Yuting Feng

Weiji Liu
email: liu.weiji@inrae.fr

Ruben Mercadé-Prieto

Xiao Dong Chen
email: xdchen@mail.suda.edu.cn

Dye-protein interactions between Rhodamine B and whey proteins that affect the photoproperties of the dye

Keywords: Rhodamine B, dye-protein interactions, alkali effect, fluorescence lifetime 1. Introduction Fluorescence-based techniques encompass different disciplines

to study varied scientific problems involving proteins. In some applications, such as protein characterization, non-covalent, extrinsic fluorescence dyes are frequently used due to their photo-sensitivities to the microenvironmental perturbations [START_REF] Groves | A method for the general identification of protein crystals in crystallization experiments using a noncovalent fluorescent dye[END_REF][START_REF] Lindgren | Detection and Characterization of Aggregates, Prefibrillar Amyloidogenic Oligomers, and Protofibrils Using Fluorescence Spectroscopy[END_REF][START_REF] Vetri | Amyloid fibrils formation and amorphous aggregation in concanavalin A[END_REF][START_REF] De | Fluorescence probing of albumin-surfactant interaction[END_REF]. The main underlying mechanisms are solvent relaxation and intramolecular charge transfer that can be affected by the environment or by the interactions between the dye and proteins [START_REF] Hawe | Extrinsic Fluorescent Dyes as Tools for Protein Characterization[END_REF][START_REF] Lakowicz | Principles of Fluorescence Spectroscopy[END_REF]. However, the photo-sensitivities of the dye can be unwelcome if the aim is to quantify the number of target proteins using the fluorescence intensity of the attached fluorophore [START_REF] Heilemann | Photoswitches: Key molecules for subdiffraction-resolution fluorescence imaging and molecular quantification[END_REF].

For example, molecular counting using quantitative fluorescence microscopy has received increasing interest in cell biology, wherein a fluorescent protein such as GFP (Green Fluorescent Protein) was genetically fused to the target protein [START_REF] Coffman | Counting protein molecules using quantitative fluorescence microscopy[END_REF]. In early studies, researches tended to use in vitro standards without considering the effect of the environment on the fluorescence intensity [START_REF] Chiu | Single-molecule measurements calibrate green fluorescent protein surface densities on transparent beads for use with 'knock-in' animals and other expression systems[END_REF][START_REF] Dundr | Quantitation of GFP-fusion proteins in single living cells[END_REF]. While many parameters including pH or halide ion concentrations were found to notably distort the results of quantitative experiments [START_REF] Chudakov | Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues[END_REF]. Consequently, it is important to understand the fluorescence properties of the fluorophore in the environment that closely mimics the desired system of study.

Quantitative fluorescence microscopy is also interested beyond the realm of life science. In food engineering, organic fluorophores are frequently used instead of GFPs.

Fluorescent labeling is accomplished by using a reactive derivative of the fluorophore that creates covalent links to the functional group of the proteins, such as amine or thiol groups [START_REF] Toseland | Fluorescent labeling and modification of proteins[END_REF][START_REF] Gonçalves | Fluorescent Labeling of Biomolecules with Organic Probes[END_REF][START_REF] Sahoo | Fluorescent labeling techniques in biomolecules: a flashback[END_REF]. One potential application of fluorescence microscopy in food science is to study the dairy fouling formed inside heat exchangers [START_REF] Sadeghinezhad | A Comprehensive Review of Milk Fouling on Heated Surfaces[END_REF]. However, in these scenarios, reliable quantitative results cannot be achieved as the fluorophore can target all proteins in the system of study [START_REF] Hagsten | Structural and compositional changes during UHT fouling removal-Possible mechanisms of the cleaning process[END_REF]. This results in the sophisticated microenvironment of the fluorophore so that the fluorescence properties of the fluorophore can differ significantly to its unconjugated state.

Rhodamine B (RhB) has been extensively used for the qualitative or quantitative purpose either in its free form, for example for thermometry or viscometry using its fluorescence intensity or lifetime [START_REF] Kitamura | Thermal Phase Transition of an Aqueous Poly(N-isopropylacrylamide) Solution in a Polymer Microchannel-Microheater Chip[END_REF][START_REF] Muller | Remote temperature measurements in femto-liter volumes using dual-focus-Fluorescence Correlation Spectroscopy[END_REF][START_REF] Paviolo | Temperature measurement in the microscopic regime: a comparison between fluorescence lifetime-and intensity-based methods[END_REF][START_REF] Benninger | Quantitative 3D mapping of fluidic temperatures within microchannel networks using fluorescence lifetime imaging[END_REF] or conjugated to macromolecules, such as proteins [START_REF] Chen | Fluorescent protein-dye conjugates : II. Gamma globulin conjugated with various dyes[END_REF][START_REF] Liu | Spatial quantification of hydrogels swelling using wide-field fluorescence microscopy[END_REF]. However, the fluorescence properties of RhB are well known to be affected by various environmental parameters, such as solvent polarity, viscosity or temperature [START_REF] Snare | The photophysics of rhodamine B[END_REF][START_REF] Casey | Effect of solvent polarity on nonradiative processes in xanthene dyes: Rhodamine B in normal alcohols[END_REF]. For example, higher temperature results in lower fluorescence intensity and shorter lifetime of RhB that facilitates the non-radiative process of its diethylamino groups, leading to a non-emissive TICT (twisted intramolecular charge transfer)-like state [START_REF] Vogel | Structural relaxation of rhodamine dyes with different N-substitution patterns: A study of fluorescence decay times and quantum yields[END_REF]. Whey proteins or polysaccharides are also reported to affect the fluorescent properties of RhB, as RhB lifetime increases at elevated protein or polysaccharide concentrations [START_REF] Mercade-Prieto | Fluorescence lifetime of Rhodamine B in aqueous solutions of polysaccharides and proteins as a function of viscosity and temperature[END_REF]. This increased lifetime cannot be explained by the macroscopic viscosity effect of the solutes, hence it is likely that the interactions between RhB and solutes (protein or polysaccharides) affect the lifetime of RhB [START_REF] Mercade-Prieto | Fluorescence lifetime of Rhodamine B in aqueous solutions of polysaccharides and proteins as a function of viscosity and temperature[END_REF].

