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266 Cell-Penetrating Peptides: Processes and Applications
Biotin
Fluorophore
Enzyme
Florophore
CPP
125I
CPP
CPP
CPP
FIGURE 12.2 (Color Figure 12.2 follows p. 14.) Summary of the various detection methods
used to quantify the internalization of CPPs by application of different labeling strategies. In
general, the CPP is labeled with a reporter group, added to adherent cells or cells in solution,
and the quantification of the CPP is carried out according to the method indicated in the figure.
• Detection of bioactivity of peptide cargo
• Detection of DNA/PNA antisense effects
• Quantification of drug activity
12.2.1 STUDIES OF CELL-PENETRATING PEPTIDES USING REPORTER GROUPS
This chapter is divided into two parts: one dealing with studies of CPPs and the
other, very briefly, with quantification of cargoes delivered by CPPs. The reporter
group, or label, is here defined as an addition to the CPP that will make it detectable.
The exception is the immunodetection of the Tat peptide in which no reporter group
is used. Detection methods used for these studies are summarized in Figure 12.2.
12.2.1.1 Radiolabeling
CPP
Quencher
-S-S ����Peptide
Detection of radioactivity by γ -counter
after oil centrifugation
Detection by labelled streptavidin
Detection by measuring
fluorescence intensity
Detection of enzyme activity
Increase in fluorescence intensity
���� reduction of the didulphide bond
Two radioisotopes, 3 H and 125 I, have been used for radioactive labeling of peptides.
Iodination is preferred because it is easier to perform and reproducible, even though
the half-life of 125 I is only 60 days compared to tritium’s 12 years. The most common
methods used for peptide iodination are Chloramine-T and the [ 125 I]-Bolton–Hunter
reaction. 9 For the Chloramine-T method, a Tyr residue is required in the sequence,
as the aromatic side chain is the acceptor of the iodine isotope. Here, a major
drawback is the possible oxidation of Trp and Cys/Met residues.
The Bolton–Hunter iodination is an acetylation of a primary N-terminal α-amino
group or an ε-amino group of Lys by N-succinimidyl-3-(4-hydroxy, 5- 125 I-iodophenyl)
propionate. In the case of a sequence containing several Lys residues, the