Measurement and Control of pH in the Aqueous Interior of Reverse Micelles

Abstract

The encapsulation of proteins and nucleic acids within the nanoscale water core of reverse micelles has been used for over three decades as a vehicle for a wide range of investigations including enzymology, the physical chemistry of confined spaces, protein and nucleic acid structural biology, and drug development and delivery. Unfortunately, the static and dynamical aspects of the distribution of water in solutions of reverse micelles complicate the measurement and interpretation of fundamental parameters such as pH. This is a severe disadvantage in the context of (bio)chemical reactions and protein structure and function, which are generally highly sensitive to pH. There is a need to more fully characterize and control the effective pH of the reverse micelle water core. The buffering effect of titratable head groups of the reverse micelle surfactants is found to often be the dominant variable defining the pH of the water core. Methods for measuring the pH of the reverse micelle aqueous interior using one-dimensional 1H and two-dimensional heteronuclear NMR spectroscopy are described. Strategies for setting the effective pH of the reverse micelle water core are demonstrated. The exquisite sensitivity of encapsulated proteins to the surfactant, water content, and pH of the reverse micelle are also addressed. These results highlight the importance of assessing the structural fidelity of the encapsulated protein using multidimensional NMR before embarking upon a detailed structural and biophysical characterization.

High-resolution NMR spectroscopy of encapsulated proteins dissolved in low-viscosity fluids

“…In most cases, it is the character of the surfactants that determines the effective pH. A simple approach for control and measurement of the pH within the reverse micelle interior has recently been developed and allows facile adjustments in order to set and optimize encapsulation conditions [20, 28]…”

Reverse Micelle Encapsulation of Proteins for NMR Spectroscopy

“…Phosphate buffers are commonly used in NMR spectroscopy, but phosphate efficiently catalyzes hydrogen exchange at side chains and should not be used in hydration experiments (Edvards, & Gottfried, 1996). Buffers such as acetate, formate, imidazole, and Tris are reasonable internal indicators of pH but do suffer limitations (Marques, et al, 2014). We generally recommend using the protein itself to monitor the pH.…”

“…This reverse micelle system has been shown to encapsulate the widest range of proteins thus far with only modest modifications of the composition of the mixture (Nucci, et al, 2014). The titratable LDAO head group provides strict control of the pH of the aqueous core of the RM when near their pK a of 4.5, with an effective buffer concentration of ~1 M under standard conditions (Marques, et al, 2014). Encapsulation of proteins within 10MAG/LDAO results in the highest structural fidelity as evaluated by chemical shift differences with bulk aqueous protein (Dodevski, et al, 2014).…”

“…Recent advances in RM encapsulation technology include the development of optimized surfactants (Dodevski, Nucci, Valentine, Sidhu, O’Brien, Pardi et al, 2014) and a means to control and monitor pH within the RM core (Marques, Nucci, Dodevski, Wang, Athanasoula, Jorge et al, 2014). While nucleic acids have been successfully examined [e.g.…”

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“… Drug loaded nanomicellar formulations encounter rapid tear dilution upon their topical administration 39 .…”