Further evidence supporting the existence of dye-protein interactions between RhB and whey proteins is that the fluorescence intensity of RITC (Rhodamine B isothiocyanate) labeled whey protein conjugates decreases dramatically at high alkaline pHs even though free RhB is pH insensitive at that pH range [START_REF] Liu | Quantification of the Local Protein Content in Hydrogels Undergoing Swelling and Dissolution at Alkaline pH Using Fluorescence Microscopy, Food and Bioprocess Technology[END_REF]. It was hypothesized that the fluorescence intensity of the dye was enhanced due to non-covalent interactions between protein and the dye, which were broken down by the alkali, thereby decreasing the fluorescence intensity at higher pH conditions.

In the present paper, we are going to further investigate the dye-protein interactions between RhB and whey proteins both using fluorescence intensity and lifetime measurements. The latter is a more robust parameter that can reveal the microenvironmental changes of the dye without disturbance of dye concentration or photo-bleaching. Both fluorescence intensity and lifetime of RhB were investigated with the presence of whey proteins at various alkaline pH. RhB was studied either in its free form or when it was covalently linked to whey proteins. Alkali is expected to destroy the non-covalent interactions between RhB and whey proteins thus decrease both the fluorescence intensity and lifetime of the dye. At extreme alkaline conditions, the lifetime of RhB is expected to resemble the value for a free dye if all dye-protein interactions are cleaved.

Experimental procedure

Materials and protein labeling

The protocol to prepare RhB-labeled whey protein solutions is similar to that reported previously [START_REF] Liu | Spatial quantification of hydrogels swelling using wide-field fluorescence microscopy[END_REF]. Briefly, whey protein isolate (WPI; Davisco, USA) of 2 wt% was homogenized and mixed with 2% (v/v) Rhodamine B Isothiocyanate (RITC; Exciton, USA) stock solution (~20 mM). The pH of the solution was adjusted carefully to 9 by slowly adding 1 M NaOH (aq) to prevent protonation of the amine group and minimize the denaturation of the protein at the same time [START_REF] Taulier | Characterization of pH-induced transitions of β-lactoglobulin: ultrasonic, densimetric, and spectroscopic studies[END_REF]. The solution was held in the dark under mild stirring for at least 16.5 h to reach maximum labeling yield.

The unreacted RITC was then removed aseptically via dialysis using a 6-8 kDa MWCO membrane (Spectra Laboratories) against 0.02% (w/v) sodium azide solution.

The concentration of WPI and RITC inside the membrane was tracked by UV/Vis absorption at 280 nm and 555 nm, respectively (SpectraMax M5, Molecular Devices).

The dialysis process normally took 65 h at a final concentration of ~1.5 wt% WPI and ~0.2 mM RITC. The dye/protein molar ratio for this dialyzed solution was ~0.2 (molecular weight of β-lactoglobulin, 18.4 kDa, was used to estimate the molecular weight of WPI). A low dye-protein ratio was aimed here to minimize inter-label interactions [START_REF] Hungerford | Effect of the labelling ratio on the photophysics of fluorescein isothiocyanate (FITC) conjugated to bovine serum albumin[END_REF].

Fluorescence measurement

The fluorescence intensity measurements were performed in conventional wide-field fluorescence microscopy (WFM) as reported in our previous work [START_REF] Liu | Spatial quantification of hydrogels swelling using wide-field fluorescence microscopy[END_REF]. In short, the WFM system consists of a wide-field fluorescence microscope (MacroZoom Z16, Leica) and a 1.0× planapochromatic objective (magnification 7.13× -115×). The dye was excited at a peak wavelength of ~530 nm (520-550 nm band, LED4D067, Thorlabs); the emission was captured using a monochrome CCD camera (ICX285ALCCD, ToupTek, 1360×1024 pixels, chip size 6.45 µm) and recorded as 8-bit images.

The fluorescence intensity of 10 μM RhB was measured at various alkaline pH with the addition of two different native WPI concentrations (i.e. 1 wt% and 5 wt%) for one hour. In order to achieve the desired pH while controlling constant concentration of protein and dye in all solutions, the amount of added NaOH (aq) was calculated using the hydrogen ion equilibrium curve [START_REF] Zhao | Understanding the alkali cold gelation of whey proteins with NaCl and SDS[END_REF]. The fluorescence intensity was firstly measured at its initial state, without adding NaOH. After that, the solution was mixed for 30 s with the addition of NaOH; an aliquot was quickly removed to a petri dish for fluorescence measurements. In control tests, MilliQ water was added instead of alkali. The fluorescence intensity of the control group was taken as the "initial state" for other cases when adding NaOH to achieve an alkaline pH. After all the fluorescence measurements, the pH of the solutions was verified via a pH meter (FE22, Mettler Toledo), showing an accuracy of ± 0.1 pH.

Fluorescence lifetime measurement

The fluorescence lifetime of the dye was obtained using Fluorescence Lifetime Imaging Microscopy (FLIM) in a two-photon microscope (Leica SP8 CARS, Germany). RhB was excited at a wavelength of 840 nm from a picoEmerald laser (APE, Germany); the emission was captured through a 10 × objective (HCX PL Fluotar, Leica, NA 0.3, WD 11 mm) and a hybrid NDD detector (Leica HyD). FLIM was performed by connecting the HyD detector to a SPC-150 TCSPC module from Becker&Hickl (Germany). Mono-exponential decay analysis was conducted using the software SPCImage 6 (Supplemental Information Figure S1) from the same company (B&H). The emitted photons were collected typically in 20 s at a laser power of ~ 30 mW on the sample (PM100D, ThorLabs).

