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Kinetics of Uptake of Cell-Penetrating Peptides 289
from the middle (TP9), did not influence the penetration efficiency of peptides, since
they internalized similarly to TP. However, the localization of these three peptides
is not identical (see Chapter 3 for details). Further shortening of the N-terminal part
by nine amino acid residues led to the loss of penetration ability. Deletions from
the N terminus or from the middle of the TP molecule decreased (TP12, TP14, and
TP11) or abolished (TP13) the internalization. Combination of deletions in the N
and C termini and also in the middle of the sequence completely inactivated the
peptide; TP15 did not internalize at all.
In order to compare rate and yield of internalization of the best penetrating short
analogues of TP, characteristic half-lives of internalization were calculated from the
first-order kinetics approximation (Equation 13.1). TP is the fastest penetrator, with
a half-life of internalization of t 0.5 = 3.4 min. Deletion analogues of TP show somewhat
slower internalization with t 0.5 of 4.3, 8.6, 6.5, and 10.7 min for TP7, TP10,
TP9, and TP12, respectively. These results are in accordance with results obtained
with analogues in which substantial parts of the TP sequence were replaced by
sequences of different other peptides or their fragments (cf. above). It was thus
demonstrated that the modifications in C-terminal moiety of TP (mastoparan part)
have more dramatic negative effect on cell penetration rate and efficacy than modifications
in N-terminal part (galanin part). The most important result, however, is
a discovery of TP10 that is an effective CPP and does not possess the ability to
interplay with the activation process of G-proteins. 9
13.4.3 PENETRATIN AND PENETRATIN ANALOGUES
Although penetratin was the first CPP described, 21 kinetic data on the internalization
of penetratin into cells are scarce. Drin et al. presented the time course of internalization
of NBD-labeled penetratin into K562 cells, 5 showing that uptake of penetratin
(at 1.6 µM initial concentration at 37°C) into the cells was rapid. The concentration
of the internalized peptide reached plateau after 1 h of incubation. From the data
(Drin et al., 5 Figure 3) the value of t 0.5 of 17 min was recalculated assuming firstorder
kinetics.
Hällbrink et al. have followed the uptake of penetratin associated with a small
peptide cargo into the Bowes cells. 1 The time course of the penetratin uptake was
roughly consistent with the first-order kinetics (1 µM initial concentration at 37°C)
and with the t 0.5 of around 56 min. It is difficult to say whether the discrepancy
between the results in Drin et al. 5 and those obtained in Hällbrink et al. 1 are due to
different cells or to the impact of cargoes.
A number of different penetratin analogues has been synthesized. 5,7,21 They have
been tested for the maximal amount internalized in different cells and vesicles, but
none of them has been used in studies of kinetics of internalization.
13.4.4 TAT AND TAT ANALOGUES
To date, more than ten Tat-derived short peptides have been shown to translocate
into the interior of different cells (see Chapter 1). Kinetic experiments were carried
out with fluorescein-labeled Tat (49–57) 22 and Tat (48–60) coupled to small peptide