Detecting ricin

Sensitive luminescent assay for ricin A-chain ribosome depurination kinetics

Matthew B. Sturm, Vern L. Schramm

Research output: Contribution to journalArticle

54 Citations (Scopus)

Abstract

Ricin is a family member of the lethal ribosome-inactivating proteins (RIP) found in plants. Ricin toxin A-chain (RTA) from castor beans catalyzes the hydrolytic depurination of a single base from a GAGA tetraloop of eukaryotic rRNA to release a single adenine from the sarcin-ricin loop (SRL). Protein synthesis is inhibited by loss of the elongation factor binding site resulting in cell death. We report a sensitive coupled assay for the measurement of adenine released from ribosomes or small stem-loop RNAs by RTA catalysis. Adenine phosphoribosyl transferase (APRTase) and pyruvate orthophosphate dikinase (PPDK) convert adenine to ATP for quantitation by firefly luciferase. The resulting AMP is cycled to ATP to give sustained luminescence proportional to adenine concentration. Subpicomole adenine quantitation permits the action of RTA on eukaryotic ribosomes to be followed in continuous, high-throughput assays. Facile analysis of RIP catalytic activity will have applications in plant toxin detection, inhibitor screens, mechanistic analysis of depurinating agents on oligonucleotides and intact ribosomes, and in cancer immunochemotherapy. Kinetic analysis of the catalytic action of RTA on rabbit reticulocyte 80S ribosomes establishes a catalytic efficiency of 2.6 × 10 8 M -1 s -1, a diffusion limited reaction indicating catalytic perfection even with large reactants.

Original languageEnglish (US)
Pages (from-to)2847-2853
Number of pages7
JournalAnalytical Chemistry
Volume81
Issue number8
DOIs
StatePublished - Apr 15 2009

Fingerprint

Ricin
Adenine
Assays
Kinetics
Ribosome Inactivating Proteins
Orthophosphate Dikinase Pyruvate
Adenosine Triphosphate
Firefly Luciferases
Peptide Elongation Factors
Cell death
Adenosine Monophosphate
Transferases
Oligonucleotides
Catalysis
Luminescence
Catalyst activity
Binding Sites
Throughput
RNA
Proteins

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Detecting ricin : Sensitive luminescent assay for ricin A-chain ribosome depurination kinetics. / Sturm, Matthew B.; Schramm, Vern L.

In: Analytical Chemistry, Vol. 81, No. 8, 15.04.2009, p. 2847-2853.

Research output: Contribution to journalArticle

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