Inhibition equivalency factors for microcystin variants in recombinant and wild-type protein phosphatase 1 and 2A assays

• Published in: Environmental science and pollution research international 2014-09, Vol.21 (18), p.10652-10660
• Main Authors: Garibo, Diana, Flores, Cintia, Cetó, Xavier, Prieto-Simón, Beatriz, del Valle, Manel, Caixach, Josep, Diogène, Jorge, Camp s, Mònica
• Format: Article
• Language: English
• Subjects: Aquatic Pollution; Atmospheric Protection/Air Quality Control/Air Pollution; Chromatography; Chromatography, Liquid; Cyanobacteria - chemistry; detection limit; Earth and Environmental Science; Ecotoxicology; Environment; Environmental Chemistry; Environmental Health; Enzyme Inhibitors - chemistry; Enzymes; Humans; inhibitory concentration 50; Kinases; Liquid chromatography; Mass Spectrometry; Mass spectroscopy; microcystin-LR; Microcystins; Microcystins - chemistry; Performance evaluation; Phosphatase; Phosphoprotein phosphatase; Protein phosphatase; Protein Phosphatase 1 - antagonists & inhibitors; Protein Phosphatase 1 - chemistry; Protein Phosphatase 1 - genetics; Protein Phosphatase 1 - metabolism; Protein Phosphatase 2 - antagonists & inhibitors; Protein Phosphatase 2 - chemistry; Protein Phosphatase 2 - genetics; Protein Phosphatase 2 - metabolism; Proteins; Research Article; Scientific imaging; screening; Toxicity; Toxins; Waste Water Technology; Water Management; Water Pollution Control; World Health Organization
• ISSN: 0944-1344; 1614-7499
• DOI: 10.1007/s11356-014-3065-7

In this work, protein phosphatase inhibition assays (PPIAs) have been used to evaluate the performance of recombinant PP1 and recombinant and wild-type PP2As. The enzymes have been compared using microcystins-LR (MC-LR) as a model cyanotoxin. Whereas PP2ARₑc provides a limit of detection (LOD) of 3.1 μg/L, PP1Rₑc and PP2AWᵢₗd provide LODs of 0.6 and 0.5 μg/L, respectively, lower than the guideline value proposed by the World Health Organization (1 μg/L). The inhibitory potencies of seven MC variants (-LR, -RR, -dmLR, -YR, -LY, -LW and -LF) have been evaluated, resulting on 50 % inhibition coefficient (IC₅₀) values ranging from 1.4 to 359.3 μg/L depending on the MC variant and the PP. The PPIAs have been applied to the determination of MC equivalent contents in a natural cyanobacterial bloom and an artificially contaminated sample, with multi-MC profiles. The inhibition equivalency factors (IEFs) have been applied to the individual MC quantifications determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, and the estimated MC-LR equivalent content has been compared to PPIA results. PPIAs have demonstrated to be applicable as MC screening tools for environmental applications and to protect human and animal health.