Heme Dissociation from Myoglobin in the Presence of the Zwitterionic Detergent N,N-Dimethyl-N-Dodecylglycine Betaine: Effects of Ionic Liquids

2.1. Materials

Horse skeletal myoglobin (95–100% lyophilized powder) and Empigen BB (Empigen BB ~30% pure liquid) were purchased from Sigma Life Sciences (St. Louis, MO, USA); myoglobin was stored at −20 °C in the dark. BMIBF₄, BMICl, EMIAc, LiBF₄ and 1,6-Diphenyl-1,3,5-hexatriene (DPH) were purchased from Sigma Chemical Company (St. Louis, MO, USA). Ionic liquids were >97% purity. NaCl was purchased from Fisher Scientific (Fair Lawn, NJ, USA).

2.2. Sample Preparation

All experiments were carried out in 2 mM sodium phosphate buffer (pH 6.9–7.2) using aqueous solutions of myoglobin, ILs, and Empigen BB in deionized water. Final myoglobin concentrations were kept at 0.15 mg/mL for all experiments excluding those examined by CD spectroscopy in the far UV region; for these experiments, myoglobin concentrations were decreased to 0.0375 mg/mL (by a factor of four) as the original 0.15 mg/mL solutions produced a signal greater than the detection range of the instrument used. Each sample contained one species of ionic liquid at a concentration of 0, 9.4, 28.1 or 56.3 mM. For absorbance and fluorescence experiments, unfolding was monitored by performing experiments in triplicate for all species and concentrations of ionic liquid 0.00, 0.10, 0.20, 0.30, 0.40, 0.50, 0.75, 1.00, 1.25, 1.50, or 1.75 mM EBB. This was achieved by adding 25 mM EBB in increasing volumes across samples. The appropriate volume of phosphate buffer was added to bring the volume of each sample to 1000 μL.

2.3. Critical Micelle Concentration Determination

Micelle formation assays were conducted using stock solutions of 1.5 M NaCl, LiBF₄, BMIBF₄, and EMIAc in water as well as 0.4 mM DPH solution, a 16 mM solution of EBB and a 2 mM sodium phosphate buffer solution, with a pH 6.9–7.2. The samples contained 0 mM, 50 mM, 100 mM, 150 mM or 300 mM of salt or ionic liquid, 2.5 µm DPH, and a serially diluted concentration of detergent ranging from 0.125–8 mM. Assays were performed in 384-well plates and DPH fluorescence was measured using a Molecular Devices SpectraMax i3x plate reader (Sunnyvale, CA, USA) with an excitation of 358 nm and an emission of 430 nm. Each datum is the average and standard deviation from 12 replicates. The data was plotted in excel and then analyzed for the CMC using linear fits to determine the onset of micelle formation and the plateau after micelle formation using an R² cutoff of 0.9. The midpoint of the fluorescence intensity transition was determined by fitting the data to cubic polynomial and subsequently taking the derivative to determine the inflection point.

The fluorescence emission spectra of DPH in the presence of EBB and without EBB as well as with and without the controls and ionic liquids were also measured. Samples contained 300 mM salt or ionic liquid, 8 mM detergent, 1.25 µM DPH in phosphate buffer. Fluorescence emission spectra were measured using a Horiba Scientific FluoroMax-4 Spectrofluorometer (Edison, NJ, USA) λ_ex = 358 nm, λ_em = 380–680 nm.

2.4. Absorbance Measurement

Myoglobin, ILs, and EBB were combined in buffer and allowed incubate for 30 min at room temperature. Samples were prepared in polystyrene cuvettes (optically clear to 340 nm) to measure absorbance spectra from 375–575 nm or in quartz cuvettes to measure absorbance spectra from 200–700 nm. Absorbance spectra were recorded using a Thermo Scientific Genesys 10S UV-Vis Spectrophotometer (Waltham, MA, USA). Dissociation of heme (H) was calculated at 409 nm by

where A is the absorbance of a sample at a given concentration of EBB, A_i represents the maximum heme absorbance, i.e., 100% bound (0 mM EBB), and A_f represents the minimum amount of bound heme (1.75 mM EBB).

