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

Abstract

Humani serum albumin (HSA) je najzastupljeniji protein plazme sa udelom od oko 50 do 60 % svih proteina plazme. Vezuje i transportuje mnoge endogene i egzogene molekule, i doprinosi antioksidativnom kapacitetu seruma jer na površini molekula poseduje jednu slobodnu tiolnu grupu ostatka Cys34. Slobodne masne kiseline (MK), koje transportuje HSA, i karbonilni stres mogli bi, kroz promenu reaktivnosti tiolne grupe, uticati na njen potencijal kao hvatača reaktivnih karbonilnih vrsta. Stoga su ciljevi ove teze bili: in vitro ispitivanje uticaja 1) vezivanja MK, različite dužine lanca i zasićenosti na (i) reaktivnost Cys34-SH grupe, (ii) njen potencijal kao hvatača reaktivnih α-oksoaldehida, tj. na stepen karbonilacije i (iii) reaktivnost tiolne grupe HSA karbonilovanog metilglioksalom (model-sistem za molekule HSA modifikovane u karbonilnom 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 u karbonilnim stresom; 4) određivanje reaktivnosti Cys34-SH grupe HSA izolovanog iz seruma dijabetičara i zdravih osoba; 5) razvijanje qTLC metode za određivanje sadržaja MK, 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) i dokozaheksaenske kiseline (DHA)) za HSA in vitro, dovodi do povećanja vrednosti konstanti 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 efekte na reaktivnost HSA-SH, a najjači efekat ispoljava polinezasićena EPA. Od zasićenih MK, najveća vrednost k dobijena je pri vezivanju MYR za HSA, što može biti posledica izuzetne komplementarnosti njenog molekula sa vezivnim mestima na molekulu HSA, koja najviše utiču na izloženost tiolne grupe rastvaraču. Između vrednosti konstanti brzine reakcije Cys34-SH, dobijenih pri vezivanju različitih MK, i izračunatih vrednosti dostupnosti Cys34 tiolne grupe rastvaraču nađen je visok stepen korelacije (r=0,927)...

Human serum albumin (HSA) is the most abundant protein of human plasma, accounting for 50-60% of total plasma proteins. HSA binds and transport many endogenous and exogenous substances, and contributes to the antioxidative pool in serum 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 changed reactivity of the thiol group, on its potential as the scavenger of the reactive carbonyl species. 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 of the 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 FAs from fish oil on the Cys34-SH group reactivity (overviewing the possibility of the modulation of the HSA scavenger properties with supplements); 3) determination of the Cys34-SH group content, correlation with the HbA1c and glucose level of persons with elevated carbonyl stress; 4) determination of the Cys34-SH group reactivity of the HSA isolated from the sera of diabetic patients and healthy persons; 5) development of qTLC method for determination of the FAs content, bound to the HSA, which was isolated from sera samples. Binding of FAs of different chain lengths and saturation (myristic (MYR), palmitic (PLM), stearic (STE), oleic (OLA), eicosapentaenoic (EPE) and docosahexaenoic acid (DHA)) to the HSA in vitro resulting in increasing of the kinetics constant (k) of the Cys34 thiol group reaction (with DTNB)(k values for HSA-FAs complexes: from 14.58±0.19 ×10-3 to 26.02±1.06 × 10-3 s-1 comparing to k of the HSA solely 7.52 ± 0.04 × 10-3 s-1), i.e. its reactivity for 2 to 3.5 times. STE and OLA show similar effects to the HSA-SH reactivity, and the strongest effect shows polyunsaturated EPA. From saturated FAs binding to the HSA, the strongest effect shows MYR, and that could be explained with extremely good fitting of the MYR molecule to the HSA FAs binding pockets which has the most influences to the exposure/accessibility of the thiol group to solvent...