ORIGINAL ARTICLE A rapid and robust assay for the determination of the amino acid hypusine as a possible biomarker for a high-throughput screening of antimalarials and for the diagnosis and therapy of different diseases Annette Kaiser • Alex R. Khomutov • Alina Simonian • Enzo Agostinelli Received: 10 November 2010 / Accepted: 17 February 2011 / Published online: 1 March 2011 Ó Springer-Verlag 2011 Abstract Eukaryotic initiation factor 5A (eIF5A) has recently been identified as a biomarker of prognostic sig- nificance and therapeutic potential for the treatment in hepatocellular carcinoma. This prompted us to establish a rapid and robust assay to determine deoxyhypusine and hypusine formed with the purified enzymes deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) from Plasmodium to develop a rapid screening assay for antimalarial drugs. The peptide hydrolysate obtained from hypusinylated eIF5A was analyzed by ultra performance liquid chromatography (UPLC) with retention times for deoxyhypusine of 7.44 min and for hypusine of 7.30 min, respectively. The limit of detection for both compounds was 0.144 ng/ll. Determination of the specific activity of Plasmodium DOHH resulted in a twofold higher specific activity than its human counterpart. Following the iron- complexing strategy of the ferrous iron which is present in the active site of Plasmodium DOHH, a series of iron chelating compounds was tested. 2,2 0 -Dipyridyl and mi- mosine abolished DOHH activity completely while 4-oxo- piperidine-carboxylates i.e. the nitrophenylether JK8-2 and EHW 437, the oxime ether of the piperidine aldehyde, showed no inhibition although they were highly active in in vitro cultures of Plasmodium and in vivo in a rodent mouse model. The method allows a high-throughput screening (HPTS) of antimalarial drugs and the evaluation of eIF5A as a biomarker. Keywords Ultra performance liquid chromatography Á Hypusine Á Plasmodium Á Analytics of a biomarker Abbreviations eIF5A Eukaryotic initiation factor 5A UPLC Ultra performance liquid chromatography HPTS High-throughput screening DHS Deoxyhypusine synthase DOHH Deoxyhypusine hydroxylase AccQ-Tag 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate 2ME 2-Mercaptoethanol OPA ortho-Phthalaldehyde PDA Photodiode-array detector Introduction During the last years there has been a considerable shift in the necessities and demands of the healthcare system worldwide. These shifts are due either to chronic diseases and ageing demographics or infectious diseases in the developing world. In the new world malaria still belongs to the sixth most important infectious diseases (World Health Organization Electronic supplementary material The online version of this article (doi:10.1007/s00726-011-0859-5) contains supplementary material, which is available to authorized users. A. Kaiser (&) Institut fu ¨r Technische Chemie und Pharmazeutische Chemie, University of Applied Sciences, Cologne, Betzdorfer Str.2, 50679 Cologne, Germany e-mail: annette.kaiser@fh-koeln.de A. R. Khomutov Á A. Simonian Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilow Street 32, 119991 Moscow, Russia E. Agostinelli Department of Biochemical Sciences ‘A. Rossi Fanelli’, SAPIENZA University of Rome and CNR, Biology and Molecular Pathology Institutes, P.le Aldo Moro 5, 00185 Rome, Italy 123 Amino Acids (2012) 42:1651–1659 DOI 10.1007/s00726-011-0859-5