Quantification of ricin, RCA and comparison of enzymatic activity in 18 Ricinus communis cultivars by isotope dilution mass spectrometry

Abstract

The seeds of the Ricinus communis (Castor bean) plant are the source of the economically important commodity castor oil. Castor seeds also contain the proteins ricin and R. communis agglutinin (RCA), two toxic lectins that are hazardous to human health. Radial immunodiffusion (RID) and the enzyme linked immunosorbent assay (ELISA) are two antibody-based methods commonly used to quantify ricin and RCA; however, antibodies currently used in these methods cannot distinguish between ricin and RCA due to the high sequence homology of the respective proteins. In this study, a technique combining antibody-based affinity capture with liquid chromatography and multiple reaction monitoring (MRM) mass spectrometry (MS) was used to quantify the amounts of ricin and RCA independently in extracts prepared from the seeds of eighteen representative cultivars of R. communis which were propagated under identical conditions. Additionally, liquid chromatography and MRM-MS was used to determine rRNA N-glycosidase activity for each cultivar and the overall activity in these cultivars was compared to a purified ricin standard. Of the cultivars studied, the average ricin content was 9.3 mg/g seed, the average RCA content was 9.9 mg/g seed, and the enzymatic activity agreed with the activity of a purified ricin reference within 35% relative activity.

Keywords: Cultivar; Mass spectrometry; Quantification; RCA; Ricin; Ricinus communis.

Fig. 1

Measurement of purified ricin and RCA standard concentrations by isotope dilution mass spectrometry. Three different tryptic digestion conditions using 4 µg, 40 µg and 40 µg with reduction and alkylation were used to digest ricin and RCA. Quantity of protein was determined using a peptide based standard calibration curve. The ricin A-chain peptides are denoted in dark gray, ricin B-chain peptide is medium gray and the RCA A-chain peptides are denoted in light gray. Error bars show one standard deviation from the mean for three replicates.

Fig. 2

Measurement of the quantity of ricin and RCA in individual cultivars in mg/g of seed as determined by isotope dilution mass spectrometry. Dark gray denotes quantity of ricin calculated from the mean of the A-chain peptides VGLPINQR and LTTGADVR, light gray denotes quantity of RCA calculated from the mean of the A-chain peptides VGLPISQR and SHLTTGGDVR. The y-axis is the quantity of ricin or RCA in mg/g seed. Asterisk denotes small seed cultivars. Each value represents the mean of three replicates; error bars show one standard deviation from the mean.

Fig. 3

Quantity of ricin in fmol in individual cultivars as calculated from individual A- and B-chain peptides. The mean quantity of A-chain peptides LTTGADVR and VGLPINQR is denoted in dark gray. The mean quantity of B-chain peptide ASDPSLK is denoted in light gray. Control samples of stock purified ricin are shown at 90 and 900 fmol. Error bars represent one standard deviation from the mean for three individual replicates.

Fig. 4

Quantification of ricin and RCA in replicate extractions from R. communis gibsonii and zanzibarensis seeds. Measured amount of ricin (dark gray) and RCA (light gray) in mg/g of seed in five individual replicate extractions from the gibsonii variety of seed and three individual extractions from the zanzibarensis seeds. Each value represents the mean of six individual IDMS replicates; error bars show one standard deviation from the mean.

Fig. 5

Quantification of ricin A-chain and B-chain in replicate extractions from R. communis gibsonii and zanzibarensis seeds. Measured amount of ricin A-chain (dark gray) and B-chain (light gray) are in fmol from five individual replicate extractions from the gibsonii variety of seed and three individual extractions from the zanzibarensis seeds. Each value represents the mean of six individual IDMS replicates; error bars show one standard deviation from the mean.

Fig. 6

Quantification of overall N-glycosidase activity for each cultivar seed extract using the adenine release assay. The N-glycosidase activity for each cultivar is reported as a percent relative to the purified ricin reference standard (Ricin_reference =100%). PBST_05 is the negative, ricin-free control. Each value represents the mean of three replicate analyses and error bars show one standard deviation from the mean.

Fig. 7

a. Separation and visualization of the ricin reference standard by one dimensional SDS-PAGE and silver staining. Molecular weight markers are in the left lane and 2, 1 and 0.5µg of the reference ricin are in the subsequent lanes. Individual ricin chains are likely located between 25 and 37 kDa b. is a duplicate gel use in the Western blot analysis. A series of immuno-reactive bands to the goat anti-ricinus communis antibody are seen between 50 and 75 kDa, 25–37 kDa and 10–20 kDa.