Uticaj nejonskih surfaktanata na strukturu i aktivnost peptidaza

Abstract

Aktivnost proteina je u korelaciji sa njihovim strukturnim konformacijama koje zavise od uslova okoline. Formulacije rastvora u kojima se proteini nalaze i uslovi pod kojima se oni čuvaju konsekventno ostvaruju odlučujući uticaj na njihovu konformaciju i aktivnost. Ispitivanje stabilnosti i aktivnosti proteina u cilju nalaženja optimalnih uslova sastava rastvora, pri različitim uslovima u k ljučujući snižene temperature, poseduje fundamentalnu naučnu važnost kao i potencijanu komercijalnu upot rebnu vrednost. Cilj ove studije je definisanje okvir a formulacija proteina, konkretno odabranih model peptidaza, u pogledu njihove strukture i optimalne aktivnosti sa posebnim naglaskom na prisustvo nejonskih surfaktanata, a zatim diferencijalno karakteri sanje procesa vezanih za konformaciju peptidaza i njihovu konačnu upotrebnu vrednost. Stabilnost i konformacija tripsina je ispitana za jone Hofmeister ove serije koji imaju stabilizujuće kosmotropske efekte na njegovu strukturu i definisan je optimum u po gledu koncentracije i vrste puferskog sistema. Infracrvenom i fluorescentnom spektroskopijom okarakterisan je uticaj serije pufera kao i sadržaj nejonskih surfaktanata Tween 20, 80 i Triton X 100 na distorzije u nativnoj strukturi tripsina i primećeno post ojanje metastabilnog intermediera stopljene globule pri uslovima snižene temperature. Definisan je optimalan sastav komponenti koji tripsinu omogućava maksimizaciju održanja strukturnog integriteta i aktivnosti do 99% nakon sedam ciklusa zamrzavanja/odmr zavanja, pri čemu je potvrđena krioprotektivna uloga nejonskih surfaktanata. Za papain i peptidaze lateksa smokve, okarakterisana je kazeinolitička, želatinolitička i kolagenolitička supstratna specifičnost klasičnim biohemi j skim metodama 2D elektroforezo m i zimografijom, kao i uticaj nejonskih surfaktanata na njihovu stabilnost infracrvenom i fluorescentnom spektroskopijom. Odabrane model peptidaze, tripsin, papain i smeša peptidaza lateksa smokve, u sinergiji sa odabranim nejonskim surfaktantima pokazali su potencijal za unapređenje metodologije detekcije mikroaerofilnih patogenih bakterija Listeria monocytogenes u pogledu povećanja osetljivosti i ekspeditivnosti postojećih protokola.

Protein activity is correlated with their structural conformations that depend on environmental conditions. Formulations of solutions in which proteins are found and the conditions under which they are stored consequently exert a decisive influence on their conformation and activity. Testing stability and activity of proteins to find the optimal solution composition, under different conditions including reduced temperatures, has fundamental scientific importance as well as potential commercial value. This study aims to define the framework for protein formulations, for specifically selected model peptidases, in terms of their structure and optimal activity with particular emphasis on the presence of nonionic surfactants, and to differentially characterize processes related to the conformation of peptidases and their final commercial usability. The stability and conformation of trypsin were examined for ions of the Hofmeister series that have stabilizing kosmotropic effects on its structure. The optimum in terms of concentration and type of buffer system was defined. The influence of the series of buffers and the nonionic surfactants Tween 20, 80, and Triton X-100 on distortions of the native structure of trypsin was characterized by infrared and fluorescence spectroscopy. The existence of a metastable intermediate - a molten globule at reduced temperature conditions was also observed. An optimal composition of components that enables trypsin to maximize the maintenance of structural integrity and activity up to 99% after seven freeze/thaw cycles was defined, while the cryoprotective role of nonionic surfactants was confirmed. For papain and fig latex peptidases, the caseinolytic, gelatinolytic, and collagenolytic substrate specificity was characterized by classical biochemical methods, 2D electrophoresis and zymography, and the influence of nonionic surfactants on their stability by infrared and fluorescence spectroscopy. The selected model peptidases trypsin, papain, and fig latex peptidase mixture, in synergy with selected nonionic surfactants, showed the potential to improve the methodology of detection of microaerophilic pathogenic bacterium Listeria monocytogenes in terms of increasing the sensitivity and expediency of existing protocols.