2.5. Fluorescence Spectroscopy

Fluorescence quenching was measured using a SpectraMax M5 or i3x plate reader (Molecular Devices) in clear, untreated UV-Star 96-well plates purchased from Greiner Bio One (655801, Kremsmünster, Upper Austria) with an optical window down to 230 nm. The excitation and emission were at 280 nm and 350 nm, respectively. The dissociation of heme (H) was similarly calculated at 350 nm by

for each sample, where I is the fluorescence emission intensity at a given concentration of EBB, I_i represents the maximum heme emission intensity, i.e., 100% bound (0 mM EBB), and I_f represents the minimum amount of bound heme (1.75 mM EBB).

Fluorescence emission spectra were collected in triplicate for samples containing 0 or 30 mM NaCl, BMICl, or BMIBF₄; 0 or 1.75 mM EBB; and 0 or 0.15 mg/mL myoglobin in quartz semi-micro cuvettes with a Fluoromax-4 Spectrophotometer (Horiba Scientific).

2.6. Circular Dichroism Spectroscopy

Circular dichroism was performed using a CD spectropolarimeter model J-810 by Jasco (Easton, MD, USA) for each concentration and species of ionic liquid, at all concentrations of EBB. Spectra were recorded between 190–260 nm to assess secondary structure and 375–500 nm to monitor dissociation of heme; from this, the signals produced under 408–410 nm light were averaged. Background correction was performed by subtracting the signal produced by each species and concentration of ionic liquid in buffer from those of the samples containing myoglobin and EBB.

2.7. Calculation of Gibbs Free Energy of Dissociation

The equilibrium constant of dissociation [K_eq] in each ionic liquid environment was calculated by

where R is the ideal gas constant (8.314 J·mol⁻¹), T is the temperature in kelvin (295 K was estimated for all experiments), and OD₄₀₉ is the peak absorbance of myoglobin solution at 409 nm. Data were analyzed in Microsoft Excel (Redmond, WA, USA) and fit to a linear trendline with an R² cutoff of 0.95. The y-intercept of each trendline was the estimated Gibbs free energy of dissociation (ΔG_dissociation). This value reflects the energy of dissociation of the heme group from the myoglobin protein. The ΔG_dissociation values were tested for statistical significance using an analysis of variance (ANOVA) and post-hoc testing for each variable using MS Excel with the XL-toolbox add-in [46].

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3.1. Protein Denaturation and Heme Dissociation by Empigen BB

Empigen BB is not a typical denaturant for proteins, and therefore it is important to characterize the interaction of this zwitterionic detergent with myoglobin and heme. As absorbance spectroscopy in the heme region is typically used to quantify myoglobin unfolding [13,42], the absorbance at 409 nm was monitored as a function of detergent concentration (Figure 2A,B). While there is a significant reduction in the heme Soret band at 409 nm (Figure 2A), this band does not disappear. However, the absorbance profile is reminiscent of a standard protein unfolding curve (Figure 2B). To ascertain whether or not Figure 2A,B indicate protein unfolding, the myoglobin secondary structure was directly characterized in the presence of EBB using CD spectroscopy in the mid-UV region (Figure 2C). Notably, there is only a minor loss of helical content upon addition of 1.75 mM EBB (~15%) which is only slightly enhanced by increasing detergent concentration up to 8 mM (Figure 2C, and see below—discussion). The CD results indicate that EBB does not fully denature the protein and therefore the loss of absorbance at 409 nm is likely due more to the dissociation of the heme group from the myoglobin than protein denaturation.

In light of these findings, we compared heme fluorescence dequenching when myoglobin was denatured by EBB with denaturation by the chaotrope guanidinium chloride (GuHCl). It has been well-demonstrated in the literature that as the distance between the native Trp residues and the heme increases, Trp fluorescence emission intensity increases. Our fluorescence results (Figure 2D) show that the Trp fluorescence significantly increases both when the protein is completely denatured by GuHCl and when it is partially denatured by EBB, indicating that the Trp-heme distance is large in both cases. These results indicate that while the protein is not completely denatured by EBB (based on the CD results), the heme is clearly removed from the protein binding pocket under these conditions. For control, we measured the absorbance of the free heme molecule in solutions with and without EBB (Figure 2E). These spectra show a similar bathochromic shift in peak position as the heme traditionally exhibits in the denaturation of myoglobin [13,42], but with different magnitude shifts relative to literature values. The heme absorbance decreases slightly with 1.75 mM EBB (and more so with 8 mM EBB).

