Ricin, denaturation

Abrin is a plant source Type 2 RIP. It is found in Abrus precatorius (rosary pea, Indian licorice, jequirity bean). The toxicology of abrin is considered to be very similar to ricin. A similar Abrus toxin is pulchellin, produced by A. pul-chellus (Millard and LeClaire, 2008). The rosary pea has been reported to be more toxic than castor beans (Griffiths et al, 1994). Species sensitivity is variable and horses are considered to be the most sensitive. The mature goat is considered to be a more resistant species and 2 g of seed/kg body weight is reported as a lethal dose. The lethal dose for cattle is reported at 600 mg of seed/kg body weight. It is likely that abrin is denatured in the rumen (Burrows and Tyrl, 2001). 

Heating a 1% (w/v) solution of ricin to >85°C for 30 min results in complete inactivation as judged by toxicity in laboratory mice (Hunt et al., 1918). Dry heat of >100°C for 60 min in an ashing oven or steam autoclave treatment at >121 °C for 1 h reduces the activity of pure ricin by >99% (Wannemacher et al., 1989). Heat inactivation of impure toxin preparations (e.g., crude ricin plant extracts) may vary. Heat-denatured ricin can undergo limited refolding (<1%) to yield active toxin. Isolated RTA and RTB are more easily inactivated by heating than is the holotoxin (Olsnes et al., 1975 Taira et al., 1978). 

Another example of the use of mass spectrometry to delect toxins is to identify ricin, a highly toxic protein that inhibits cell protein synthesis. Ricin is produced from the seeds of Ricinus communis plants (known conunonly as castor beans) [73]. Structurally, ricin is made of A- and B-chains linked by a disulfide bridge. The toxicity of ricin is due primarily to the A-chain, which acts as an RNA A-glycosidase, which leads to ribosome incapacitation and ultimately to cell death. Ricin was identified from crude castor bean extracts using LC-MS/MS. The extract was denatured, reduced, and alkylated prior to trypsin digestion. Ricin identification was based on the detection of marker peptides in the digest. These markers include T5, T7, Til, T12, and T13 from the A-chain and T3, T5, T14, T19, and T20 from the B-chain. MS/MS can provide the amount and sequence of each marker for irrefutable evidence. For quick screening of ricin in crude extracts, MALDI-MS can be used to provide the molecular mass profile of the marker peptides. 

To detect ricin, we studied its inhibitory effects on luciferase expression by adding a series of concentrations of ricin into the IVT reactions in the array device. To achieve a lower detection limit, 4-hr protein expression was used, though ricin detection can be achieved in as short as 5 minutes. Ricin sample size was 2 pL. As shown in Figure 5a, the expression yield of luciferase, indicated by luminescence, decreases with the concentration of ricin (solid circles). However, the expression yield remained the same when the ricin is heat denatured and its toxicity is deactivated (open circles). The error bar of each data point indicates the standard deviation that is obtained from three repeat experiments. The calibration curve is obtained by plotting the detection... 

Figure 5 (a) The inhibitory effects of ricin on the production yield of luciferase. The expression yield of luciferase is plotted as a function of the concentration of ricin (solid circles) and heat-denatured ricin (open circles), (b) Calibration curve for ricin detection. (Reproducedfrom reference 14. Copyright 2006American Chemical Society)... 

Fig. 59. Dep>endence of the first-order rate constant, ki, on pH and temperature for the denaturation of ricin (Levy and Benaglia, 1950).

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