The fluorescence lifetime of free RhB (τRhB) or whey protein conjugated RhB (τWPI-RITC) was measured at different WPI concentrations and various alkaline pH. The quantities of added NaOH were also calculated using the hydrogen ion equilibrium curve to achieve same dye concentration for all solutions (i.e. 10 μM) so as to avoid the effect of dye concentration on the lifetime [START_REF] Kristoffersen | Testing Fluorescence Lifetime Standards using Two-Photon Excitation and Time-Domain Instrumentation: Rhodamine B, Coumarin 6 and Lucifer Yellow[END_REF]. In order to minimize the temperature effect on the lifetime measurements, all solutions were allowed to equilibrate to room temperature for at least one hour before any experiment was conducted. The temperature of the solutions was recorded using thermocouples and found to be 20 ±1 °C; this temperature perturbation corresponds to a lifetime variability at ~4% (Figure S2). The addition of alkali into the solution showed no effect on the solution temperature.

For lifetime measurements, the calculated amount of NaOH was quickly added into protein solutions and mixed for 30 s. An aliquot of the mixture was injected into a glass cuvette and well sealed prior to subsequent measurement. Each time, the lifetime of the sample was analyzed right after the imaging; the experiment was stopped until stable values were achieved. In each set of the experiment, MilliQ water was added instead of alkali for control tests. The lifetime of the dye in the control group was taken as the "initial state" for other cases when adding NaOH to reach an alkaline pH. Notice that in some cases when the pH and the protein concentration were both high enough, alkali cold gelation could happen quickly after the mixing with NaOH, while this gelation showed no effect on the lifetime measurements [START_REF] Zhao | Effect of N-Ethylmaleimide as a Blocker of Disulfide Crosslinks Formation on the Alkali-Cold Gelation of Whey Proteins[END_REF].

Tryptophan fluorescence of whey proteins

WPI solutions were prepared using 50 mM phosphate buffer (PBS) at different alkaline pH. Different amounts of RhB were added in order to achieve elevated RhB concentrations while maintaining constant protein concentration at 0.05 wt%. The fluorescence emissions of the solutions were recorded between 310 and 400 nm using a spectrophotometer upon excitation at 295 nm. Solutions were usually incubated at 25 °C or up to 55 °C for at least one hour before measurement. Prolonged heat treatment (80 °C, 24 h) was performed to obtain a fully denatured whey protein solutions [START_REF] Mahmoudi | Light-Scattering Study of the Structure of Aggregates and Gels Formed by Heat-Denatured Whey Protein Isolate and β-Lactoglobulin at Neutral pH[END_REF]. The effect of RhB on the tryptophan fluorescence of denatured WPI was also investigated at neutral pH and room temperature using the same procedure.

Note that the contribution of RhB on the total emission was verified to be less than 0.1%.

Rearrangement of RhB between aggregated and native proteins

Free RhB was mixed with native whey proteins to achieve a similar composition to the solutions before dialysis (Section 2.1). The mixtures were then heated at 80 °C for at least 24 h to induce fully denaturation of the proteins; the solutions were cooled down to room temperature prior to adding fresh native whey proteins to reach a final protein ratio between unheated and heated at 0 (by adding MilliQ water), 1, 2, and 3.

After that, they were incubated at room temperature for at least 4 hours before adjusting pH to 4.2 followed by centrifugation at 12000 rpm to separate the precipitated aggregates. Preliminary experiments showed that pH 4.2 was sufficient to separate the unheated and heated proteins, where it was possible to precipitate ≥ 80% of heated whey proteins and ≤ 10% of unheated WPI. After centrifugation, the precipitate was redissolved using 0.1 M NaOH (aq). The amount of RhB and WPI in both the supernatant and dissolved precipitates was monitored using UV/Vis spectroscopy. A similar protocol was applied for WPI-RITC.

Statistical Analysis

Sigmoidal regressions and the best-fitted parameters were calculated using the SigmaPlot v12.5 (Systat, Inc., USA). Welch t tests were applied to check the statistically difference between calculcated pK transitions.

Results and Discussions

Effect of protein concentration on RhB lifetime at neutral pH

Previous literature has reported that whey protein is able to increase the fluorescence intensity of RhB regardless if the dye is in free form or covalently linked to whey proteins (WPI-RITC) [START_REF] Liu | Spatial quantification of hydrogels swelling using wide-field fluorescence microscopy[END_REF]. In this paper, we further investigate this phenomenon by measuring the fluorescence lifetime of the dye as it is a preferred parameter to characterize the micro-environment of the fluorophore without disturbance from dye concentration, photobleaching or scattering effect [START_REF] Waters | Accuracy and precision in quantitative fluorescence microscopy[END_REF]. The fluorescence decay of RhB in water can be well fitted using a mono-exponential decay model as presented elsewhere [START_REF] Mercade-Prieto | Fluorescence lifetime of Rhodamine B in aqueous solutions of polysaccharides and proteins as a function of viscosity and temperature[END_REF]. The fluorescence lifetime of RhB (τRhB) alone was obtained at 1.7 ns, which is consistent with what has been reported at 1.74 ns in a 20 °C water solution [START_REF] Boens | Fluorescence Lifetime Standards for Time and Frequency Domain Fluorescence Spectroscopy[END_REF]. When covalently linked to whey proteins, the lifetime of RhB (τWPI-RITC) significantly increases to ~2.3 ns even though the protein concentration is lower than 0.1 wt%. This increase of lifetime of the dye after labeling with protein has long been recognized for fluorescein or rhodamine dyes, revealing a dramatic change of its molecular environment [START_REF] Chen | Fluorescent protein-dye conjugates : II. Gamma globulin conjugated with various dyes[END_REF][START_REF] Brismar | Spectra and fluorescence lifetimes of lissamine rhodamine, tetramethylrhodamine isothiocyanate, texas red, and cyanine 3.18 fluorophores: influences of some environmental factors recorded with a confocal laser scanning microscope[END_REF].

