HPLC separation and analysis method to evaluate molecules involved in the arginine-ornithine cycle in Phytomonas

L. FRACCAROLI; PS. TORRES; MD. RUIZ; MS. MARCORA; V. DE PINO; C. CARRILLO

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencia; 2017

Reunión de la Sociedad Argentina de Protozoología

Phytomonas spp. are trypanosomatid parasites that infect a varietyof plants and can cause diseases that affect regional economies,mainly in South America. Little is known about Phytomonas biology;however, recently published genomic data provide new venues forresearch. We are interested in the study of arginine ? ornithine cyclewith special focus on the enzymes involved in the conversion of arginine-citrulline-ornithine.To confirm our previous evidences about the presence of NOS andODC and the absence of ADC and AGMATINASE enzymes in thiscycle, we set up a method for separation and analysis of productsand intermediates using high-performance liquid chromatography(HPLC) that involves precolumn derivatization with o-phthaldialdehyde,C18 analytical column and fluorescence detection. We testedtwo mobile phase solutions (MP): 1- MPA: 0.1M sodium acetate,pH7.2/MPB: 100% methanol and 2- MPA: 25mM phosphate bufferpH7.5/MPB: acetonitrile:methanol:water 45:45:10, both at three differenttemperatures (15, 25 and 40ºC). Under these different conditionswe separated a mixture of standard aminoacids: arginine,ornithine, citrulline, agmatine, lysine, glycine and threonine. Wefound that mobile phase 2 at 40ºC was the best condition to obtainindividual peaks for each aminoacid with a detection limit of at least1uM. As a first in vivo approach, Phytomonas Jma was cultured ina semi-defined media (SDM-79) and extracts obtained from 2 and10x106 cells were deproteinized and neutralized. The HPLC chromatogram,under the selected conditions, showed the seven aminoacidpeaks well resolved and a low background signal. Furtheranalysis will be made to analyze the aminoacid profile under differentculture conditions.In conclusion, we have set up a HPLC protocol that will be usefulto analyze Phytomonas metabolism. This method could also beused to analyze aminoacids in a variety of biological samples suchas physiological fluids, tissue extracts and different cell types.