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270 Cell-Penetrating Peptides: Processes and Applications
exposed to diazotized 2-nitroaniline, which selectively modifies Lys side chains. As
the reagent does not cross the plasma membrane, only peptides exposed to the
extracellular medium are modified, resulting in increased hydrophobicity and subsequent
increased HPLC retention time. This allows separation of intracellular and
surface-bound peptide.
This procedure has the advantage that the reagent is highly reactive even at 0°C,
so that the results are not biased by efflux processes because they are suppressed at
this temperature. An additional advantage is that the various metabolites of the
peptide can be isolated and characterized by mass spectroscopy. Furthermore, the
use of a fluorescence detector in the HPLC equipment increases the sensitivity of
this analysis.
12.2.1.5 Immunodetection
In order to quantify the uptake of Tat (37–72), Vivés and colleagues used both
fluorescein labeling and direct immunodetection, using a monoclonal antibody
against the Tat basic cluster. 18 Quantification was done by FACS analysis. This
detection by the monoclonal antibody is a direct quantification of the CPP, and not
via a reporter group.
12.2.1.6 Detection of Enzyme Activity
Internalization of RNAse A, β-Galactosidase, horseradish peroxidase, and
Pseudomonas exotoxin A by Tat fragment (residues 37–72) was studied by Fawell
et al. 3 The uptake was monitored colorimetrically and by cytotoxicity measurements.
Quantification was carried out by measuring absorbance of the respective substrate
produced by the CPP internalized enzymes.
12.2.1.7 Fluorogenic Construct Assay
Resonance energy transfer (RET) quenching is a process in which a molecule with
an absorbance spectra overlapping the emission spectra of a flourophore quenches
the fluorescence when the molecules are in close proximity. Several such fluorophore-quencher
pairs have been found, e.g., DABCYL/EDANS 19 and Abz/3-nitro-
Tyr. 12 The amount of quenching is dependent on the distance between the molecules
as well as the spectral overlap.
The Abz flourophore is compatible with Fmoc and t-Boc chemistries. It is
resistant to photobleaching and has a well characterized amino acid-like quencher,
3-nitro-Tyr, which is stable under both main types of peptide chemistries.
We have used a short fluorescently labeled peptide, with the sequence: Abz-Cys-
LKANL as cargo in delivery quantification. 2 The CPPs contained a Cys-residue used
for covalent attachment of the cargo–peptide via a disulfide bond, and a 3-nitro-Tyr
extension acting as a quencher to the Abz group, which resulted in a reductionsensitive
fluorogenic construct (Figure 12.2). The cellular uptake of the construct is
registered as an increase in the fluorescence intensity when the disulfide bond of
the CPP–S-S cargo construct is reduced in the intracellular milieu.