A Novel Stereospecific Bioluminescent Assay for Detection of Endogenous D-Cysteine

The presence of endogenous D-stereoisomers of amino acids in mammals dispels a long-standing dogma about their existence. D-Serine and D-aspartate function as novel neurotransmitters in mammals. However, the stereoisomer with the fastest, spontaneous in vitro racemization rate, D-cysteine, has not been reported. We utilized a novel, stereospecific, bioluminescent assay to identify endogenous D-cysteine in substantial amounts in the eye, brain, and pancreas of mice. D-Cysteine is enriched in mice embryonic brains at day E9.5 (4.5 mM) and decreases progressively with development (mu M levels). D-Cysteine is also present in significantly higher amounts in the human brain white matter compared with gray matter. In the luciferase assay, D-cysteine conjugates with cyano hydroxy benzothiazole in the presence of a base and reducing agent to form D-luciferin. D-Luciferin, subsequently, in the presence of firefly luciferase and ATP, emits bioluminescence proportional to the concentration of D-cysteine. The assay is stereospecific and allows the quantitative estimation of endogenous D-cysteine in tissues in addition to its specificity for D-cysteine. Future efforts aimed at bioluminescent in vivo imaging of D-cysteine may allow a more noninvasive means of its detection, thereby elucidating its function.

Automated summary

The study reveals the presence of endogenous D-cysteine in mammals and introduces a novel bioluminescent assay for its quantitative estimation in tissues. The findings highlight the enrichment of D-cysteine in embryonic brains and its higher levels in human brain white matter compared to gray matter.