TAT Peptide and Its Conjugates: Proteolytic Stability
- 14 July 2009
- journal article
- research article
- Published by American Chemical Society (ACS) in Bioconjugate Chemistry
- Vol. 20 (8), 1531-1537
- https://doi.org/10.1021/bc900081e
Abstract
The proteolytic cleavage of TATp, TATp-PEG1000-PE conjugate (TATp-conjugate), and TATp as TATp-conjugate in mixed micelles made of TATp-conjugate and PEG5000-PE (2.5% mol of TATp-conjugate, TATp-Mic) were studied by HPLC with fluorescent detection using fluorenylmethyl chloroformate (FMOC) labeling and by MALDI-TOF MS analysis. The cleavage kinetics were analyzed in human blood plasma and in trypsin-containing phosphate buffered saline (PBS), pH 7.4, to simulate the proteolytic activity of human plasma. The trypsinolysis of free TATp, TATp-conjugate, and TATp-Mic revealed that the main initial fragmentation is an endocleavage at the carboxyl terminus resulting in an Arg-Arg (RR) dimer. The trypsinolysis followed pseudo-first-order kinetics. The cleavage of the free TATp was relatively fast with a half-life of a few minutes (t1/2 ∼ 3.5 min). The TATp-conjugate showed more stability with about a 3-fold increase in half-life (t1/2 ∼ 10 min). TATp in TATp-Mic was highly protected against proteolysis with an over 100-fold increase in half-life (t1/2 ∼ 430 min). The shielding of TATp by PEG moieties in the proposed TATp-Mic is of great importance for its potential use as a cell-penetrating moiety for multifunctional “smart” drug delivery systems with detachable PEG.Keywords
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