Characterization of Conjugated Protease Inhibitors
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Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1888 Characterization of conjugated protease inhibitors ERIKA BILLINGER ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 ISBN 978-91-513-0835-7 UPPSALA urn:nbn:se:uu:diva-398909 2020 Dissertation presented at Uppsala University to be publicly examined in B42, BMC, Husargatan, Uppsala, Thursday, 13 February 2020 at 09:15 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: Professor Boris Turk (Jozef Stefan Institute, Department of Biochemistry, Molecular and Structural Biology). Abstract Billinger, E. 2020. Characterization of conjugated protease inhibitors. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1888. 86 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0835-7. 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. Keywords: Serine protease inhibitors, conjugation, immobilization, leupeptin analogues, potato serine protease inhibitor, soluble carriers, inorganic carriers. Erika Billinger, Department of Chemistry - BMC, Box 576, Uppsala University, SE-75123 Uppsala, Sweden. © Erika Billinger 2020 ISSN 1651-6214 ISBN 978-91-513-0835-7 urn:nbn:se:uu:diva-398909 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-398909) Education is not the learning of facts, but the training of the mind to think - Einstein LIST OF PAPERS This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Billinger, E., Johansson, G. Kinetic studies of serine protease in- hibitors in simple and rapid ‘active barrier’ model systems: Dif- fusion through an inhibitor barrier. Analytical Biochemistry, 2018, 546, 43–49. II Billinger, E., Zuo, S., Lundmark, K., Johansson, G. Light scat- tering determination of the stoichiometry for protease-potato ser- ine protease inhibitor complexes. Analytical Biochemistry, 2019, 582, 113357. DOI: 10.1016/j.ab.2019.113357 III Billinger, E.*, Zuo, S., Johansson G. Characterization of serine protease inhibitor from Solanum tuberosum conjugated to solu- ble dextran & particle carriers. ACS Omega, 2019, 4, 18456- 18464. DOI: 10.1021/acsomega.9b02815 *Corresponding author. IV Billinger, E., Viljanen, J., Bergström Lind, S., Johansson, G. In- hibition properties of free and conjugated leupeptin analogues. 2019. Submitted. Reprints were made with permission from the respective publishers. Further permissions related to paper III excerpted should be directed to the ACS. Publications not included in this thesis. V Kjellander, M., † Billinger, E., † Ramachandraiah, H., Boman, M., Bergström Lind, S., Johansson, G. A flow-through nanoporous alumina trypsin bioreactor for mass spectrometry peptide finger- printing. Journal of Proteomics, 2018, 172, 165-172. † These authors contributed equally. CONTRIBUTION REPORT I. Participated in formulating the research idea, planned and per- formed all experimental work, wrote the initial draft of the manu- script and shared the writing of the manuscript. II. Planned the overall experimental work, performed parts of it, par- ticipated in writing the manuscript. III. Participated in formulating the research idea, planned the initial scope, performed most experimental procedures, wrote a draft of the manuscript and organized the writing of the manuscript. Served as corresponding author. IV. Participated in formulating the research idea, planned and per- formed most experimental work, wrote the initial draft of the man- uscript and shared the writing of the manuscript. CONTENTS INTRODUCTION .....................................................................................11 ENZYMES ...........................................................................................12 Proteases ..........................................................................................14 The catalytic site ...............................................................................16 Recognition sites for the substrate .....................................................17 Kinetics of serine protease reaction ...................................................17 Inhibition ..........................................................................................21 SERINE PROTEASE INHIBITORS .....................................................25 Potato serine protease inhibitor .........................................................25 Peptide aldehydes .............................................................................26 CONJUGATION ..................................................................................29 Amide formation ..............................................................................31 Reductive amination .........................................................................32 Carriers ............................................................................................34 TOOLS SECTION ................................................................................39 Static light scattering (SLS) ..............................................................40 Solid-phase peptide synthesis (SPPS) ................................................43 Mass spectroscopy (MS) ...................................................................46 MY WORK ...............................................................................................49 PAPER I ...............................................................................................50 Kinetic studies of serine protease inhibitors in simple and rapid ‘active barrier’ model systems ..........................................................50 One-layer model ...............................................................................50 Two-layer model ..............................................................................52 Conclusions ......................................................................................53 PAPER II ..............................................................................................54 Light scattering determination of the stoichiometry for protease- potato serine protease inhibitor complexes .......................................54 The active sites .................................................................................54 Trypsin-PSPI interaction ...................................................................56 α-chymotrypsin-PSPI interaction ......................................................57 Elastase-PSPI interaction ..................................................................58 Conclusions ......................................................................................59 PAPER III.............................................................................................60 Characterization of serine protease inhibitor from solanum tuberosum conjugated to soluble dextran and particle carriers .........60 Prefractionation & oxidation of dextran ............................................60 Conjugation of PSPI .........................................................................61