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. 2018 Oct 29;8(4):126.
doi: 10.3390/biom8040126.

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

Eric M Kohn  1   2 , Joshua Y Lee  3   4 , Anthony Calabro  5 , Timothy D Vaden  6 , Gregory A Caputo  7   8
Affiliations

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

Eric M Kohn et al. Biomolecules. .

Abstract

We have investigated myoglobin protein denaturation using the zwitterionic detergent Empigen BB (EBB, N,N-Dimethyl-N-dodecylglycine betaine). A combination of absorbance, fluorescence, and circular dichroism spectroscopic measurements elucidated the protein denaturation and heme dissociation from myoglobin. The results indicated that Empigen BB was not able to fully denature the myoglobin structure, but apparently can induce the dissociation of the heme group from the protein. This provides a way to estimate the heme binding free energy, ΔGdissociation. As ionic liquids (ILs) have been shown to perturb the myoglobin protein, we have investigated the effects of the ILs 1-butyl-3-methylimidazolium chloride (BMICl), 1-ethyl-3-methylimidazolium acetate (EMIAc), and 1-butyl-3-methylimidazolium tetrafluoroborate (BMIBF₄) in aqueous solution on the ΔGdissociation values. Absorbance experiments show the ILs had minimal effect on ΔGdissociation values when compared to controls. Fluorescence and circular dichroism data confirm the ILs have no effect on heme dissociation, demonstrating that low concentrations ILs do not impact the heme dissociation from the protein and do not significantly denature myoglobin on their own or in combination with EBB. These results provide important data for future studies of the mechanism of IL-mediated protein stabilization/destabilization and biocompatibility studies.

Keywords: detergents; ionic liquids; myoglobin; protein folding.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Molecular structures (A) 3D structure of myoglobin from PDBID: 1WLA. The heme group is colored magenta while the two native Trp residues are yellow. (B) Chemical structures of salts, ionic liquids (ILs), and the Empigen BB (EBB) detergent.
Figure 2
Figure 2
Spectroscopic analysis of myoglobin unfolding. (A) Heme absorbance at 409 nm (A409) as a function of EBB concentration; (B) Absorbance spectra of myoglobin with 0 mM EBB (black), 1 mM EBB (gray), or 1.75 mM EBB (red); (C) Representative circular dichroism (CD) spectra for myoglobin with: 0 mM EBB (black), 1.75 mM EBB (green), 8 mM EBB (blue); (D) Fluorescence intensity (I) of Trp emission from myoglobin in buffer (black), denatured with 1.75 mM EBB (blue), or denatured with 3 M GuHCl (green). Fluorescence data are the averages and standard deviations of five samples; (E) Absorbance spectra of 50 µM heme B in phosphate buffer (black) or buffer supplemented with 1.75 mM EBB (green) or 8 mM EBB (blue). All samples were in 2 mM sodium phosphate, pH 7.
Figure 3
Figure 3
Dissociation of heme from myoglobin monitored by absorbance spectroscopy—Denaturation of myoglobin (0.15 mg/mL) by EBB was monitored by the loss of heme absorbance at 409 nm. Experiments were performed in the presence of 0 (black), 9.4 mM (green), 28.1 mM (blue), 56.3 mM (red) ionic liquids or salts. (A) NaCl; (B) LiBF4; (C) BMICl; (D) EMIAc; (E) BMIBF4. Normalization was performed by setting the absorbance at 409 nm to 1 at 0 detergent concentration. Panel (F) shows representative absorbance spectra for myoglobin with: 0 mM BMIBF4 0 mM EBB (black), 0 mM BMIBF4 1.75 mM EBB (green), 56.3 mM BMIBF4 0 mM EBB (blue), 56.3 mM BMIBF4 1.75 mM EBB (red). All data in panels (A–E) are averages of three independent samples and error bars represent the standard deviation of the replicates.
Figure 4
Figure 4
Dissociation of heme monitored by fluorescence spectroscopy—Denaturation of myoglobin (0.15 mg/mL) by EBB was monitored by the relief of heme quenching of native Trp fluorescence at 350 nm by when excited by 280 nm light. Experiments were performed in the presence of 0 (black), 9.4 mM (green), 28.1 mM (blue), 56.3 mM (red) ionic liquids or salts. (A) NaCl; (B) LiBF4; (C) BMICl; (D) EMIAc; or (E) BMIBF4. Normalization was performed by setting the fluorescence at 350 nm to 1 at 0 detergent concentration. Panel (F) shows representative fluorescence spectra for myoglobin with: 0 mM BMIBF4 0 mM EBB (black), 0 mM BMIBF4 1.75 mM EBB (green), 56.3 mM BMIBF4 0 mM EBB (blue), and 56.3 mM BMIBF4 1.75 mM EBB (red). All data in panels (A–E) are averages of three independent samples and error bars represent the standard deviations of the replicates.
Figure 5
Figure 5
Denaturation of myoglobin monitored by CD spectroscopy—Denaturation of myoglobin (0.15 mg/mL) by EBB was monitored by change in CD signal around the heme transition. Experiments were performed in the presence of 0 (black), 9.4 mM (green), 28.1 mM (blue), or 56.3 mM (red). (A) NaCl; (B) LiBF4; (C) BMICl; (D) EMIAc; or (E) BMIBF4. Normalization was performed by setting the CD signal at 409 nm to 1 at 0 detergent concentration. Panel (F) shows representative CD spectra for myoglobin with (green) and without EBB (black) in buffer or with (red) or without (blue) EBB in the presence of 56.3 mM BMIBF4.
Figure 6
Figure 6
Gibbs free energy analysis of heme dissociation by EBB in the absence or presence of ionic liquids. Gibbs free energy of disassociation (ΔGdissociation) was calculated from the absorbance data represented in Figure 2 and Figure 3. All ΔGdissociation values shown are the average of at least three individual experiments and the standard deviation of those values. An analysis of variance (ANOVA) was performed on the original data set and no statistical difference was found between any IL species or concentration of IL.
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