magistrsko delo
Milica Janković (Author), Marko Novinec (Mentor), Marko Dolinar (Thesis defence commission member), Iztok Turel (Thesis defence commission member)

Abstract

Za pravilno delovanje živih organizmov je bistvenega pomena stroga regulacija encimske aktivnosti, saj nezadostno ali prekomerno delovanje encimov povezujemo z razvojem številnih patoloških stanj. Kot ena izmed za raziskave zanimivih skupin encimov se je izkazala skupina papainu podobnih proteaz in sicer so to cisteinski katepsini. Katepsin B je cisteinska peptidaza s katalitično diado sestavljeno iz cisteinskega in histidinskega ostanka. Izraža se v vseh tipih celic in večinoma nespecifično razgrajuje proteine in peptide. Njegova aktivnost je povezana z dolgim seznamom bolezni kot so nevrološka, avtoimunska, skeletna, kardiovaskularna obolenja ter nastanek in metastaziranje različnih malignih tumorjev. Zaradi tega so katepsini zelo priljubljena tema številnih raziskav na temo popolne ali delne inhibicije encimske aktivnosti. Kot inhibitorji aktivnosti katepsina B se dinamično preučujejo različne proteinske molekule, organske molekule, kovinski ioni in kovinski kompleksi. V okviru magistrske naloge smo preučevali inhibitorni vpliv kavne kisline v kombinaciji s kovinskimi ioni na aktivnost katepsina B. V prejšnjih raziskavah so sicer ugotovili inihbitorne učinke tako kavne kisline kot nekaterih kovinskih ionov na aktivnost katepsina B, vendar pa smo v tej študiji preverili vpliv kompleksov kavne kisline in izbranih kovinskih ionov na aktivnost encima ter ugotovili, ali ima kompleks močnejši inhibitorni učinek na aktivnost katepsina B kot posamezni komponenti. V začetnih poskusih sem preverila učinek kavne kisline v prisotnosti nekaterih kovinskih ionov, da bi določila, kateri ion najmočneje inhibira aktivnosti katepsina B v kombinaciji s kavno kislino. Kot najboljši so se izkazali železovi(III) ioni. Vpliv kavne kisline in železovih ionov na encimsko aktivnost pri treh različnih pH vrednostih sem spremljala s fluorometričnim zasledovanjem hidrolize sintetičnega substrata Z-FR-AMC, rezultate analizirala s programom GraphPad Prism in določila vrednosti konstant inhibicije in EC50 ter mehanizem delovanja in situ pripravljenega kompleksa. Ugotovili smo, da so inhibitorni učinki kavne kisline izboljšani, ko v reakcijske mešanice dodamo ustrezno količino železovih(III) ionov. Določene vrednosti EC50 za inhibicijo katepsina B z in situ pripravljenim kompleksom pri različnih pH so 6,9 ± 5,3 µM (pH 4,5), 7,1 ± 2,3 µM (pH 5,5), 24,9 ± 5,4 µM (pH 7,4) in so nižje v primerjavi z EC50 vrednostmi pri inhibiciji s posameznimi komponentami. Kompleks se je pokazal kot inhibitor encimske aktivnosti, ki deluje po mehanizmu linearne akompetitivne inhibicije s konstanto akompetitivne inhibicije Kiu= 4,27± 0,29 µM. Poskusila sem tudi sintetizirati in okarakterizirati kompleks kavne kisline in železovega(III) iona v množinskem razmerju 3:1, vendar je sinteza in posledično tudi karakterizacija kompleksa pokazala le delni uspeh. Problemi so bili bodisi v čistosti ali pa v topnosti nastalih produktov. Zagotovo pa je nadaljevanje raziskav v smeri sinteze kompleksa kavne kisline in kovinskih ionov in priprave kristalne strukture kompleksa smiselno, saj bi tako dobili boljši vpogled in bolj jedrnato razlago o načinu delovanja kompleksa kavne kisline in železovih ionov pri inhibiciji katepsina B.

Keywords

encimska inhibicija;kavna kislina;kovinski ioni;kovinski kompleksi;encimska kinetika;kinetične meritve;magistrska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [M. Janković]
UDC: 577.15(043.2)
COBISS: 132942851 Link will open in a new window
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Other data

Secondary language: English
Secondary title: Effect of caffeic acid and iron(III) ions and their complexes on cathepsin B activity
Secondary abstract: The strict regulation of the enzyme activity is essential for the proper functioning of all living organisms, due to the known connection of the extreme, either minimum or maximum, activity of the enzymes to the numerous pathological conditions. A group of papain-like proteases, namely cysteine cathepsins, has awoken a special interest amongst researchers. Cathepsin B is a cysteine peptidase with a catalytic dyad consisting of a cysteine and a histidine residue. It is expressed in all types of cells and it commonly degrades proteins and peptides non-specifically. The activity of the cathepsin B is closely associated with a long list of conditions such as neurological, autoimmune, skeletal, cardiovascular diseases and the formation and metastasis of various malignant tumors. For this reason, cathepsins are a very popular subject of many studies of complete or partial inhibition of enzyme activity. Different protein molecules, organic molecules, metal ions and metal complexes are dynamically studied as inhibitors of the activity of the cathepsin B. During the research for the Master thesis, we have studied the inhibitory effect of the caffeic acid in combination with iron(III) ions on the activity of cathepsin B. In previous studies, the inhibitory effects of both these elements separately have been proven. However, our research focuses on understanding of the effect of the complex of caffeic acid and the selected metal ion on the activity of enzyme, and determing whether the complex, rather then it's separated components, presents a more powerful inhibitory effect on cathepsin B's activity. In the initial experiments, I have checked the effect of caffeic acid in the presence of different metal ions, in order to determine which ion most strongly inhibits the activity of cathepsin B when combined with caffeic acid. Iron(III) ions were proven to be the best choice. I have measured the effect of the caffeic acid and iron(III) ions on the activity of the enzyme in three different pH values by flourometric tracking of the hydrolysis of the synthetic substrate Z-FR-AMC. Further on I analized the data with GraphPad Prism program and determined the values of the inhibition constants and the EC50 and the mechanism of action of the in situ prepared complex. We found that the inhibitory effects of caffeic acid are improved when an appropriate amount of iron(III) ions is added to the reaction mixtures. Determined values of the EC50 for the inhibition of cathepsine B with the in situ prepared complex in different pH are 6,9 ± 5,3 µM (pH 4,5), 7,1 ± 2,3 µM (pH 5,5), 24,9 ± 5,4 µM (pH 7,4), and are lower when compared to the EC50 values of the inhibition by individual components of the complex. The complex was proven to be an enzyme activity inhibitor acting by a uncompetitivne inhibition mechanism, with the constant of uncompetitive inhibition Kiu= 4,27± 0,29 µM. I have also attempted to synthesize and characterize the complex of caffeic acid and iron(III) ion in the ratio 3:1, however the synthesis and, consequently, the characterization of the complex showed only partial success. The problems appeared either in the purity or in the solubility of the formed products. Certainly, continuing research in the direction of the synthesis of the complex of caffeic acid and metal ions and the preparation of the crystal structure of the complex makes sense, because this may provide a better insight and a more concise explanation of the mode of action of the complex of caffeic acid and iron(III) ions in the inhibition of cathepsin B.
Secondary keywords: cathepsin B;enzyme inhibition;caffeic acid;metal ions;kinetic measurements;metal complex;Katepsini;Encimski inhibitorji;Univerzitetna in visokošolska dela;
Type (COBISS): Master's thesis/paper
Study programme: 1000377
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, smer Biokemija
Pages: 61 str.
ID: 16608102