期刊
ANALYTICA CHIMICA ACTA
卷 751, 期 -, 页码 176-181出版社
ELSEVIER
DOI: 10.1016/j.aca.2012.08.049
关键词
Cytokinin nucleotides; Capillary electrophoresis; Isopentenyltransferase; Cytokinin biosynthesis
资金
- Czech Ministry of Education, Youth, and Sports [MSM 6198959216, LC06034]
- Centre of Region Hana for Biotechnological and Agricultural Research [ED0007/01/01]
- Grant Agency of the Czech Republic [522/08/0920]
A capillary zone electrophoresis (CZE) method for separation of adenosine and N-6-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 mu mol L-1). Good repeatability of migration times (within 1.3%), peak areas (within 1.8%) and linearity (R-2 >0.999) was achieved over the concentration range 5-1000 mu mol 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-QqTOE-MS. Dephosphorylation of ATP was observed as a parallel reaction. (C) 2012 Elsevier B.V. All rights reserved.
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