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Characterization of conjugated protease inhibitors
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC. (Gunnar Johansson)ORCID iD: 0000-0003-1710-9128
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2020. , p. 86
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1888
Keywords [en]
Serine protease inhibitors, conjugation, immobilization, leupeptin analogues, potato serine protease inhibitor, soluble carriers, inorganic carriers.
National Category
Natural Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-398909ISBN: 978-91-513-0835-7 (print)OAI: oai:DiVA.org:uu-398909DiVA, id: diva2:1377333
Public defence
2020-02-13, B42, BMC, Husargatan, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2020-01-23 Created: 2019-12-11 Last updated: 2020-03-05
List of papers
1. Kinetic studies of serine protease inhibitors in simple and rapid 'active barrier' model systems: Diffusion through an inhibitor barrier
Open this publication in new window or tab >>Kinetic studies of serine protease inhibitors in simple and rapid 'active barrier' model systems: Diffusion through an inhibitor barrier
2018 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 546, p. 43-49Article in journal (Refereed) Published
Abstract [en]

A model based on gelatin for protease activity studies was designed. The model is also extended to study the efficiency of inhibitors in a separate protective layer covering the layer containing the target substrate. A good correlation between protease concentration and the size of erosion wells formed in a plain gelatin layer was observed. Similarly, increased concentration of inhibitors gave a systematic decrease in well area. Kinetic analyses of the two-layer model in a spectrophotometric plate reader with a fixed concentration of substrate in the bottom layer displayed a strict dependence of both inhibitor concentration and thickness of the top "protective" layer. An apparent, but weaker inhibition effect was also observed without inhibitors due to diffusional and erosion delay of enzyme transport to the substrate-containing layer.

Place, publisher, year, edition, pages
ACADEMIC PRESS INC ELSEVIER SCIENCE, 2018
Keywords
Active barrier, Protease screening, Inhibitor screening, Gelatin model substrate, Protease inhibitors
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-352702 (URN)10.1016/j.ab.2018.01.022 (DOI)000429623500008 ()29408179 (PubMedID)
Available from: 2018-06-08 Created: 2018-06-08 Last updated: 2019-12-11Bibliographically approved
2. Light scattering determination of the stoichiometry for protease-potato serine protease inhibitor complexes
Open this publication in new window or tab >>Light scattering determination of the stoichiometry for protease-potato serine protease inhibitor complexes
2019 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 582, article id 113357Article in journal (Refereed) Published
Abstract [en]

The interaction between pancreatic proteases and a serine protease inhibitor purified from potato tubers was investigated by chromatography-coupled light scattering measurements. The molar mass distribution in the chromatogram was compared to theoretical values calculated for the different possible combinations of complexes and free components by three different approaches, namely section analyses of the chromatograms, full mass average determination and mass distribution analysis. This revealed that the inhibitor was able to bind trypsin in a 2:1 complex, whereas the data for chymotrypsin clearly showed a limitation to 1:1 complex regardless of the molar ratio in the injected samples. The same experiment carried out with elastase and the potato inhibitor gave only weak indications of complex formation under the conditions used.

Place, publisher, year, edition, pages
Elsevier, 2019
Keywords
Light scattering, Potato tuber protein, Serine and cysteine inhibitors, Serine protease, Complex formation, Absolute mass
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-396981 (URN)10.1016/j.ab.2019.113357 (DOI)000518156800006 ()31276650 (PubMedID)
Available from: 2019-11-12 Created: 2019-11-12 Last updated: 2020-04-02Bibliographically approved
3. Characterization of Serine Protease Inhibitor from Solanum tuberosum Conjugated to Soluble Dextran and Particle Carriers
Open this publication in new window or tab >>Characterization of Serine Protease Inhibitor from Solanum tuberosum Conjugated to Soluble Dextran and Particle Carriers
2019 (English)In: ACS Omega, ISSN 2470-1343, Vol. 4, no 19, p. 18456-18464Article, review/survey (Refereed) Published
Abstract [en]

A serine protease inhibitor was extracted from potato tubers. The inhibitor was conjugated to soluble, prefractionated dextran and titanium dioxide and zinc oxide nanoparticles. Conjugation to dextran was achieved by periodate oxidation of the dextran, followed by Schiff base coupling to inhibitor amino groups, and finally reduction, whereas the conjugation to the oxide particles was carried out by aminosilanization and carbonyldiimidazole activation. The inhibitory effect of the conjugated inhibitor was compared to that of free inhibitor in solution and with gelatin gel as a direct substrate. A certain degree of inhibitory activity was retained for both the dextran-conjugated and particle-conjugated inhibitors. In particular, 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.

National Category
Organic Chemistry
Research subject
Chemistry
Identifiers
urn:nbn:se:uu:diva-396972 (URN)10.1021/acsomega.9b02815 (DOI)000495089100053 ()31720549 (PubMedID)
Available from: 2019-11-12 Created: 2019-11-12 Last updated: 2019-12-11Bibliographically approved
4. Inhibition properties of free and conjugated leupeptin analogues
Open this publication in new window or tab >>Inhibition properties of free and conjugated leupeptin analogues
(English)In: Article, review/survey (Other academic) Submitted
Abstract [en]

New synthetic leupeptin analogues Ahx-Phe-Leu-Arg-COOH and Ahx-Leu-Leu-Arg-COOH were prepared by solid-phase peptide synthesis using the Fmoc strategy. The structures were confirmed by mass spectrometry (MS). Determination of the inhibitory properties against trypsin revealed that these tripeptide inhibitors were tight binding inhibitors to their target enzyme, similar to the natural occurring leupeptin and with Ki values generally in the micromolar range. The Ahx-Phe-Leu-Arg-COOH analogue was conjugated to inorganic oxide nanoparticles and agarose gel beads. In all the conjugates, the inhibitory activity was present and in the same range as for the free peptides. 

Keywords
Leupeptin analogues, solid-phase peptide synthesis, inorganic carriers, conjugation
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-398908 (URN)
Available from: 2019-12-11 Created: 2019-12-11 Last updated: 2019-12-11

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