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268 Cell-Penetrating Peptides: Processes and Applications
additionally, its hydrophobicity and size can alter the properties of the peptide. The
2-aminobenzoic acid fluorophore (Abz, Figure 12.1) is small, stable, and photoresistent,
12 and does not alter the hydrophobicity of the peptide to the same extent as
fluorescein and rhodamine do. However, Abz is not easily visualized in fluorescence
microscopy due to its deep blue color (λ em 420 nm), but it is useful for spectrometry
detection.
The coupling of the fluorophore to the CPP is usually carried out by covalent
linkage to the N-terminal α-amino group or ε-amino group of Lys of the peptide.
For fluoresceinyl-modification, the succinimidyl ester (Molecular Probes, Holland)
can be used in a five-fold excess to the peptidyl-resin in t-Boc peptide chemistry.
In solid phase peptide synthesis with Fmoc chemistry, the fluorescein isothiocyanate
was applied in the presence of diisopropylethyl amine (DIEA). The Abz group can
be introduced by a routine amino acid-coupling step. The labeling is preferably
carried out before cleavage of the peptide from the solid support to ensure specific
linkage of the fluorophore. Labeling during solid phase synthesis has the further
advantage of avoiding the time and low yield of labeling the peptide in solution,
because it is critical to remove any unreacted dye, which can greatly complicate
subsequent experiments. 13
Flow cytometry or fluorescence-activated cell sorting (FACS) is a convenient way
of quantifying the content of fluorescently labeled peptide. This method has been used
by Garcia–Echeverria et al. for detecting FITC labeled-penetratin. 14 FACS is a sensitive
method; less than 100 fluorophores per particle or cell have been detected by this
method. 14 After CPP treatment of the attached cells, they are treated with trypsin and
fixed in a 10% paraformaldehyde solution prior to cell sorting analysis.
Confocal fluorescence microscopy quantification of fluorophore-labeled CPP
was described by Scheller et al. 15 and also in Chapter 4. An on-line protocol was
employed that would avoid biasing the internalization results by outflow of the
peptides. Briefly, cells are seeded on coverslips and treated with peptide. Three
regions of interest are scanned in the cytosol and one in the nucleus of three selected
cells. The fluorescence resulting from the incubation media is removed as background
fluorescence. After a 30-min assessment, cell viability is checked by addition
of tryphan blue. 15
The NBD (7-nitrobenz-2-oxo-1, 3-diazol; NBD) fluorophore group, stable in
Fmoc cleavage conditions, 16 has been used to label CPPs. 7 The main advantage with
this label is the sensitivity toward reduction agents, such as dithionite, that can be
used to inactivate extracellular and membrane-bound label. Moreover, the NBD
group changes fluorescence characteristics in a hydrophobic environment toward an
emission blue shift (from 537 to 530 nm) as well as an increase in intensity. This
property makes it possible to measure membrane partition coefficients of the labeled
peptide.
EXAMPLE 12.2
QUANTIFICATION OF BIOTIN/ABZ/FITC-LABELED CPP
Either the N terminus or the ε-amino group of a Lys residue could be used for
labeling with biotin, Abz, or FITC. In the case of labeling pVEC, a novel CPP, 17 the