In the presence of additional WPI both τRhB and τWPI-RITC increase, eventually reaching a plateau value as shown in Figure 1. This increased lifetime of τRhB due to additional WPI agrees with the work reported by Mercade-Prieto, et al. [START_REF] Mercade-Prieto | Fluorescence lifetime of Rhodamine B in aqueous solutions of polysaccharides and proteins as a function of viscosity and temperature[END_REF] who discovered an increased τRhB at elevated concentrations of whey protein and polysaccharides and concluded that the increase of RhB lifetime cannot be explained with an increased macroscopic viscosity caused by higher protein or polysaccharides concentration. Consequently, both the increase of τRhB and τWPI-RITC is probably due to a larger extent of dye-protein interactions. It is important to note that, the fluorescence decay of RhB with the addition of WPI can also be successfully fitted by a mono-exponential decay model, suggesting there are no free RhB molecules when WPI is present (i.e. they are all bound to the protein, Fig. S3-4). An inspection of the τWPI-RITC values reveals a limited effect of protein concentration on protein conjugated RhB: τWPI-RITC quickly rises to the plateau at a relatively lower protein concentration and results with shorter lifetime value (i.e. 2.6 ns at 5 wt% WPI). This shorter lifetime implies less significant dye-protein interactions between WPI-RITC and proteins compared to that of free dye. This phenomenon is analogous to what has been found by Vieira Ferreira, et al. [START_REF] Vieira Ferreira | Ultraviolet/Visible Absorption, Luminescence, and X-ray Photoelectron Spectroscopic Studies of a Rhodamine Dye Covalently Bound to Microcrystalline Cellulose[END_REF] who reported a higher value of RhB lifetime when the dye was physically adsorbed onto microcrystalline cellulose (3.2 ns) than chemically linked to its polymer chain (2.5 ns). They deduced this by the formation of nonplanar conformer when the dye was covalently bound to cellulose while without labeling reaction, the dye is able to stable in a more planar form. In our case, the shorter τWPI-RITC can be the result of the blocking effect of the protein that RhB is linked to which inhibits the dye-protein interactions between the bound RhB to other proteins.

Effect of alkali on the fluorescence properties of RhB with WPI

Although RhB is pH insensitive in the tested alkaline range, it has been reported that the fluorescence intensity of WPI-RITC decreases in alkaline conditions [START_REF] Liu | Quantification of the Local Protein Content in Hydrogels Undergoing Swelling and Dissolution at Alkaline pH Using Fluorescence Microscopy, Food and Bioprocess Technology[END_REF]. It was hypothesized that the reduction of the fluorescence intensity of WPI-RITC is mainly caused by the break-down reactions of alkali on the dye-protein interactions between RhB and its linked protein. Therefore, one could imagine a similar effect of alkali to destroy the dye-protein interactions between free RhB and whey proteins thus quench the fluorescence intensity of the dye.

Figure 2(a) supports this idea by showing the fluorescence intensity evolution of free RhB (IRhB) right after the addition of NaOH with a whey protein concentration of 5 wt%. At all pH conditions tested (except neutral and extreme alkaline conditions such as pH 14), IRhB decreased immediately after the addition of alkali (within two minutes), and increase gradually with time. This fluctuation of fluorescence with time could be related to alkaline denaturation of whey proteins as a uniform pH of the solution can be achieved quickly [START_REF] Mercadé-Prieto | Caustic-induced gelation of beta-lactoglobulin[END_REF]. Because of this, only the time-stable data of fluorescence was used to quantify the effect of alkali on RhB fluorescence. The fluorescence intensity ratio α, defined as the ratio of fluorescence intensities obtained at alkaline conditions and neutral, was fitted using the sigmoidal expression following:

= 0 + ∆ I * ∆ I (eq.1)
where α0, ∆α, I * , ∆pI are fitting parameters listed in Table 1. The values of last measured α (60 min in this case) are plotted against pH in Figure 1(b). The fluorescence intensities decrease to lower ratio values at higher protein concentrations. This is reasonable as there are more dye-protein interactions in more concentrated solutions, leading to a higher reduction of IRhB. The calculated transitions I * shows independence on the protein concentration: 12.2±0.1 with 1 wt% WPI and 12.04±0.04 at a protein concentration of 5 wt%.

The fluorescence lifetime of free RhB (τRhB) after adding alkali with the presence of WPI is also recorded as shown in Figure 3. Unlike the fluorescence intensity, the lifetime of RhB shows an increasing trend at mild pH conditions (pH ≤12.5). At these pH, the τRhB keep increasing with time until reaching a maximum value, while the increment reduces as pH goes higher, resulting in a similar τRhB at pH 12.5 compared to that at neutral at a protein concentration of 1 wt% (Other protein concentration results see Figure S5). Because of this, τRhB can go even higher than the value at neutral pH, while with more concentrated whey protein solution such as 10 wt% as shown in Fig. 3 point and considered as time-stable. Our results confirm that the pH does not have a strong effect on free RhB, as τRhB changes little in all alkaline conditions tested (variability ≤1 %). The decrease of τRhB with alkali when proteins are present can be also well fitted using a four-parameter sigmoidal function as:

= 0 + ∆ τ * ∆ τ (eq.2)
where τ0, ∆τ, τ * , ∆pτ are fitting parameters as summarized in Table 1. In general, results from lifetime agree with that obtained from fluorescence: τRhB varies little at pH lower than 12 and have a sharp transition with a pK ~12 (i.e. τ * ,). These transitions are independent with protein concentration and show no statistical differences with those observed from fluorescence. These pK values are close to that observed for alkaline denaturation of heat-induced aggregated β-lactoglobulin (BLG), whereas a value of 10.6 was observed for unaggregated BLG [START_REF] Taulier | Characterization of pH-induced transitions of β-lactoglobulin: ultrasonic, densimetric, and spectroscopic studies[END_REF][START_REF] Mercadé-Prieto | The pH threshold in the dissolution of beta-lactoglobulin gels and aggregates in alkali[END_REF]. This pK shift was proposed to be caused by the non-covalent interactions between protein aggregates, hence it is hypothesized by analogy that similar non-covalent bonds between the protein and dye were destroyed by the alkali and cause the reduction of fluorescence intensity and lifetime. However, even at the extreme alkaline condition as pH 14, τRhB remains at relatively higher values compared to its unconjugated state (i.e. 1.7 ns), suggesting there are still abundant dye-protein interactions. 1); regression parameters are reported in Table 1.

