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

Robin Roychaudhuri, Moataz M. Gadalla, Timothy West, Solomon H Snyder

Research output: Contribution to journalArticlepeer-review

Abstract

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 (μM 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.

Original languageEnglish (US)
Pages (from-to)3257-3262
Number of pages6
JournalACS Chemical Neuroscience
Volume13
Issue number23
DOIs
StatePublished - Dec 7 2022

Keywords

  • bioluminescent
  • d -amino acids
  • d -cysteine
  • endogenous
  • luciferase
  • mammals
  • neurotransmitter
  • racemization
  • serine racemase
  • stereospecific

ASJC Scopus subject areas

  • Biochemistry
  • Physiology
  • Cognitive Neuroscience
  • Cell Biology

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