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

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.

Effects of Ionic Liquid Alkyl Chain Length on Denaturation of Myoglobin by Anionic, Cationic, and Zwitterionic Detergents

“…Experiments using the zwitterionic detergent N,N -dimethyl- N -dodecylglycine betaine showed that the inclusion of a series of RTILs had no impact on the partial denaturation and heme-loss from myoglobin. This result was intriguing since some of the same RTILs were shown previously to enhance myoglobin denaturation by guanidinium HCl (GuHCl) and urea [10,12,21]. These differences in behavior highlight the still unresolved questions of the chemical mechanism by which different RTILs stabilize or destabilize protein structure and activity.…”

“…There are unanswered questions at both the basic, fundamental levels up through the increased complexity of in vivo and ecological investigations. A number of literature reports in recent years have focused on characterizing the specific interactions of RTILs with various classes of biomolecules or individual molecules including proteins, nucleic acids, polysaccharides, and lipids [7,10,11,12,13]. These studies often target the mechanism of interaction between a subset of RTILs with a specific target [10,14,15].…”

“…These studies often target the mechanism of interaction between a subset of RTILs with a specific target [10,14,15]. Previous work from our lab has shown the functional impact of incorporating RTILs with both proteins and lipids [10,11,12,14].…”

“…Myoglobin is a small, monomeric, heme-containing protein involved in oxygen storage in mammalian muscle tissue. The heme incorporated in the protein structure yields an intense absorption band at ~409 nm that disappears upon protein denaturation [12]. As such, myoglobin has been a widely characterized model to study protein folding and unfolding thermodynamics.…”

Correlating Lipid Membrane Permeabilities of Imidazolium Ionic Liquids with Their Cytotoxicities on Yeast, Bacterial, and Mammalian Cells

“…ILs have many potential applications in biochemistry and biomedical technology such as drug delivery, enzymatic reaction enhancement, and use as biomaterials. The biomass solubilization [1,2,3,4] and protein structural stabilization and destabilization mechanisms [5,6,7,8,9] of ILs in aqueous solution have been characterized extensively. These studies have primarily focused on either the molecular diversity of IL pairs and the differential effects ion pairs have on activity or the mechanisms by which different IL species disrupt protein structure/function.…”