Promene reaktivnosti tiolne grupe Cys34 humanog serum-albumina pri vezivanju masnih kiselina in vitro i u karbonilnom stresu

Humani serum albumin (HSA) je najzastupljeniji protein plazme sa udelomod oko 50 do 60 % svih proteina plazme. Vezuje i transportuje mnoge endogene iegzogene molekule, i doprinosi antioksidativnom kapacitetu seruma jer na površinimolekula poseduje jednu slobodnu tiolnu grupu ostatka Cys34. Slobodne masne kiseline(MK), koje transportuje HSA, i karbonilni stres mogli bi, kroz promenu reaktivnostitiolne grupe, uticati na njen potencijal kao hvatača reaktivnih karbonilnih vrsta. Stogasu ciljevi ove teze bili: in vitro ispitivanje uticaja 1) vezivanja MK, različite dužinelanca i zasićenosti na (i) reaktivnost Cys34-SH grupe, (ii) njen potencijal kao hvatačareaktivnih α-oksoaldehida, tj. na stepen karbonilacije i (iii) reaktivnost tiolne grupeHSA karbonilovanog metilglioksalom (model-sistem za molekule HSA modifikovane ukarbonilnom stresu); 2) vezivanja MK ribljeg ulja na reaktivnost Cys34-SH grupe(sagledavanje mogućnosti modulacije svojstava HSA pomoću suplemenata); 3)određivanje sadržaja Cys34-SH grupe, korelacija sa sadržajem HbA1c i glukoze ukarbonilnim stresom; 4) određivanje reaktivnosti Cys34-SH grupe HSA izolovanog izseruma dijabetičara i zdravih osoba; 5) razvijanje qTLC metode za određivanje sadržajaMK, vezanih za HSA, koji je izolovan iz realnih uzoraka.Vezivanje MK (različite dužine lanca i zasićenosti: miristinske (MYR),palmitinske (PLM), stearinske (STE), oleinske (OLA), eikozapentenske (EPA) idokozaheksaenske kiseline (DHA)) za HSA in vitro, dovodi do povećanja vrednostikonstanti brzine reakcije (k) Cys34 tiolne grupe (i DTNB) (k vrednosti kompleksa HSAMK:od 14,58±0,19 x 10-3 do 26,02±1,06 x 10-3 s-1 u odnosu na k HSA: 7,52±0,04 x 10-3 s-1), odnosno njene reaktivnosti za 2 do 3,5 puta. STE i OLA ispoljavaju slične efektena reaktivnost HSA-SH, a najjači efekat ispoljava polinezasićena EPA. Od zasićenihMK, najveća vrednost k dobijena je pri vezivanju MYR za HSA, što može biti posledicaizuzetne komplementarnosti njenog molekula sa vezivnim mestima na molekulu
Human serum albumin (HSA) is the most abundant protein of humanplasma, accounting for 50-60% of total plasma proteins. HSA binds and transport manyendogenous and exogenous substances, and contributes to the antioxidative pool inserum because of Cys34 free thiol group on the surface of its molecule. Free fatty acids(FAs), transported by HSA, and carbonyl stress could influence, through changedreactivity of the thiol group, on its potential as the scavenger of the reactive carbonylspecies. Therefore, the aims of this thesis were: in vitro examination of the influence 1)of different (in terms of chain length and saturation) FAs binding on the: (i) reactivity ofthe Cys34-SH group, (ii) its potential as the scavenger of the reactive α-oxoladehydes,and (iii) on the reactivity of the thiol group of HSA carbonylated with methylglyoxal(the model-system for HSA molecules modified during carbonyl stress); 2) of FAsfrom fish oil on the Cys34-SH group reactivity (overviewing the possibility of themodulation of the HSA scavenger properties with supplements); 3) determination of theCys34-SH group content, correlation with the HbA1c and glucose level of persons withelevated carbonyl stress; 4) determination of the Cys34-SH group reactivity of the HSAisolated from the sera of diabetic patients and healthy persons; 5) development of qTLCmethod for determination of the FAs content, bound to the HSA, which was isolatedfrom sera samples.Binding of FAs of different chain lengths and saturation (myristic (MYR),palmitic (PLM), stearic (STE), oleic (OLA), eicosapentaenoic (EPE) anddocosahexaenoic acid (DHA)) to the HSA in vitro resulting in increasing of the kineticsconstant (k) of the Cys34 thiol group reaction (with DTNB)(k values for HSA-FAscomplexes: from 14.58±0.19 ×10-3 to 26.02±1.06 × 10-3 s-1 comparing to k of the HSAsolely 7.52 ± 0.04 × 10-3 s-1), i.e. its reactivity for 2 to 3.5 times. STE and OLA showsimilar effects to the HSA-SH reactivity, and the strongest effect shows polyuns