Effect of alkali on the fluorescence properties of WPI-RITC with WPI

From the previous section, we have confirmed the capacity of alkali to destroy dye-protein interactions between free RhB and whey proteins. The apparent pK transitions are similar to those reported for WPI-RITC systems [START_REF] Liu | Quantification of the Local Protein Content in Hydrogels Undergoing Swelling and Dissolution at Alkaline pH Using Fluorescence Microscopy, Food and Bioprocess Technology[END_REF]. This consistency suggests similar non-covalent dye-protein interactions regardless of the dye is free or covalently linked to the protein. after mixing with NaOH with supplemented 5 wt% native WPI. IWPI-RITC decrease rapidly after the addition of alkali in a similar manner to IRhB, while the fluorescence stays more stable at higher pH ranges (pH ≥12.5). For comparison, the variations of WPI-RITC lifetime (τWPI-RITC) with time after adding NaOH are also shown in Figure 4(b). However, the lifetime results again disagree with what has been obtained from the intensity as τWPI-RITC are more stable at pH≤ 12.5 while keep decreasing at higher pH. Figure 4(c) summarizes the effect of alkaline pH on τWPI-RITC; data was also fitted using equation 2. The computed pK of the sigmoidal transition using τWPI-RITC are also close to 12 while slightly higher values were obtained with more concentrated protein conditions (~13).

As mentioned previously, if the lifetime of the dye is solely increased due to the dye-protein interactions, both τRhB or τWPI-RITC are expected to return to the value close to its free form (i.e. ~ 1.7 ns). Results showed in Fig. 2 to 4 confirm the hypothesis that the non-covalent bonds between RhB and whey proteins are capable to increase the fluorescence intensity and lifetime of the dye, however, none of them is back to the status at its free from. One exception can be seen for WPI-RITC when additional protein is absent, τWPI-RITC can reach ~1.8 ns at pH 14, which is just slightly higher than the expected value. In this case, as the effect of alkali on the dye can be excluded (τRhB does not change significantly at pH 14 as shown in Fig. 3), it can be concluded that most of the dye-protein interactions were cleaved at such condition. Another evidence showing the ability of alkali to destroy dye-protein interactions is that the increase of lifetime both for τRhB or τWPI-RITC due to the addition of whey proteins decrease at more alkaline pH (Figure S6).

In order to quantify the effect of alkali destroying dye-protein interactions, the value of free RhB lifetime in water (τw) was subtracted from τRhB or τWPI-RITC before normalizing to τw. These normalized values indicate the effect of dye-protein interactions on RhB lifetime, hence the decrease of the values should be related to the loss of dye-protein interactions that cleaved by alkali. One needs to take attention to the term "destroy" here, as the fluorescence decay of RhB can only be successfully fitted using a mono-exponential decay model in alkaline conditions tested (whatever free RhB or WPI linked ones), indicating that none of protein-bound RhB molecules was released to be free. If any reduction of dye-protein interactions corresponds to a release of free RhB molecules, a bi-exponential decay of RhB fluorescence should be found (i.e. there are two RhB lifetimes corresponding to two different RhB components). Results show a dramatically different impact of alkali on dye-protein interactions between free RhB system and conjugated dye one. For example, in WPI-RITC system as shown in Fig. 5(b), the normalized values keep decreasing at higher pH conditions, indicating a stronger effect of alkali on destroying dye-protein interactions as the NaOH concentration increases. While for free RhB as shown in Fig. 5(a), slightly alkaline pH can even result in higher values compared to that at neutral, suggesting more dye-protein interactions are involved (e.g. pH˂ 12 and [WPI]≤ 5 wt%). This opposite effect of pH on dye-protein interactions in free RhB system can be deduced from two competing forces: on one hand, the break-down capacity of NaOH is relatively low at such conditions while on the other hand, proteins start undergoing structure disruption and expose their buried residues and hydrophobic regions to the solvent [START_REF] Taulier | Characterization of pH-induced transitions of β-lactoglobulin: ultrasonic, densimetric, and spectroscopic studies[END_REF]. This might induce more interactions between the protein and the dye thus increase the lifetime of RhB [START_REF] Waissbluth | Alkaline denaturation of β-lactoglobulins. Activation parameters and effect on dye binding site[END_REF].

There is one noticeable difference of alkali on dye-protein interactions in WPI-RITC system: when additional WPI is absent, around 80% (compare normalized values at pH 14 and at neutral) of dye-protein interactions were destroyed. However, there is only ~60% loss of interactions with the presence of additional proteins. More interestingly, this loss is consistent in concentrated protein solutions ([WPI]≥ 5 wt%).

These results show different dye-protein interactions between RhB and its linked protein to additional proteins.

