The structure and diffusion behaviour of the neurotransmitter γ-aminobutyric acid (GABA) in neutral aqueous solutions

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Highlights

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    Diffusion coefficients and densities of binary aqueous solutions of γ-aminobutyric acid (GABA).

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    Dependence on both shape and size of GABA on its diffusion.

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    Interactions intramolecular and the solute-water interactions in these systems.

Abstract

GABA (γ-aminobutyric acid) is a non-protein amino acid with important physiological properties, and with considerable relevance to the food and pharmaceutical industries. Particular interest has focused on its role as an inhibitory neurotransmitter in the mammalian cerebral cortex. In this paper, we report density and mutual diffusion coefficients of GABA in non-buffered aqueous solutions (0.001–0.100) mol·dm−3 at 298.15 K. Under these conditions, 1H and 13C NMR spectroscopy and pH measurements show that it is present predominantly as a monomeric zwitterionic species. Diffusion coefficients have been computed assuming that this behaves as the binary system GABA/water. From density and intermolecular diffusion coefficients measurements, the molar volume, hydrodynamic radii, Rh, diffusion coefficients at infinitesimal concentration, D0, activity coefficients and the thermodynamic factors, FT, have been estimated. Within experimental error, the hydrodynamic volume calculated from this is identical to the molar volume obtained from density measurements. From the NMR spectra and literature data, it is suggested that this amino acid diffuses in aqueous solution as a curved, coil-like hydrated zwitterionic entity.

Introduction

Amino acids and their derivatives are both biologically important compounds, relevant to the understanding of the behaviour of proteins and the role of solvent structure in their folding and denaturation processes, and also enjoy nutritional, cosmetic, biomedical and pharmaceutical relevance [1]. Although many studies concentrate on the α-amino acids, where amine and carboxylate substituents are on the same carbon atom, a number of other amino acids, where the substituents are on different carbon atoms, also play important biological roles. Among them, γ-aminobutyric acid (GABA), a four carbon non-protein amino acid (Scheme 1), widely distributed in plants, animals and microorganisms, plays a relevant role as a major inhibitory neurotransmitter in the synapses of the central nervous systems [2].

GABA is also used in the treatment of several diseases related with sleeplessness, depression, and autonomic disorders (caused by, e.g., Parkinson‘s disease) [3]. In addition, the anti-inflammatory and anti-oxidant properties of GABA have led to an increase in its use in the food and pharmaceutical industries [4], [5]. It is worth noting that a wide range of traditional foods produced by microbial fermentation contain the safe and eco-friendly GABA, providing new health-benefited products enriched with this amino acid.

Both the neurotransmitter behaviour and the development of new products or formulations containing GABA-require knowledge of its release/migration properties, in vitro and in vivo. Clear differences exist in diffusion in biological systems, such as extracellular space, compared with pure water due to effects such as medium inhomogeneity, anisotropic transport and molecular crowding [6], [7], [8]. However, modelling of behaviour in these systems, and applications of GABA in the food pharmaceutical areas, depend on the understanding of its behaviour in aqueous solution. Following our previous studies on a set of amino-acids [9], [10], [11], [12], [13], [14], [15], [16], inter-diffusion coefficients of γ-aminobutyric acid have been measured over the concentration range from (0.001 to 0.10) mol·dm−3 at 298.15 K using the Taylor dispersion technique. These have been studied in non-buffered aqueous solutions at the natural pH (around 7) to avoid problems of dealing with multicomponent systems, and the diffusion coefficients have been computed assuming a binary GABA/water system. γ-Aminobutyric acid can exist in three different protonated forms (Scheme 1); NMR studies confirm that under these conditions, the dominant species is the electrically neutral, but dipolar, zwitterionic structure, where both carboxylic and amino groups are charged. From the diffusion results, values have been estimated for the diffusion coefficients at infinite dilution, D0, the hydrodynamic radii, Rh (using the Stokes-Einstein equation), and the thermodynamic activity coefficients, through application of the Nernst–Hartley equation. This leads to a better understanding of the thermodynamics of these amino acids in aqueous solutions. The effect of the viscosity of the medium on the diffusion behaviour is discussed, and provides the possibility of applying the data to media other than pure water. For computing activity coefficients, density measurements have been made, and molar volumes calculated from these. The molar volume for GABA in water at neutral pH is identical, within experimental error, to the calculated hydrodynamic volume, indicating the same zwitterionic structure is present, suggested by NMR measurements to have the curved, coil-like conformation illustrated in Scheme 2.

Section snippets

Materials

The γ-aminobutyric acid (GABA) (Sigma-Aldrich Ultra mass fraction purity >0.99; CAS 56-12-2: molar mass = 103.120 g·mol−1) was used as received (Table 1). The solutions for the diffusion and density measurements were prepared using Millipore-Q water. For the NMR measurements, solutions were prepared with D2O (Aldrich >0.99) and the pH was adjusted by addition of DCl (Aldrich >0.99) or NaOD (Aldrich >0.99). All solutions were freshly prepared at T = 298.15 K before each experiment. The pH∗ value

Volumetric properties of γ-aminobutyric acid in water at 298.15 K

The experimental density values of GABA in aqueous solutions at 298.15 K for different concentrations are collected in Table 2 and plotted against the molal concentration in Fig. 1.

These values were well fitted using least-squares regression, by the equation:ρ/(g·cm-3)=0.99710+0.02950m(R2=0.9976)as shown by both the regression coefficient value, R2, and the good agreement between our estimated value of the pure water density value, ρ0, (i.e., 0.99710 g·cm−3) and those found in the literature [23].

Conclusions

We have measured mutual diffusion coefficients and densities of binary aqueous solutions of γ-aminobutyric acid (GABA), at finite concentrations from (0.001 to 0.100) mol·dm−3 at 298.15 K. The slight decreasing with concentration found for the diffusion coefficient of GABA is probably due to interactions inside the GABA molecule, as well as zwitterions-water interactions.

1H and 13C NMR spectra were measured at pH = 1.2; 4.5; 7.3 and 12.2, to characterize the conformation of GABA in these aqueous

Acknowledgements

The authors are grateful for funding from “The Coimbra Chemistry Centre” which is supported by the Fundação para a Ciência e a Tecnologia (FCT), Portuguese Agency for Scientific Research, through the projects UID/QUI/UI0313/2013 and COMPETE Programme (Operational Programme for Competitiveness). NMR data were obtained at the UC-NMR facility which is supported in part by FEDER – European Regional Development Fund through the COMPETE Programme and by National Funds by the FCT, through grants

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