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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Kinetic Studies on Cellular Uptake of PolyargininePeptide Using FRETAkihiro Ambo, Yasuyuki Suzuki, Motoko Minamizawa, andYusuke SasakiDepartment of Biochemistry, Tohoku Pharmaceutical University, Sendai, 9818558, JapanIntroductionCell-penetrating peptides (CPPs) are known to be able to pass through the plasmamembrane and are used as tools for delivery of hydrophilic molecules such as peptides,proteins, and oligonucleotides [1]. Endocytic mechanisms involving clathrin-mediated,caveolae-mediated, macropinocytosis, or a non-endocytic mechanism involving directtranslocation have been suggested for cellular uptake of CPPs in live cells [2]. In eitheruptake mechanism, the cationic portion of CPPs is thought to initially interact with anionicsubstances present at the extracellular surface of the plasma membrane, such as proteoglycans.However, their transport mechanisms are still not clearly understood.In the present study, we attempted to determine the cellular uptake of CPPs into cellsusing conjugated peptides by the fluorescence resonance energy transfer (FRET) technique.The FRET-peptide consists of FAM-Gab-Cys-(cargo or CPP)-NH 2 and Dabcyl-Gab-Cys-(CPP or cargo)-NH 2 , which are covalently linked via an S-S bond (Table 1). The use ofFRET-peptides has the advantage that undelivered peptides do not need to be removedfrom cells during the cellular uptake determination, and thus minimizes artifacts arisingfrom incomplete washing of the undelivered peptide. We kinetically examined the cellularuptake of conjugated FRET-peptides into Jurkat cells under different incubation conditionsby FACS analysis.Table 1. Sequences of FRET-peptidesAbbr.SequencesTOF-MS (m/z, M+H + )foundcalcd.FRET-CR9rev-FRET-CR9FRET-CDR9Dabcyl-Gab-Cys-(Arg) 9 -NH 2 2698.95 2698.35|FAM-Gab-Cys-His-His-NH 2FAM-Gab-Cys-(Arg) 9 -NH 2 2698.93 2698.35|Dabcyl-Gab-Cys-His-His-NH 2Dabcyl-Gab-Cys-(Arg) 6 -D-Arg-(Arg) 2 -NH 2 2698.77 2698.35|FAM-Gab-Cys-His-His-NH 2Results and DiscussionWe designed and synthesized three FRET-peptides as listed in Table 1. Cys-His-His-NH 2was employed as the cargo, which itself is not able to pass across the plasma membrane(data not shown). The conjugate peptides did not emit fluorescence due to quenching of theFAM group by the Dabcyl group, but after reduction with 1 mM glutathione, which existsabundantly in the cytosol, equally high fluorescence was measured with FRET-CR9 andrev-FRET-CR9. As shown in Figure 1, intracellular fluorescence intensities of threeFRET-peptides rapidly increased for up to 15 min and then decreased after prolongedperiods. The permeability of FRET-CDR9 was high, at least 1.4-fold better than that of520