3.2. Spectroscopic Analysis of Heme Dissociation from Myoglobin with Ionic Liquids

Considering EBB does not cause full denaturation of the protein but is sufficient to remove the heme, the effect of low concentrations of ILs on this process was monitored using absorbance spectroscopy, specifically using the heme absorption, or Soret band, at 409 nm. As the heme is lost from the binding pocket there is a concomitant loss of absorptivity and a slight red shift in the Soret band [42,43,47]. Control experiments indicated that the effects were complete in under 10 min of incubation with the detergent, so all samples were allowed to equilibrate with the Empigen BB for 30 min before measurement to ensure equilibrium was achieved. The absorbance of 0 mM detergent sample was normalized to 100% bound and the fraction of bound/unbound heme was calculated from the absorbance measurements similar to previously reported [42,43].

Myoglobin was exposed to increasing concentrations of Empigen BB in the presence of a fixed concentration of IL or salt. The results of these experiments are shown in Figure 3. The results demonstrate several important points. First, the EBB removes the heme with a typical sigmoidal, two state transition that is reminiscent of myoglobin denaturation by SDS and other denaturants [48]. It is important to note that the results from experiments shown in Figure 2 indicate this transition is more likely the result of heme dissociation, not denaturation. With respect to IL/salt concentration, increasing the concentration of the ionic additive did not cause significant shifts in the dissociation curves, indicating the process is generally insensitive to the ionic strength of the solution. Most interestingly, the IL BMIBF₄ did not significantly affect the dissociation profile with EBB. This is an important finding as it was previously shown that high concentrations of BMIBF₄ can denature myoglobin in the absence of other chaotropes [42,43]. However, the absorbance spectrum of the samples containing detergent and BMIBF₄ exhibited a lower absorbance than the samples treated with detergent alone (Figure 3F). This supports the idea that the destabilizing effect of BMIBF₄ is concentration dependent, and that myoglobin folding is insensitive to low concentrations of BMIBF₄, however, there may be additional BMIBF₄-heme interactions that affect absorptivity but not the folding stability of the protein. These absorbance spectroscopy results were also utilized to determine the ΔG_dissociation associated with the EBB induced dissociation of heme (see below).

The EBB-induced heme dissociation was next investigated by changes in emission intensity of the intrinsic tryptophan. In the folded state, the iron at the center of the heme group efficiently quenches Trp fluorescence emission through a heavy atom quenching mechanism [49,50]. As the protein unfolds, the Trp-heme distance increases as the heme is lost, relieving the quenching effect, resulting in an increase in Trp emission intensity. Based on the crystal structure of myoglobin (PDBID 1WLA), in the folded state the two native Trp residues are ~23 Å and ~16 Å from the heme (measured from heme iron to Trp indole nitrogen). This dequenching assay can be more sensitive to subtle changes in folding or to heme dissociation as even small changes in distance between quencher and fluorophore can have significant effects on quenching efficiency. The results in Figure 2 show that when myoglobin is exposed to EBB, the dissociation of heme is sufficient to relieve the heme-Trp quenching.

The results of the dissociation experiments monitored by Trp fluorescence are shown in Figure 4. The Trp intensity increases upon addition of EBB indicating that the distance between the heme and the Trp residues is increasing. Again, as was observed for the absorbance measurements, the Trp intensity increases are similar in profile between samples with no salt/IL and those with 9.4 mM, 28.1 mM, and 56.3 mM IL/salt included in the sample. These results indicate that treatment with Empigen BB results in a final conformation in which the heme is significantly far from the native Trp residues.

Circular dichroism spectroscopy is a widely utilized method for evaluating protein folding because of the distinctive spectral shapes associated with protein secondary structures in the far UV spectral region. However, due to the strong absorption of the imidazolium-based ILs in the far UV region, traditional protein structural analysis by CD is unreliable. For this reason, the signal produced by heme was used, as the restricted chiral environment provided by myoglobin generates a positive CD band to protein-associated heme [42]. This approach can also be used to investigate the removal or dissociation of the heme group from the myoglobin protein, as the removal of heme from the binding pocket decreases this signal regardless of protein secondary structure or loss thereof.