Quenching effect of RhB on the tryptophan fluorescence of whey proteins

So far we have shown the existence of dye-protein interactions that can be destroyed by alkali, and hence decrease the fluorescence and lifetime of RhB. To go further, these dye-protein interactions were investigated from the perspective of proteins. Tryptophan (Trp) is the dominant intrinsic fluorophore in proteins, whose fluorescence is typically utilized to characterize the conformational transitions of the proteins. A series of fluorescence dyes including RhB were able to interact with the Trp residues within bovine serum albumin (BSA) and quench the Trp fluorescence through photon-induced electron transfer (PET) [START_REF] Hu | Study on the interaction between methyl violet and bovine serum albumin by spectral analyses[END_REF][START_REF] Hu | Spectroscopic studies on the interaction between methylene blue and bovine serum albumin[END_REF][START_REF] Zhang | Studies of the interaction between Sudan I and bovine serum albumin by spectroscopic methods[END_REF][START_REF] Zhang | Spectroscopic studies on the interaction of Congo Red with bovine serum albumin[END_REF][START_REF] Zhang | Interaction of malachite green with bovine serum albumin: Determination of the binding mechanism and binding site by spectroscopic methods[END_REF][START_REF] Shaikh | Spectroscopic investigations on the mechanism of interaction of bioactive dye with bovine serum albumin[END_REF][START_REF] Cai | Probing site-selective binding of rhodamine B to bovine serum albumin[END_REF]. In these scenarios, Trp serves as an electron donor and the dye-Trp quenching occurs either through molecular collisions, a process called "dynamic quenching" or formation of a stacked dye-Trp complex that is unable to fluorescence, named "static quenching" [START_REF] Vaiana | Fluorescence quenching of dyes by tryptophan: interactions at atomic detail from combination of experiment and computer simulation[END_REF][START_REF] Doose | A Close Look at Fluorescence Quenching of Organic Dyes by Tryptophan[END_REF]. The effect of RhB on the fluorescence spectra of whey protein Trp is shown in Figure 6 The quenching effect of RhB on the Trp fluorescence of whey proteins can be described by the Stern-Volmer equation:

F0/F = 1 + sv[Q] ( eq. 3)
where F0 and F are the steady-state fluorescence intensities with the absence and presence of the quencher, respectively. Ksv is the Stern-Volmer quenching constant and [Q] is the concentration of the quencher, which is RhB in our case.

Irreversibility of RhB binding on whey proteins

It has been proved the capacity of alkali to destroy the dye-protein interactions between RhB and whey proteins, however, in free RhB system, most of them survive even at extreme alkaline conditions (Fig. 5). Hence, it is conceivable that the binding of RhB upon Trp residues plays a significant role in the dye-protein interactions due to its high resistance to alkali. If so, the binding of RhB on whey proteins might be irreversible as most of RhB molecules form RhB-Trp complexes. To verify this hypothesis, it was designed to test the possibility of RhB rearranging between whey proteins. To differentiate proteins, WPI was firstly incubated with RhB followed with heat treatment to thermally denature whey proteins and create protein aggregates;

these aggregated proteins can be easily distinguished from additional native WPI as they precipitate at pH 4.2. The thermal treatment is not considered to significantly affect the stability of Trp-RhB complex due to its high thermal tolerance.

The protein and dye content in the soluble and precipitated portions, for both WPI-RhB and WPI-RITC mixtures, is summarized in Figure 7. Large amounts of native WPI were added to facilitate the rearrangement of the dye between native and aggregated proteins if any. The acidification process can consistently precipitate ~80% of the heated whey proteins and around 10% of native WPI. The expected values of precipitated WPI portion when mixing with 1, 2 and 3 times of native whey proteins are 45%, 33%, and 28%, respectively. These results are consistent with the experimental data shown in Fig. 7(a) and (c). Without adding native WPI, there is a noticeable difference between WPI-RhB and WPI-RITC system: the portion of precipitated dye and protein are similar in the case of WPI-RITC, indicating homogenous labeling of RITC on whey proteins. However, only ~60% dye goes to deposit even though more than 80% protein precipitates in WPI-RhB.

Considering the distribution of dye content, no statistical differences of RhB portion were observed both for WPI-RhB and WPI-RITC mixtures when adding different amounts of native WPI. Therefore, if the percentage of precipitated fluorescence dye remains unchanged with the addition of native WPI, the rearrangement of RhB between aggregated protein and native one is unlikely to occur.

These results strongly suggest an irreversible binding of RhB upon aggregated whey proteins, which might due to the formation of Trp-RhB complexes.

Discussion

In this paper, we have accumulated experimental evidence supporting the existence of dye-protein interactions and the ability of alkali to destroy them. Alkali not only destroys the existed interactions between RhB and whey proteins but simultaneously induce the base-denaturation of proteins. These reactions complicate the dye-protein interactions so that the dye-protein interactions could even be enhanced, particularly at mild alkaline conditions. The phenomenon of dye molecules possesses high affinity to whey proteins (e.g. BSA) and shows preferential binding sites are considered to be related to their carcinogenic action in vivo [START_REF] Wu | Binding of the Environmental Pollutant Naphthol to Bovine Serum Albumin[END_REF][START_REF] Jisha | Site-Selective Binding and Dual Mode Recognition of Serum Albumin by a Squaraine Dye[END_REF]. In particular, Cai, et al. [START_REF] Cai | Probing site-selective binding of rhodamine B to bovine serum albumin[END_REF] discovered that RhB binds specifically at site I and site II of BSA, provably through hydrophobic, hydrogen bonding and electrostatic interactions. Moreover, Millan, et al.

[51] studied the molecular docking of RhB on the lysozyme protein, where the docked RhB was postulated to lie within the hydrogen bonding distance with Trp 63 and Ala 107. It was proposed that van der Waal forces, hydrogen bonding, and electrostatic forces were involved in the lysozyme-RhB interaction. Lysozyme was proved to have a high level of sequence identity with bovine α-lactalbumin along with their conformational structures [START_REF] Mckenzie | Lysozyme and α-Lactalbumin: Structure, Function, and Interrelationships[END_REF]. Consequently, RhB might interact with WPI, by analogy, through hydrogen bonding and electrostatic interactions which are well known to be broken down by NaOH.

The quenching effect of RhB on the Trp fluorescence of WPI provides a different aspect on the dye-protein interactions. This phenomenon is only studied in free RhB system, and it is postulated that RhB forms a non-fluorescent complex with Trp residues in WPI. While this might not be the case for WPI-RITC system, as RhB is unlikely to be in van der Waals interaction with Trp residues when it is covalently linked to the lysine residues [START_REF] Sawyer | The core lipocalin, bovine β-lactoglobulin[END_REF]. Supporting evidence can be found in WPI-RITC system that most of dye-protein interactions were cleaved at pH 14 when additional WPI is absent (Fig. 5(b)).

