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Quantification of Cell-Penetrating Peptides and Their Cargoes 267
reaction yields heterogeneously labeled products, which makes it difficult to estimate
specific activity. For the studies of transportan, peptide iodination was carried out
by the Chloramine T method. 6
EXAMPLE 12.1
PREPARATION AND INTERNALIZATION OF 125 I-LABELED TRANSPORTAN PEPTIDES
Peptide iodination was carried out by the Chloramine T method. 10 Na 125 I (specific
activity 16.1 mCi/mg, concentration 0.1 mCi/mL) was mixed with 5 eq. of the
peptide, and phosphate buffer was added in tenfold excess. 5 The Chloramine T
solution was prepared at a concentration of 2 mg/ml in phosphate buffer, pH 7.4.
After 2 min of incubation, adding sodium metabisulfit (2.4 mg/ml in phosphate
buffer, pH 7.4) stopped the reaction. The peptides were purified on a reversed-phase,
column SEP-PAK 51910 (Millipore) using stepwise gradient of acetonitrile in water.
The iodination was performed using 1 mCi Na 125 I and 4.9 nmol of biotinylated
peptide for transportan and transportan 2, while transportan 3 was iodinated using
5 mCi of Na 125 I and 12.6 mmol, respectively. The fractions with the highest specific
activity of radiolabeled peptides were used in the uptake experiments. The radiolabeled
peptides, in HEPES-buffered Krebbs Ringer solution, were added to Bowes
melanoma cells in suspension. The incubation was made on a mildly shaking waterbath
at 37ºC. To separate the extracellular peptide, the cell suspensions were centrifuged
through a mixture of 40% dioctyl phalate and 60% dibutyl phalate and
washed in an acidic solution (0.2 M acetic acid in 0.5 M NaCl, pH 2.5). The fractions
were counted in a γ-counter (Packard, Meriden, CT). As a control of the intactness
of the cells during the incubation time, only Na 125 I, instead of labeled peptide, was
added.
12.2.1.2 Biotinylation and Cell-ELISA
Biotin (Figure 12.1) has been widely used as a reporter group on CPPs, as it is
considered a less disturbing modification compared to the widely used fluorophores,
which can interact with plasma membranes by themselves. Indirect immunofluorescence
detection of biotin is more time consuming than direct visualization of a
chromophore, but enables greater flexibility of methods (see Example 12.2).
Fisher and colleagues used a cell-ELISA for quantification of biotinyl–penetratin.
11 In short, 50,000 cells per well were seeded and, after overnight incubation,
biotinylated penetratin (40 µM) was added. Cells were rinsed, fixed, and permebilized
with 3% Tween 20. Endogenous alkaline phosphatase activity was blocked by
1% bovine serum albumin (BSA) in phospahate-buffered saline (PBS) at 65°C for
60 min. Alkaline phosphatase labeled-streptavidin was added for 30 min at room
temperature and developed by substrate addition; absorbance was measured at
405 nm.
12.2.1.3 Fluorescence Microscopy, Spectrophotometer, and FACS
Today, a wide variety of fluorophores is in use, with fluorescein perhaps the most
common (Figure 12.1). However, fluorescein is sensitive to photobleaching and,