Heme dissociation was also monitored by the loss of the CD signal at 409 nm as a function of EBB in the presence of varying concentrations of ILs or salts. Similar to the results from the absorbance measurements, the CD results showed the sigmoidal dissociation profiles (Figure 5). Consistent with the absorbance and fluorescence measurements, the ILs and salts did not have a dramatic impact on the dissociation event. Under all conditions tested, the heme dissociated from the myoglobin with the same, broad profile regardless of the overall ionic strength of the solution or the identity of the IL or salt used in the experiment. Additionally, the complete loss of CD signal at 409 nm is consistent with the fluorescence dequenching results indicating a complete loss of the heme from the protein (Figure 1C).

In aggregate, the spectroscopic results indicate that the dissociation of heme from myoglobin by EBB is generally insensitive to the addition of a variety of ILs and salts, and to modest changes in the ionic strength of the solution environment. The lack of sensitivity to ionic strength is not surprising considering the hydrophobicity-driven mechanism of detergent mediated unfolding that is also the likely driver of heme displacement from the binding pocket. The range of ionic strengths tested in this work is relatively modest considering the typical ionic strengths in biological environments, so the protein stability is not likely to be significantly affected. More interestingly, the heme dissociation profile was not significantly affected by the inclusion of ILs, especially BMIBF₄ which has been shown to destabilize myoglobin, enhancing the denaturing effects of GuHCl. The results presented in Figure 3, Figure 4 and Figure 5 do not indicate any significant impact of BMIBF₄ (or the other ILs) on heme dissociation, indicating there is likely no additional protein denaturation caused by the inclusion of the ILs.

3.3. Thermodynamic Analysis of Heme Dissociation from Myoglobin

The spectroscopic experiments described allows for further quantification of the dissociation of heme from myoglobin by EBB. Assuming that the loss in heme absorbance and CD signal is attributable primarily to heme dissociation and not protein unfolding, we can use the H-values (Equations (1) and (3)) to compute heme-dissociation free energies. Using the data collected, the ΔG_dissociation was calculated for each of the IL/salt conditions tested. As reported previously for denaturation of myoglobin, the A₄₀₉ values from the absorbance spectroscopy experiments can be used to calculate the equilibrium constant (K_eq) from the transition region of unfolding curve and then the ΔG_dissociation can be directly calculated [42,43]. Previously, similar transitions were used to determine ΔG_unfolding for the complete denaturation of the protein, but in this case, the transition more accurately reflects the ΔG_dissociation of the heme from the protein. The values for ΔG_dissociation under all conditions are shown in Figure 6. The values of ΔG_dissociation were ~8000 kJ/mol in most cases, regardless of the IL identity or concentration. The values were compared using an ANOVA method which showed no statistical significance based on IL identity or IL concentration.

3.4. Critical Micelle Concentration Determination

Many reports have focused on ILs ability to form micelles and the investigation of the denaturing mechanism in relation to a micelle- or detergent-like mechanism [51]. Considering this, the first experiments performed were to ensure that the ILs used in this study did not impact the formation of micelles by monitoring the CMC in the presence of ILs. It is widely known that the CMC of some detergents, such as SDS which is common in protein unfolding studies, are dramatically affected by the ionic strength of the environment [52,53], which is a concern for IL studies and any others where ionic strength is a variable. The environmentally sensitive fluorophore (DPH) exhibits a large increase in fluorescence emission intensity when incorporated in a nonpolar environment, such as the hydrophobic core of a micelle, and thus the polarity driven intensity change has been used in many studies to monitor the formation of micelles [52].

The CMCs for EBB in the presence of varying concentrations of IL or control salts are shown in Table 1 using high IL or salt concentrations. Based on literature, three approaches were used to fit the data: onset of fluorescence increase, which represents the beginning of micelle formation, plateau of fluorescence increase which represents saturation or nearly complete micelle formation, and midpoint of the sigmoidal increase in fluorescence. In all cases, the differences between 0 mM IL or control and the highest concentration of IL or control salt are within the standard deviations of the measurements. Using the midpoint method, all of the CMCs determined were in the range between 0.95–2.6 mM while the onset of micelle formation method ranging between 0.28–0.51 mM in all cases. The results demonstrate that the ILs and salts used do not significantly affect the CMC of Empigen BB and therefore the denaturation of myoglobin. Representative spectra of DPH above and below CMC values for EBB are shown in Supplemental Figure S1.