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Light scattering determination of the stoichiometry for protease-potato serine protease inhibitor complexes
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC. (Gunnar Johansson)
2019 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 582, p. 113357-Article 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
2019. Vol. 582, p. 113357-
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-396981DOI: 10.1016/j.ab.2019.113357OAI: oai:DiVA.org:uu-396981DiVA, id: diva2:1369614
Available from: 2019-11-12 Created: 2019-11-12 Last updated: 2019-12-11
In thesis
1. Characterization of conjugated protease inhibitors
Open this publication in new window or tab >>Characterization of conjugated protease inhibitors
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
Serine protease inhibitors, conjugation, immobilization, leupeptin analogues, potato serine protease inhibitor, soluble carriers, inorganic carriers.
National Category
Natural Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-398909 (URN)978-91-513-0835-7 (ISBN)
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: 2020-01-23

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