Izolacija in biokemijska karakterizacija paraloga proteina Vaa-snaclec-3/2 iz strupa modrasa (Vipera ammodytes ammodytes)

Snedec, Andraž (author), Križaj, Igor (mentor)

Modras (Vipera ammodytes ammodytes) je najbolj strupena in medicinsko najbolj pomembna evropska kača. Velik delež njegovega strupa predstavljajo lektinu tipa C podobni proteini (snacleci), ki z vezavo na trombocitne receptorje in plazemske proteine vplivajo na hemostazo. Vaa snaclec-3/2 je najbolje raziskan snaclec iz modrasovega strupa. Zaradi reverzibilne trombocitopenije, ki jo inducira po vezavi na trombocitni receptor GPIb, je Vaa-sanclec-3/2 zanimiva molekula za razvoj bolj varnih antikoagulantnih zdravil. Poleg Vaa-sanclec-3/2 je bilo v strupu modrasa identificiranih še pet drugih snaclecov s še neznano funkcijo. Štirje med njimi zaradi nizke zastopanosti v strupu in podobnih fizikalno kemijskih lastnosti še niso bili izolirani v homogeni obliki. Cilj diplomske naloge je bil iz modrasovega strupa izolirati vsaj enega od teh. Kompleksno zmes proteinov v modrasovem strupu smo najprej ločili po velikosti z gelsko filtracijo. Za nadaljnje čiščenje tarčnih snaclecov smo uporabili ionsko-izmenjevalno kromatografijo. Preizkusili smo različne pogoje ločevanja s kationsko- in anionsko izmenjevalno kromatografijo, a pogojev za uspešno ločbo nismo uspeli najti. S kationsko-izmenjevalno kromatografijo pri pH 4,5 pa smo od molekul snaclec vendarle uspeli odstraniti večino drugih komponent strupa, kar predstavlja obetavno izhodišče tako za pripravo homogenih izooblik Vaa-snaclec, kot za pripravo nekaterih drugih komponent strupa, ki še niso označene. V diplomskem delu smo uporabili tudi javno dostopen program za kvantitativno modeliranje povezave med strukturo in lastnostmi spojine (QSPR; ang. Quantitative Structure-Property Relationship), ki temelji na strojnem učenju. Z njegovo pomočjo smo napovedali elucijske profile Vaa-snaclecov pri kationsko-izmenjevalni kromatografiji v odvisnosti od pH mobilne faze. Napovedi so bile podobne nekaterim empiričnim rezultatom. Določili smo vrednost parametra, pri katerem naj bi se Vaa snacleci ločili med seboj, žal pa je bil čas, ki je bil na razpolago za izvedbo diplomskega dela, prekratek, da bi teoretično napoved preizkusili tudi v praksi.

• Language: Slovenian
• Keywords: kačji strup, Vipera ammodytes ammodytes, lektinu tipa C podobni proteini, QSPR
• Work type: Bachelor thesis/paper
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2026
• PID: 20.500.12556/RUL-186925
• Publication date in RUL: 07.09.2026

Secondary language

• Language: English
• Title: Isolation and Biochemical Characterisation of the Vaa-snaclec-3/2 Protein Paralog from the Nose-Horned Viper (Vipera ammodytes ammodytes) Venom

Abstract

The nose-horned viper (Vipera ammodytes ammodytes, Vaa) is the most venomous and medically significant European snake. A large proportion of its venom consists of C-type lectin-like proteins (snaclecs), which affect haemostasis by binding to platelet receptors and plasma proteins. Vaa-snaclec-3/2 is the most studied snaclec from the Vaa venom. Due to the reversible thrombocytopenia it induces upon binding to the platelet GPIb receptor, Vaa-snaclec-3/2 is an interesting molecule for the development of safer anticoagulant drugs. In addition to Vaa-sanclec-3/2, five other snaclecs with unknown functions have been identified in the Vaa venom. Four of these have not yet been isolated in a homogeneous form due to their low abundance in the Vaa venom and similar physicochemical properties. The aim of this research was to isolate at least one of them. First, the complex protein mixture of Vaa venom was separated by size using size exclusion chromatography. Ion-exchange chromatography was then used for further purification. Although various separation conditions were tested using cation and anion exchange chromatography, conditions that would enable the separation of Vaa snaclecs were not successfully identified. However, using cation exchange chromatography at pH 4.5, we did manage to remove most of the other venom components from the snaclec mixture. Cation exchange chromatography at pH 4,5 therefore represents a promising starting point not only for the preparation of homogeneous Vaa-snaclec isoforms, but also for the preparation of certain other venom components that have not yet been identified. In this thesis, we also utilised a publicly available Quantitative Structure-Property Relationship (QSPR) model, developed using machine learning, to predict the elution profiles of Vaa-snaclecs in cation exchange chromatography at different mobile phase pH values. Some of the predictions coincided with the preliminary empirical results. The model enabled us to determine the parameter value at which the Vaa-snaclecs could separate from one another. Unfortunately, the time available for the thesis was too short to test the theoretical prediction in practice.

• Keywords: snake venom, Vipera ammodytes ammodytes, C-type lectin-like proteins, QSPR