Characterization of conjugated protease inhibitors

The overall theme of this thesis is a step by step approach for the design and characterization of conjugated protease inhibitors. This involves both a new assay method for protease activity and protease inhibition (paper I), a study of the stoichiometry for protease inhibitor interaction (paper II), design of inhibitory peptides (paper IV) and the construction of inhibitor conjugates (paper III & IV). (I) A model based primarily on erosion in gelatin for protease activity and inhibition studies was designed. The model was also extended to a separate protective layer covering the layer containing the target substrate. A good correlation between protease concentration and rate of erosion was observed. Similarly, increased concentration of inhibitors gave a systematic decrease in the erosion rate. Kinetic analyses of a two-layer model with substrate in the bottom layer displayed a strict dependence of both inhibitor concentration and thickness of the top “protective” layer. (II) The binding stoichiometry between pancreatic proteases and a serine protease inhibitor purified from potato tubers was determined by chromatography-coupled light scattering measurements. This revealed that the inhibitor was able to bind trypsin in a 2:1 complex, whereas the data for a-chymotrypsin clearly showed a limitation to 1:1 complex. The same experiment carried out with elastase and the potato inhibitor gave only weak indications of complex formation under the conditions used. (III) A serine protease inhibitor was extracted from potato tubers and conjugated to soluble, prefractionated dextran or inorganic particles. A certain degree of inhibitory activity was retained for both the dextran-conjugated and particle-conjugated inhibitor. The apparent Ki value of the dextran-conjugated inhibitor was found to be in the same range as that for free inhibitor. The dextran conjugate retained a higher activity than the free inhibitor after 1 month of storage at room temperature. Conjugation to oxide particles improved the heat stability of the inhibitor. (IV) New synthetic Leupeptin analogues, Ahx-Phe-Leu-Arg-COOH & Ahx-Leu-Leu-Arg-COOH, were synthesized with solid-phase peptide synthesis using Fmoc strategy. These tripeptide inhibitors were tight binding inhibitors to the target enzyme trypsin, similar to the natural occurring leupeptin. The phenylalanine containing synthetic analogue was conjugated to inorganic particles and agarose gel beads. In all cases, the inhibitory activity was well preserved