Trp is well known to serve as electron donors and efficiently quench oxazine and rhodamine fluorophores through PET [START_REF] Vaiana | Fluorescence quenching of dyes by tryptophan: interactions at atomic detail from combination of experiment and computer simulation[END_REF][START_REF] Marmé | Highly sensitive protease assay using fluorescence quenching of peptide probes based on photoinduced electron transfer[END_REF]. Hence, it is proposed that Trp, being the donor here, transfer the electron to the acceptor RhB, resulting in non-radiative dissipation of excited-state energy (mainly via rotation of diethylamino groups). The efficiency of photon-induced charge separation can be evaluated by the change of its free energy, ∆Gcs, by using Rehm-Weller formula as given below [START_REF] Rehm | Kinetics of Fluorescence Quenching by Electron and H-Atom Transfer[END_REF].

∆Gcs = Eox-Ered-E0,0+C (eq. 5)
where Eox and Ered are the first one-electron oxidation potential of the donor (0.64 V at pH 7 for Trp, [START_REF] Jovanovic | Electron-transfer reactions of tryptophan and tyrosine derivatives[END_REF]) and reduction potential of the acceptor (-0.8 V for RhB, [START_REF] Fischer | Photoinduced electron transfer quenching of rhodamine B in polymer films[END_REF]), respectively. E0,0 is the energy of the zero-zero transition to the lowest excited singlet state of the fluorophore (Trp in this case, 3.9 eV, [START_REF] Kazakov | Photonics of UO22+complexes with tryptophan: 2. Competition of electron phototransfer from higher excited electronic and vibration-rotation levels of tryptophan to UO22+with nonradiative relaxation[END_REF]), and C is the solvent dependent Coulombic interaction energy, which can be neglected in the moderately polar environment [START_REF] Doose | Fluorescence Quenching by Photoinduced Electron Transfer: A Reporter for Conformational Dynamics of Macromolecules[END_REF]. The calculation shows a negative value of free energy ∆Gcs from Trp to RhB (-2.46 V), indicating an intense exergonic reaction. This result supports our previous hypothesis for a PET quenching mechanism. Furthermore, a more profound quenching effect of RhB on Trp of aggregated WPI is found, showing a value of Ksv at 2.34×10 4 M -1 , indicating that the aggregation of whey proteins does not inhibit the interaction between RhB and Trp 19 within protein aggregates (Fig. S8).

Conclusion

In this work, the fluorescence properties, including fluorescence intensity and lifetime of RhB and WPI-RITC are investigated at various alkaline conditions with the addition of native whey proteins. The non-covalent interactions between RhB and WPI exist no matter the dye is unconjugated or covalently attached to the proteins.

These dye-protein interactions increase both the fluorescence intensity and lifetime of RhB but are suppressed at alkaline pH. However, alkali-induced denaturation of proteins also occurs which complicates the dye-protein interactions. In particular, the lifetime of RhB could even increase at mild alkaline pHs. Results show different dye-protein interactions were created when the dye is free or covalently linked to proteins. In the unconjugated system, RhB was found to quench the Trp fluorescence of WPI, leading to the formation of non-fluorescent complex. While this is unlikely to happen when the dye is already covalently linked to protein. This RhB-Trp complex might play a significant role in the dye-protein interactions of unconjugated system due to its high stability in alkaline conditions. The formation of RhB-Trp could be also responsible for the irreversible binding of RhB upon heat-induced whey protein aggregates. This behavior might suggest a cheaper alternative for protein labeling in some applications such as in food processing when whey proteins are generally treated with heat.

Figure 1 .

 1 Figure 1. The effect of additional native whey protein on the fluorescence lifetime of free RhB (τRhB) and whey protein conjugated RhB (τWPI-RITC) at room temperature. The final dye concentration was fixed at 10 μM, and for WPI-RITC the conjugated [WPI] was lower than 0.1 wt%. Error bars denote standard deviation (SD) in one FLIM image.

  (b), this phenomenon was not observed. At low protein concentrations (≤5 wt%), when pH goes even higher than pH 13, τRhB increases rapidly in the first 20 minutes and starts to decrease gradually and reaching stable values with a much longer time. However, at more concentrated protein solutions such as 10 wt% WPI, τRhB at pH ≥12.5 keep decreasing before reaching stable values. Note that these lifetime behaviors are inconsistent with what was observed from fluorescence. This discrepancy could be due to the variability of fluorescence measurement from WFM, which could be affected by various factors such as scattering effect.

Figure 3 (

 3 Figure 3(c) summarizes the effect of pH on the τRhB at all alkaline conditions tested and at different WPI concentrations. The values of τRhB are the last measured

Figure 2 .

 2 Figure 2. (a) Evolution of normalized RhB fluorescence intensity against time after the addition of NaOH to the desired pH with supplemented 5 wt% native WPI. (b) Effect of pH on fluorescence ratio α at different native whey protein concentrations (shown in legend). α in (b) are values taken at the last measured moment when IRhB is stable (i.e. 60 min). Error bars denote the SD using three different magnification in WFM. Solid lines represent the sigmoidal fit of data to Eq. (1); regression parameters

Figure 3 .

 3 Figure 3. Fluorescence lifetime of RhB (τRhB) at different time after adding NaOH to reach an alkaline pH with additional native WPI of (a) 1 wt% and (b) 10 wt%. (c) Effect of pH on τRhB at various additional native WPI concentrations (shown in legend). τRhB in (c) are taken at the last measured time. Error bars present SD of all pixels in the FLIM image.

Figure 4 (

 4 Figure 4(a) depicts the normalized fluorescence intensity of WPI-RITC (IWPI-RITC)

Figure 4 .

 4 Figure 4. Fluorescence intensity (a) and lifetime (b) of WPI-RITC with 5 wt% native WPI at different mixing time with NaOH. (c) pH effect on fluorescence lifetime of WPI-RITC at different supplemented native WPI concentrations. Error bars in (a) denote SD using three different magnifications in WFM and in (c) they are SD of all pixels in one FLIM image.

