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Two new methods for ricin detection
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ANALYTICAL CHEMISTRY /
MAY 1, 2009
gested with trypsin and analyzed by LC/ MS/MS to confirm the identity of the toxin and to rule out false-positive nuclease activity.
SHUTTERSTOCK
Ricin, a protein extracted from the castor bean, is one of nature’s most potent toxins. A single castor bean contains sufficient ricin to kill ⬎1000 people if the purified toxin is injected or inhaled. Therefore, ricin is considered a major bioterrorism threat. That’s why two groups of researchersOSuzanne Kalb and John Barr at the U.S. Centers for Disease Control and Prevention, and Matthew Sturm and Vern Schramm at the Albert Einstein College of MedicineOindependently developed assays for the sensitive detection of ricin’s enzymatic activity. Their results are reported in two new AC papers (DOI 10.1021/ac802769s; 10.1021/ ac8026433). Castor beans are the source of castor oil, which has many industrial, automotive, and medicinal applications. But castor oil production generates large quantities of ricin as a waste material, and this ready availability is one reason that the toxin is considered such a threat. Ricin exerts its deadly effect by catalyzing the depurination of 28S ribosomal RNA (rRNA). This depurination, which releases a single adenine from an RNA motif known as the sarcin⫺ricin loop, disrupts the binding of elongation factor 2 to 28S rRNA. As a result, protein synthesis is inhibited, and the cell dies. Although several methods for the detection of ricin protein have been reported, these assays generally do not discriminate between inactive and active ricin. According to Barr, “We wanted to develop a method that was very sensitive and selective for the total analysis of ricin, which includes the biological activity and a structural component.” Kalb and Barr’s MS-based technique incorporates three layers of selectivity to detect both ricin presence and activity in the same sample. First, ricin was isolated from food and clinical samples by immunoaffinity capture on antibody-coated beads. Then, ricincatalyzed depurination of a DNA mimic of the 28S rRNA substrate was detected by MALDI TOFMS. Finally, immunopurified ricin from the activity assay was di-
The castor bean plant produces ricin, a potent toxin and potential bioterrorism threat.
In the activity assay, MS peaks that corresponded to depurinated substrate were detected in milk, apple juice, human serum, and human saliva spiked with 1⫺5 pmol ricin but were not detected in samples lacking ricin. The compositional analysis identified ricin peptides and allowed discrimination between ricin isoforms and between ricin and a less toxic homolog. “One of the challenges with ricin is to differentiate between toxic and much less toxic forms,” says Barr. “Being able to discriminate between these forms with the structural analysis can give you additional information on the risk factors associated with the sample.” Sturm and Schramm took a different approach to ricin detection with an assay that couples RNA depurination to light production by firefly luciferase. Small RNA targets or intact eukaryotic ribosomes served as ricin substrates. According to Schramm, “We wanted to detect ricin activity acting on a physiological
substrateOin other words, the ribosome.” To detect the ricin-catalyzed release of a single adenine from a ribosome, the investigators converted the adenine to adenosine monophosphate (AMP) and then to ATP with the enzymes adenine phosphoribosyl transferase and pyruvate orthophosphate dikinase (PPDK). Finally, ATP was used by luciferase to produce light. The emitted light, which was proportional to adenine concentration, was measured with a luminometer. “The beauty of this system is that luciferase regenerates AMP, which can be converted back to ATP by PPDK,” says Schramm. “So it’s a cyclic amplification that allows us to detect adenine at the femtomole scale.” To remove contaminating AMP and ATP from isolated ribosome preparations, the investigators passed the ribosomes through a size exclusion spin column. The luminescent assay revealed that the depurinating activity of purified ricin on rabbit reticulocyte 80S ribosomes approaches catalytic perfection. “We can do real-time kinetics and get rigorous kinetic constants like turnover number and inhibition constant using the actual substrate of ricin, mammalian ribosomes,” says Schramm. Both groups envision many applications for their ricin detection assays. Kalb and Barr plan improvements to their method to enhance the detection and quantitation of ricin in food and clinical samples. “We’re working on getting a very accurate quantitative method using MS isotope dilution techniques,” says Barr. “We’re also trying to improve the sensitivity for the structural components and to improve the speed.” Schramm says that his group is looking for ways to combat ricin poisoning. “Our real focus is to make transition-state analog inhibitors of ribosome-inactivating proteins as rescue agents for bioterrorism or cancer therapy,” he explains. “We can use this assay to screen inhibitor libraries and to establish the power of some inhibitors we’ve made.” Barr and Kalb’s paper appears in the March 15 issue of Analytical Chemistry. —Laura Cassiday
10.1021/AC900458V 2009 AMERICAN CHEMICAL SOCIETY
Published on Web 03/20/2009
