title: A tyrosine-containing analog of mu-conotoxin GIIIA as ligand in the receptor binding assay for paralytic shellfish poisons.
abstract: Development of novel analytical tools to detect marine biotoxins has been warranted in view of the apparent global pervasiveness of algal-derived shellfish poisoning, and the limitations of existing methods. Here, we describe the initial phase in the development and evaluation of a tyrosine-containing analog of μ-conotoxin (μ-CTX) GIIIA as an alternative to saxitoxin (STX) in a receptor binding assay (RBA) for paralytic shellfish poisons. The peptide analog was synthesized and characterized for structure and bioactivity. The major product of oxidation elicited paralytic symptoms in mice at a minimum dose of 1.31 mg kg(-1) (i.p.). Mass spectrometry analysis of the bioactive peptide gave a molecular mass of 2637.52 Da that was close to the predicted value. Iodination via chloramine-T produced non-, mono- and di-iodinated peptides (respectively, NIP, MIP and DIP). Competition assays against (3)H-STX revealed higher Ki and EC50 (P < 0.0001, ANOVA) indicating reduced affinity for the receptor, and limited displacement of receptor-bound STX. However, subsequent use of MIP may extend the application of RBA to detect small changes in toxin levels owing to its likely enhanced displacement by STX. This may be useful in analyzing samples with toxicities near the regulatory limit, or in establishing baseline values in high risk environments.
authors: Philippine Nuclear Research Institute, Commonwealth Avenue, Diliman, Quezon City 1101, Philippines; Marine Science Institute, Velasquez St., University of the Philippines, Diliman, Quezon City 1101, Philippines. Electronic address: adlmendoza@pnri.dost.gov.ph.
authors: Aileen D L
authors: AD
authors: Mendoza
authors: Philippine Nuclear Research Institute, Commonwealth Avenue, Diliman, Quezon City 1101, Philippines. Electronic address: ezsombrito@gmail.com.
authors: Elvira Z
authors: EZ
authors: Sombrito
authors: Marine Science Institute, Velasquez St., University of the Philippines, Diliman, Quezon City 1101, Philippines. Electronic address: lulycruzj@yahoo.com.
authors: Lourdes J
authors: LJ
authors: Cruz
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001667:Binding, Competitive; D001681:Biological Assay; D020916:Conotoxins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D005506:Food Contamination; D005513:Food Inspection / Q000379:methods*; D054879:Halogenation; D006801:Humans; D008024:Ligands; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D009124:Muscle Proteins / Q000378:metabolism*; D009498:Neurotoxins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D010455:Peptides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D010679:Philippines; D017207:Rats, Sprague-Dawley; D012530:Saxitoxin / Q000032:analysis* / Q000378:metabolism / Q000633:toxicity; D012758:Shellfish / Q000032:analysis; D057096:Shellfish Poisoning / Q000209:etiology / Q000378:metabolism; D014316:Tritium
chemical_list: D020916:Conotoxins; D008024:Ligands; D009124:Muscle Proteins; D009498:Neurotoxins; D010455:Peptides; D014316:Tritium; C046101:conotoxin GIII; D012530:Saxitoxin
keywords: Mu-conotoxin GIIIA; Paralytic shellfish poisoning; Receptor binding assay; Saxitoxin