Figure 5 .

 5 Figure 5. The effect of pH on the normalized fluorescence lifetime of (a) unconjugated (a) and (b) whey protein conjugated RhB at various alkaline conditions with supplementation of different amounts of whey proteins (shown in legend). The normalization was performed using the lifetime of free RhB in water (τw = 1.7 ns).

  (a). Trp fluorescence of native whey proteins shows a maximum emission peak at ~335 nm when excited at 295 nm; this maximum peak resembles the values for the major component of whey proteins, BLG [39]. BLG contains 2 Trp residues: Trp 19 present in a hydrophobic pocket, wheres Trp 61 is located at the surface of the protein, near the pocket. The former was recognized to have a full contribution to the Trp fluorescence of BLG, hence the Trp fluorescence of whey protein is considered mainly from Trp 19 of BLG [50]. The addition of RhB leads to a significant decrease in the Trp fluorescence intensity without causing a shift of the peak, suggesting that RhB can readily access the hydrophobic interior of BLG and interact with Trp 19.

Figure 6

 6 (b) displays the typical Stern-Volmer plot of Trp fluorescence at evaluated RhB concentrations. The plot shows a linear relationship between the quencher concentration and the fluorescence ratio F0/F. This linearity indicates that the quenching mechanism of RhB on Trp fluorescence of whey proteins is either via static or dynamic quenching mechanism [6]. The best-fitted quenching constant Ksv was found at 1.48×10 4 M -1 at neutral pH, close to that found for quenching of RhB on Trp fluorescence of lysozyme, where the quenching occurs through static quenching leading to the formation of a non-fluorescent RhB-Trp complex [51]. Typically, static and dynamic quenching can be distinguished by their differing dependence on the temperature and viscosity or preferably lifetime measurement. Higher temperatures generally result in faster diffusion and hence larger amounts of collisional quenching, while it will result in the dissociation of weakly bound complexes and consequently smaller amounts of static quenching. Our results show little temperature dependence of RhB quenching effect at temperature up to 55 °C (Fig. S7(a)), which might suggest a strong affinity of RhB on Trp 19 of BLG. The accessibility of RhB for Trp 19 can be determined using the modified Stern-Volmer equation: Ka is the Stern-Volmer constant of the accessible fraction and fa is the fraction of the initial fluorescence that is accessible to the quencher. The modified Stern-Volmer is plotted in the inset of Figure 6(b) showing a value of fa -1 around 1, confirming that Trp 19 of BLG is fully accessible to RhB. Additionally, the quenching effect of RhB on Trp fluorescence was also performed at various alkaline conditions in order to verify whether alkali can destroy the interactions between RhB and Trp 19.Surprisingly, our results show little effect of alkali on the RhB quenching behavior at alkaline pH up to 14 (Fig.S7(b)). In conclusion, it is proposed that RhB can form complex with Trp 19 residue of BLG and quench its fluorescence. This complex is fairly stable and can survive at high temperatures and also extreme alkaline conditions. These strong dye-protein interactions could explain why τRhB remain with high values even at pH 14 with the presence of WPI (Fig.3(c)).

Figure 6 .

 6 Figure 6. (a) Tryptophan fluorescence spectra of 0.05 wt% WPI with elevated RhB concentration (shown in legend) in a 50 mM PBS buffer at pH 7. (b) Stern-Volmer plot and modified Stern-Volmer plot (shown in inset) for RhB quenching on Trp fluorescence of whey proteins at pH 7; the emission was obtained at its peak wavelength (i.e. 331 nm). The solid line shows the linear fit of the data. Error bars denote SD of triplicates.

Figure 7 .

 7 Figure 7. Distribution of whey protein and RhB content in a mixture of native WPI and RhB linked WPI aggregates at four different molar ratios of added native proteins after acidic precipitation at pH 4.2. (a) and (b) present the system when RhB is covalently bound to WPI (i.e. WPI-RITC) and in (c) and (d) RhB is unconjugated. Error bars show SD of three individual experiments.

Table 1

 1 Sigmoidal regression parameters of data sets in Figure2to 4 for eq. 1 and 2.Standard errors of each parameter are also shown.

				[WPI]
	Dye	Figure#		(wt%)	α0	∆α	I *	∆pI
				1	0.79±0.01 0.14±0.01	12.2±0.1	-0.16±0.06
		2(b)	IRhB
				5	0.67±0.01 0.21±0.01 12.04±0.04 -0.17±0.05
				[WPI] (wt%)	τ0	∆τ	τ *	∆pτ
	RhB
				1	2.01±0.02 0.27±0.03 11.93±0.12 -0.39±0.11
		3(c)
				5	2.35±0.02 0.36±0.03 11.88±0.09 -0.18±0.08
			τRhB
				10	2.44±0.03 0.39±0.04 11.94±0.12 -0.38±0.12
				15	2.40±0.03 0.45±0.05 12.08±0.13 -0.55±0.13
				0	1.81±0.05 0.53±0.08 12.29±0.18 -0.60±0.19
				1	1.95±0.04 0.45±0.08 11.57±0.16 -0.62±0.19
	WPI-
		4(c)	τWPI-RITC	5	1.5±0.6	1.0±0.6	13.5±1.3	-1.3±0.5
	RITC
				10	1.91±0.05 0.58±0.06	12.9±0.13	-0.8±0.1
				15	1.9±0.1	0.6±0.1	13.1±0.25 -0.72±0.15

Acknowledgments

This work was supported by the project funding from the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institution and the "Jiangsu Specially-Appointed Processors Program" of China, the Youth Fund of Natural Science Foundation of Jiangsu Province of China (No. BK20140343).We are also grateful for the financial supports from the National Key Research and Development Program of China (International S&T Cooperation Program, ISTCP, 2016YFE0101200).

Conflicts of interest

There are no conflicts of interest to declare.