this complex spectroscopic behavior of indole ring still remains a great challenge for the scientists. In 1957, Teale and Webber studied the fluorescence spectra of L-Trp in neutral water and reported the fluorescence maxima to be at 348 nm.13 Several experimental and computational techniques were employed to investigate the changes in fluorescence maxima, spectral width and quantum yield of L-Trp in different environment.1,2,8,13–16 Fluorescence decay of aqueous L-Trp at pH 7 was shown to follow biexponential decay by Rayner and Szabo.17 The effect of pH and temperature on the decay kinetics of aqueous L-Trp was also reported in detail.16,18 With the recent advances in employing enzymes in synthetic chemistry, investigation of proteins and enzymes in non-aqueous media has experienced an upsurge.19,20 4 ACS Paragon Plus Environment

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Non-aqueous enzymology offers potential advantages, such as, increased solubility of substrates, enhanced thermal stability, easier recovery of enzymes, and suppression of sidereactions involving water, etc.19,21,22 Since the emergence, green neoteric solvents mainly deep eutectic solvents (DESs) have attracted the attention of the research community as an alternative to organic solvents in various biochemical and biocatalysis applications.23–25 Therefore, understanding the photophysics of L-Trp in these solvents will not only offer a new alternative to aqueous media, but may also help in exploring protein behavior in extreme environments. DESs are prepared by simply mixing two (or more) non-toxic and inexpensive components, most commonly a hydrogen-bond donor (HBD, e.g., glycerol, urea) and a hydrogen-bond acceptor (HBA, a quaternary ammonium salt, such as, choline chloride).23 The interactions (mostly H-bonding) between these two components result in the depression of freezing point leading to the formation of DESs that are thermodynamically-stable liquids at room temperature.26 Several investigations on the application of DESs as media in various enzymatic activity, protein stability, biological preservations and in promoting amphiphile selfassembly of phospholipids and surfactants have been explored recently.27–31 Although, DESs have been demonstrated to offer potential non-aqueous environment where protein structure can be preserved, detailed understanding of the overall conformational and structural dynamics of proteins within DESs is still awiated.25,29 In order to fully understand protein behavior in DESs, it is essential to first study the properties of simple L-Trp unit. Towards this end, we present the first detailed investigation of the photophysical properties of L-Trp in two common and popular representative DESs - Reline and Glyceline over a temperature range of 298.15-358.15 K. Photophysical properties, such as, absorbance and emission maxima, steady-state anisotropy, fluorescence quantum yield, excited-state intensity and anisotropy decay, and their temperature dependence of L-Trp have potential to



offer key insights to the protein dynamics in these neoteric solvents. Further, we also report the outcomes of our investigation of fluorescence quenching of L-Trp within Reline by acrylamide, a well-established electron/charge acceptor quencher; acrylamide is often used to probe the protein conformation during the quenching of intrinsic L-Trp fluorescence.32–35

Experimental Section Materials. The DESs, Reline and Glyceline, were prepared by mixing choline chloride (ChCl, >99% from Sigma-Aldrich) with urea (>99% from Sigma-Aldrich), and glycerol ( ≥ 99.5%, spectrophotometric grade from Sigma-Aldrich), respectively, in a molar ratio 1 : 2 followed by stirring under heating (∼80 °C) until a homogeneous, colorless liquid was formed. Liquid Reline and Glyceline obtained under ambient conditions thus prepared were rigorously dried under vacuum for at least 24 h. A Karl-Fisher titrator was subsequently used to measure the water content of Reline and Glyceline prior to their use. DESs were dried further until the water content was found to be

Photophysical Behavior and Fluorescence Quenching of L-Tryptophan

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