new phosphatase isolated from soy bean used in microcystin-LR assay

4. ANDRINOLO D, OLIVER C, BAUZA L, CARELLA N, GIANNUZZI L, ECHENIQUE R, CANEO M. A

Rio de Janeiro. Brasil

Congreso; International Conference on Cyanobacteria 5-10 August 2007; 2007

UFRJ

Microcystins are produced by different species of Cyanobacteria, and it has described more of 50 chemical variants. The regular molecular target is protein phosphatases such as 1A and 2B, both present in liver. In effect, liver is the main organ target for microcystin, being probably related to primary liver cancer. Because of these effects and the intermittent presence in tap water is necessary to have methods to measure these toxins in fast, and if it’s possible, cost-effective manner. Among the methods to evaluate microcystins it can be mentioned the inhibition phosphatases assay, the most expensive HPLC/DAD and HPLC/MS, and several immunologic assays based on ELISA. The inhibition of phosphatases by microcystin is an economic method to analyze many samples in short periods of time, such as summer blooms. The objective of this work was to develop a inhibition phosphatase assay from a vegetal protein source. According to previous work in our laboratory, the phosphatase A2 (e.g. PP2A) purified from soy bean shows a measurable inhibition produced by microcystin LR. The enzyme was thoroughly purified from roots of soy beans using homogenization with mortar on ice bath, centrifugation (10,000 g, 4°C), chromatography in DEAE-Sephacel and Agarose-Heparin. Purification steps were followed by SDS-PAGE (Tris-Glycine, Laemmli system). Enzymatic activity was evaluated using the hydrolysis of pnitrophenol phosphate and measuring spectrophotometrically p-nitrophenol in alkaline medium. The method was applied on artificial water samples and samples from Rio de la Plata. Response was linear in the range of 0,1 a 10 ppm of microcystin LR. Every microcystin shows different inhibition, so the proposed assay only can be expressed as equivalent to “microcystin LR”. In addition, this method is easily applicable in laboratories with minimum equipment and useful to detect the beginning of a toxic bloom