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286 Cell-Penetrating Peptides: Processes and Applications
Measuring the radioactivity of [ 125 I]–transportan after internalization in Bowes
cells showed that more than 50% of the maximal internalization of TP is achieved
in the approximate interval of 3 min between adding TP to the suspension and
centrifugation used to separate free and bound label, during which time all solutions
were kept on ice. At all concentrations under study (5 to 500 nM), the time course
of uptake is similar. Incubation of the cell suspension at 37°C with labeled transportan
first induces fast increase of TP concentration in the cells. In 15 to 25 min,
maximal intracellular concentration is achieved.
The maximal uptake of labeled transportan depends slightly on the peptide
concentration in the solution. The relative uptake is higher at lower concentrations:
16.3 and 9.2% of the total amount of peptide at 5 and 500 nM, respectively. Taking
into the account volumes of the cells and the surrounding solution, it was estimated
that the concentration of [ 125 I]–transportan inside the cells is at least twofold higher
than the concentration of free ligand outside them. The fraction of the label internalized
into the cells did not persist on the maximal level, but rather slowly decreased
over the period studied. In parallel to the decrease of radioactivity inside cells, the
fraction of radioactivity outside started to increase. The decrease of radioactivity
inside the cells could be interpreted as outflow of radioactively labeled fragments
of the degraded TP from the cells. Interestingly, immunochemical staining showed
no decrease in label in the cells after 4 h.
One explanation to this discrepancy is the possible cleavage of the peptide bond
between Gly 12 and Lys 13 that would leave the [ 125 I] label on Tyr 9 and the biotin label
on Lys 13 on different peptide fragments; it is possible that fragments retaining
radioactivity behave differently from fragments with the biotin attached. The cleavage
of the parent peptide galanin between amino acids Gly 12 and Pro 13 by membranebound
proteases in spinal cord material has been demonstrated, supporting this
interpretation. 19 Additionally, it has been shown that [ 125 I]–biotinyl–transportan is
degraded when incubated with membranes.
The observed time course of the radioactivity in the cells was simulated by the
following kinetic model:
k1 k3 k4 A B C D (Scheme 13.2)
k2 k5 where A and B represent [ 125 I]–biotinyl–transportan outside and inside the cells, C
and D are radioactive peptide fragment (or fragments) inside and outside the cells,
and k denotes the first-order rate constants for corresponding processes. It is reasonable
to speculate that the binding of the peptide to the membrane surface is fast
enough for it to be ignored as an independent step, at least in this time resolution.
Thus, this scheme is close to the scheme shown in Figure 13.1. The amount of
radioactive peptides in the cells (B + C) and the corresponding rate constants were
calculated by a numerical treatment of the experimental data using a modified
regression computer program of Stojan. 18