A fluorescent-labeled microcystin-LR terbium cryptate

Abstract

com o grupo éster do criptato de térbio. A formação do produto foi acompanhada por cromatografia líquida de alta eficiência (CLAE) em 238 nm e 310 nm. A presença de microcistina-LR na molécula marcada foi confirmada através de ensaio com enzima ligada a imunoabsorvente (ELISA) e por reação protéica com ácido bicincônico. O espectro de luminescência do criptato e da molécula conjugada também foram confirmados.A new fluorescent labeled compound of microcystin-LR with terbium cryptate was obtained by initial conjugation of microcystins-LR with aminoethanethiol followed by the reaction with the ester group of terbium cryptate. The product formation was followed by high performance liquid chromatography (HPLC) at 238 nm and 310 nm. The presence of microcystin-LR in the labeled molecule was confirmed by enzyme linked immunosorbent assay ELISA and by protein reaction with bicinhonic acid. Luminescence spectra of cryptate and the conjugated molecule were carried through as well. Keywords: terbium cryptate, microcystin-LR, cyanobacteria IntroductionMicrocystins are a group of stable heptapeptide hepatotoxins produced by freshwater cyanobacteria genera that lower the water quality leading to an increase in the risk of intoxication for animals and humans. Species of genera Microcystis, Anabaena, Aphanizomenon, Planktothrix (Oscillatoria), and Nostoc are frequently described as producers of microcystins. 1 The eutrophication of ponds, rivers and other freshwater ecosystems allows cyanobacteria to flourish, leading to contamination of the water. Microcystins are potent inhibitors of protein phosphatases 1 (PP 1 ) and 2A (PP 2A ), which are regulatory enzymes present in the cytosol of mammalian cells. 3 The general structure of microcystins is composed of cyclo-(D-Ala-X-D-MeAsp-ZAdda-D-Glu-Mdha), where X and Z could be various kinds of L-amino acids; D-MeAsp is D-erythro-β-methylaspartic acid; Mdha is N-methyldehydroalanine; and Adda is the unusual amino acid 2S,3S,8S,9S-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4E,6E-dienoic acid. 4 The most extensively studied form is microcystin-LR that contains L-leucine and L-arginine in the two main variant positions. In the search for more sensitive markers, some chelates of lanthanides had already been obtained and tested in various biological assay models. [25][26][27] Time-resolved fluoroimmunometric assay (TR-FIA) is a method based on the luminescence emission that is measured after a delay has elapsed from a pulsed excitation, enabling the short-lived background fluorescence to be excluded. 28Soini et. al.29 labeled anti-rabbit IgG with europium chelate as a model to detect rabbit IgG for human smooth muscle myosin in a histological section; Bonin et al. 30 used a complex of europium and samarium for the detection of diphteria antitoxins in serum using the ELISA method and Xu et al.31 labeled anti-β-LH and monoclonal anti-β-FSH antibodies with europium and samarium, respectively. The use of luminescent lanthanide as a substitute for labeled enzyme (e.g. horseradish peroxidas...

Lanthanides and Algae

“…They are used in sensitive immunoassays to suppress the background [122] or as very sensitive fluorescence probes [123]. An example of their use is the labeling of the cyanotoxin microcystin [124,125].…”

Facile and rapid access to linear and truncated microcystin analogues for the implementation of immunoassays

“…To our knowledge, there are no reports in the literature for fluorescent labelling of this unusual b-amino acid. Several fluorescent probes of MC-LR have been prepared but the labelling strategies are based on the chemoselective derivatisation of the side-chain of an amino acid other than Adda: (1) conjugation under basic conditions of a tetramethylrhodamine 1,3-diketone derivative to the arginine side-chain 27 or (2) a two-step process involving a Michael type addition of an aminothiol (i.e., cysteamine or cysteine) to the a,b-unsaturated carbonyl of the Mdha residue followed by the reaction of its amino group with N-hydroxysuccinimidyl (NHS) of a terbium cryptate 28 or isothiocyanate derivative of fluorescein (FITC). 29 Consequently, we have explored the synthesis of an aldehyde-reactive Adda-based fluorescent "tripod" through the four-step sequential derivatisation of an in-house-developed heterotrifunctional cross-linking reagent (Scheme 4).…”