uu.seUppsala University Publications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Inhibition properties of free and conjugated leupeptin analogues
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC. (Gunnar Johansson)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
(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 [en]
Leupeptin analogues, solid-phase peptide synthesis, inorganic carriers, conjugation
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-398908OAI: oai:DiVA.org:uu-398908DiVA, id: diva2:1377305
Available from: 2019-12-11 Created: 2019-12-11 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

Open Access in DiVA

No full text in DiVA

By organisation
Department of Chemistry - BMC
Natural Sciences

Search outside of DiVA

GoogleGoogle Scholar

urn-nbn

Altmetric score

urn-nbn
Total: 4 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf