Abstract
GABA (γ-amino butyric acid) is a biologically active four carbon non-protein amino acid found in prokaryotes and eukaryotes. Glutamate is a five carbon α-amino acid which can be converted to GABA catalyzed by the enzyme glutamate decarboxylase. In this protocol, we describe the procedure for extraction and quantification of GABA and glutamate from cells of the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). Apart from Synechocystis, this protocol has already been successfully tested for the cyanobacterium Nostoc punctiforme and for the green alga Tetraspora sp. CU2551. The protocol can also be used for the analyses of other primary amino acids. We have successfully employed this protocol in our studies of GABA and glutamate analyses in Synechocystis (Kanwal and Incharoensakdi, 2016).
Keywords: GABA assay, Glutamate assay, Amino acid analysis, Cyanobacteria, Synechocystis
Background
Amino acid analysis has been done previously by employing a number of methods that include the detection using thin layer chromatography or ion exchange separation coupled with post-column derivatization with ninhydrin method or also using HPLC by fluorescent detection. However, several challenges are encountered in those detection methods such as poor sensitivity, degradation of derivatives, formation of multiple derivatives and longer retention times causing higher solvent consumption. Furthermore, the UV detection methods alone could not be used owing to optically inactive nature of amino acids.Here, we describe the detailed procedure for extraction and analyses of amino acids such as GABA and glutamate in Synechocystis. The method used for amino acids separation and detection is high performance reversed-phase liquid chromatography followed by UV detection of pre-column OPA (o-phthaldehyde) derivatization of amino acids adopted from Henderson et al. (2006). This method offers several advantages over previous methods by improving the sensitivity of detection. Reversed-phase chromatography allows faster separation of amino acids, whereas UV detection offers improved sensitivity. OPA derivatization of amino acids results in stable derivatives that make the UV detection of amino acids possible. The method also offers the detection and quantification of amino acids within 26 min, which consequently reduces solvent consumption.
Materials and Reagents
Equipment
Procedure
Data analysis
Figure 2. Representative chromatograms of separated amino acids extracted from the cyanobacterium Synechocystis sp. PCC 6803. A. Chromatogram of standard glutamate and GABA for the concentration of 2 nmol/ml; B. Chromatogram of amino acid separation from Synechocystis sp. PCC 6803 extract. Data were obtained from Shimadzu LC solution software and were confirmed by three independent experiments.
Notes
Recipes
Acknowledgments
The method for GABA and glutamate extraction was adapted from Bieleski and Turner (1966) with some modifications. The HPLC-based method for the detection and quantification of GABA and glutamate is a modification of protocol published by Henderson et al. (2006). This work was supported by research grants from Chulalongkorn University (CU) Ratchadaphiseksomphot Endowment Fund (Food and Water Cluster, CU-58-011-FW) and from the Thailand Research Fund (IRG5780008). Simab Kanwal thanks CU Graduate School for providing post-doctoral fellowship.
References
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