Analysis of cytokinin nucleotides by capillary zone electrophoresis with diode array and mass spectrometric detection in a recombinant enzyme in vitro reaction |
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Authors: | Tibor Bé res,Marké ta Gemrotová ,Petr Tarkowski,Markus Ganzera,Ví tězslav Maier,David Friedecký ,Marco A. Dessoy,Ludger A. Wessjohann,Luká &scaron Spí chal,Miroslav Strnad,Karel Doležal |
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Affiliation: | 1. Laboratory of Growth Regulators, Institute of Experimental Botany ASCR, Šlechtitel? 11, 783 71 Olomouc, Czech Republic;2. Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Šlechtitel? 11, 783 71 Olomouc, Czech Republic;3. Institute of Pharmacy, Pharmacognosy, University of Innsbruck, Innrain 52, 6020 Innsbruck, Austria;4. Regional Centre for Advanced Technologies and Materials, Department of Analytical Chemistry, Faculty of Science, Palacký University in Olomouc, 17. listopadu 12, Olomouc 771 46, Czech Republic;5. Department of Clinical Biochemistry, Laboratory for Inherited Metabolic Disorders, University Hospital and Palacký University, I.P. Pavlova 6, 775 20 Olomouc, Czech Republic;6. Leibniz Institute of Plant Biochemistry, Department of Bioorganic Chemistry, Weinberg 3, D-06120 Halle (Saale), Germany |
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Abstract: | A capillary zone electrophoresis (CZE) method for separation of adenosine and N6-isopentenyladenosine (cytokinin) nucleotides was developed, optimized and validated. Aqueous solutions of several amino acids were evaluated as the background electrolyte constituents. Separation of six nucleotides in less than 20 min with high theoretical plate number (up to 400 000 for isopentenyladenosine triphosphate) was achieved using a 100 mM sarcosine/ammonia buffer at pH 10.0. The detection limits of the CZE-UV method are in the low micromolar range (0.69–1.27 μmol L−1). Good repeatability of migration times (within 1.3%), peak areas (within 1.8%) and linearity (R2 > 0.999) was achieved over the concentration range 5–1000 μmol L−1. The method was used to assay the activity of the recombinant Arabidopsis thaliana isopentenyltransferase 1 (AtIPT1). Baseline separation of isopentenylated nucleotides by CE–ESI-MS using a volatile buffer (30 mM ammonium formate; pH 10.0) was accomplished. The identities of the reaction products – isopentenyladenosine di- and triphosphate were confirmed by HPLC-QqTOF-MS. Dephosphorylation of ATP was observed as a parallel reaction. |
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Keywords: | Cytokinin nucleotides Capillary electrophoresis Isopentenyltransferase Cytokinin biosynthesis |
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