[Highly toxic type â ¡ ribosome-inactivating proteins ricin and abrin and their detection methods: a review].

Type Ⅱ ribosome-inactivating proteins (RIPs) are an important class of protein toxins that consist of A and B chains linked by an interchain disulfide bond. The B-chain with lectin-like activity is responsible for binding to the galactose-containing receptors on eukaryotic cell surfaces, which is essential for A-chain internalization by endocytosis. The A-chain has N-glycosidase activity that irreversibly depurinates a specific adenine from 28S ribosomal RNA (28S rRNA) and terminates protein synthesis. The synergistic effect of the A-B chain inactivates the ribosome, inhibits protein synthesis, and exhibits high cytotoxicity. Ricin and abrin that are expressed by the plants Ricinus communis and Abrus precatorius, respectively, are typical type Ⅱ RIPs. The toxicity of ricin and abrin are 385 times and 2885 times, respectively, more that of the nerve agent VX. Owing to their ease of preparation, wide availability, and potential use as a bioterrorism agent, type Ⅱ RIPs have garnered increasing attention in recent years. Ricin is listed as a prohibited substance under schedule 1A of the Chemical Weapons Convention (CWC). The occurrence of ricin-related bioterrorism incidents in recent years has promoted the development of accurate, sensitive, and rapid detection and identification technology for type Ⅱ RIPs. Significant progress has been made in the study of toxicity mechanisms and detection methods of type Ⅱ RIPs, which primarily involve qualitative and quantitative analysis methods including immunological assays, mass spectrometry analysis methods, and toxin activity detection methods based on depurination and cytotoxicity. Immunoassays generally involve the specific recognition of antigens and antibodies, which is based on oligonucleotide molecular recognition elements called aptamers. These methods are fast and highly sensitive, but for highly homologous proteins in complex samples, they provide false positive results. With the rapid development of biological mass spectrometry detection technology, techniques such as electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) are widely used in the identification of proteins. These methods not only provide accurate information on molecular weight and structure of proteins, but also demonstrate accurate quantification. Enzyme digestion combined with mass spectrometry is the predominantly used detection method. Accurate identification of protein toxins can be achieved by fingerprint analysis of enzymatically digested peptides. For analysis of protein toxins in complex samples, abundant peptide markers are obtained using a multi-enzyme digestion strategy. Targeted mass spectrometry analysis of peptide markers is used to obtain accurate qualitative and quantitative information, which effectively improves the accuracy and sensitivity of the identification of type Ⅱ RIP toxins. Although immunoassay and mass spectrometry detection methods can provide accurate identification of type Ⅱ RIPs, they cannot determine whether the toxins will retain potency. The widely used detection methods for activity analysis of type Ⅱ RIPs include depurination assay based on N-glycosidase activity and cytotoxicity assay. Both the methods provide simple, rapid, and sensitive analysis of type Ⅱ RIP toxicity, and complement other detection methods. Owing to the importance of type Ⅱ RIP toxins, the Organization for the Prohibition of Chemical Weapons (OPCW) has proposed clear technical requirements for the identification and analysis of relevant samples. We herein reviewed the structural characteristics, mechanism of action, and the development and application of type Ⅱ RIP detection methods; nearly 70 studies on type Ⅱ RIP toxins and their detection methods have been cited. In addition to the technical requirements of OPCW for the unambiguous identification of biotoxins, the trend of future development of type Ⅱ RIP-based detection technology has been explored.

Isolation of antitumor proteins abrin-A and abrin-B from Abrus precatorius.

Two toxic proteins were purified from the seeds of Abrus precatorius by DEAE-A 50 and Sepharose 4B chromatography. One of them does not bind on the Sepharose 4B column (Abrin-b) and the other (Abrin-a) is eluted with 0.2 M galactose. The amino acid compositions and tryptic maps of these two proteins were similar, but not identical. The molecular weights estimated by SDS-gel electrophoresis were 67,000 for abrin-b as compared with 65,000 for abrin-a. In the presence of mercaptoethanol, both abrin-a and abrin-b gave rise to two bands. The lethal doses of abrin-a and abrin-b for mice recorded within 48 h were 10 and 25 microgram per kg of body weight respectively. Abrin-a at 0.8 microgram per ml concentration level agglutinated human 0-type erythrocytes, whereas abrin-b showed no such activity. Abrin-a at 5 microgram per ml concentration level agglutinated both the Sarcoma 180 cells and Ehrlich ascites tumor cells, but it required 150 microgram per ml for abrin-b. Both these two proteins at a sublethal dose could inhibit the growth of Ehrlich ascites tumor cells which were injected simultaneously with these proteins. 131I-abrin-a and 131I-abrin-b were able to bind Sarcoma 180 cells, and the binding of abrin-a could be inhibited by lactose, raffinose, galactose and rhamnose, but none of 15 sugars tested inhibited the binding of abrin-b.