The 1999-2000 Beckman Scholars: Cindy V. Ly
Beckman research project in the Shear group:
Sensitive and selective strategy for probing D-amino acids in biological microenvironments
Contrary to prevailing dogma regarding dependence of biological systems on L-amino acids, trace levels of the D-enantiomers of some amino acids have been detected in neurological tissue. The objective of my research was to develop a sensitive and selective method that would allow analysis of D-amino acid neuromodulators at cellular and sub-cellular levels, enabling us to explore the potential role of these molecules in neuronal activity.
An enzymatic approach was established in which D-amino acid oxidase catalyzes the production of H2O2 from D-alanine; peroxidase then mediates the oxidation of tyramine, in the presence of H2O2, to produce a fluorescent dimer. Thus, D-alanine levels correspond stoichiometrically to the amount of dimerized tyramine produced. Detection was achieved using capillary-zone electrophoresis coupled to multi-photon excited fluorescence (CZE-MPE). CZE-MPE is an analytical system well-suited to this application because CZE accommodates volume-limited samples in the nanoliter to picoliter range and permits separation of these samples into their component parts; MPE fluorescence provides sensitive and simultaneous detection of multiple chromophores in a biological matrix using a single laser source.
Analysis of reactions performed off-column gave concentration and mass detection limits of 1.4 +/- 0.6 microM and 90 +/- 40 amole, respectively. However, on-column methods in which a portion of the capillary is used as a microreactor are more suited to analysis of biological microenvironments because dilution of the analyte is limited and only minimal reagent is needed. On-column approaches yielded detection limits of 13 +/- 5 microM and 400 +/- 100 amole. No significant response is observed when assay reagents are incubated with a mixture of other neuronally relevant amino acids, including D-aspartate, glycine, D-glutamate, D-serine, and L-alanine; therefore, this assay is selective for D-alanine. Although D-amino acid oxidase is reported to have activity with several other D-amino acids, evidence suggests that these alternative substrates are not biologically prevalent.
Although close to publication, several experimental parameters must be adjusted before drafting a manuscript for a journal such as Analytical Chemistry. Firstly, our sensitivity can be improved significantly by using optical filters that maximize collection of emission and minimize collection of background (e.g., laser scatter). Furthermore, reducing the capillary diameter from 6 micrometers, presently used, to 1 micrometer or smaller would lead to substantial gains in mass detection limits. In addition, a multi-component analysis will be performed to demonstrate the ability of the technique to simultaneously analyze various neuroactive compounds.
In estimating the sensitivity that will be required to analyze possible D-alanine content at a subcellular level, we can begin by considering the total alanine content of a cell: neurons from the land snail Helix aspersa, for example, have been reported to contain alanine at picomole quantities and millimolar concentrations.1 It is not currently known what fraction of this total alanine may exist as the D-enantiomer; the established approach may be adequate for analysis at this cellular scale. However, by making the suggested system modifications, we expect improvements that would make our detection strategy conducive to subcellular determination of D-amino acids for analysis of secretory and storage vesicles within a single-cell.
1. Oates, Mary D., Cooper, Bruce R., Jorgenson, James W., Analytical Chemistry, 1990, 62, 15731577.
Created and maintained by Ruth Shear. Comments to author at DrRuth@mail.utexas.edu
Created Mon Mar 22nd 1999. Last modified Thu, Mar 10, 2011.