20.500.12556/RUL-162534 Izolacija serinske proteinaze VaaSP-6 iz strupa modrasa in karakterizacija njenih lastnosti Isolation of serine proteinase VaaSP-6 from the nose-horned viper venom and characterization of its properties Serinske proteaze iz kačjih strupov so kompleksni in multifunkcionalni encimi, ki primarno vplivajo na hemostazo. Različne vrste serinskih proteaz v strupu povzročajo antikoagulantne in prokoagulantne učinke. Molekule, ki imajo slednje lastnosti, so manj zastopane. Iz predhodnih raziskav strupa modrasa (Vipera ammodytes ammodytes) poznamo cDNA zaporedje serinske proteaze VaaSP-6 in s tem njeno aminokislinsko zaporedje, vendar njene biološke aktivnosti in molekulskega mehanizma še ne. Glede na visoko sekvenčno podobnost, predvidevamo, da je molekulski mehanizem njenega delovanja podoben sorodnemu encimu VaaSP-VX. Le-ta aktivira faktorja strjevanja krvi V in X (FV in FX) ter tako deluje prokoagulantno. V magistrskem delu smo iz strupa modrasa uspešno izolirali proteazo VaaSP-6 z uporabo velikostno izključitvene kromatografije, kationsko izmenjevalne kromatografije in tekočinske kromatografije visoke ločljivosti z obrnjeno fazo ter njeno prisotnost potrdili z masno spektrometrijo. Z dvodimenzionalno gelsko elektroforezo smo ji določili izoelektrične točke v območju pH 4,5–7,5 in navidezno molekulsko maso 35 kDa. Raziskali smo njen vpliv na proces strjevanja krvi s testiranjem aktiviranega parcialnega tromboplastinskega časa, protrombinskega časa in trombinskega časa. Pokazali smo, da proteaza VaaSP-6, za razliko VaaSP-VX, podaljša aktivirani parcialni tromboplastinski čas in protrombinski čas t.j. deluje antikoagulantno. Dodatno smo pokazali, da ne razgrajuje fibrinogena, in ne aktivira FX. Snake venom serine proteases are complex and multifunctional enzymes that primarily affect haemostasis. Different groups of serine proteases in the venom can have either anticoagulant or procoagulant effects. Molecules that have the latter property are rare. From previous studies on the venom of the nose-horned viper (Vipera ammodytes ammodytes) we already had the cDNA sequence of the protease VaaSP-6 and thus its amino acid sequence, but its biological and molecular mechanisms were not yet known. Due to the high sequence similarity, we speculate that its molecular mechanism of action is similar to that of VaaSP-VX, which can activate the blood coagulation factors V and X (FV and FX). It therefore exerts a procoagulant effect in the blood system. In this Master's thesis, we successfully isolated the VaaSP-6 protease using size exclusion chromatography, cation exchange chromatography and reversed-phase high-pressure liquid chromatography. Mass spectroscopy was used to identify the protease. Using two-dimensional gel electrophoresis, we determined several isoelectric points in the pH range of 4.5–7.5 and an apparent molecular mass of 35 kDa. We investigated its influence on the process of blood coagulation by measuring the activated partial thromboplastin time, the prothrombin time and the thrombin time. We were able to show that VaaSP-6, in contrast to VaaSP-VX, prolongs the activated partial thromboplastin time and prothrombin time and thus acts as an anticoagulant. We have also shown that it does not degrade fibrinogen and does not activate FX. Serinske proteaze VaaSP-6 prokoagulanti toksinologija modras Serine proteinases VaaSP-6 procuagulants toxinology nose-horned viper true false false [J. Počič] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2024-09-25 07:18:02 2024-09-25 07:18:07 2024-10-16 13:40:42 0000-00-00 00:00:00 2024 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 543.645.4:612.014.46(043.2) 246779 211800835 42016.pdf 42016.pdf 1 6825F022B46A85A3FAB33031609AAA43 864c3467f30585b37d8d2a44b20675ea31a5dfb9892754fd2ea09276135549cd 6fb407b4-7afd-11ef-b232-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=191879 Biotehniška fakulteta 0 0 0