magistrsko delo
Povzetek
Proteini so biološke makromolekule, zgrajene iz aminokislin. Razlikujejo se po številu in
zaporedju v polipeptidno verigo povezanih aminokislin (primarna struktura). Le-to vpliva
na zvitje proteina v njegovo nativno obliko (sekundarna, terciarna, kvartarna struktura).
Vloga proteinov v živih organizmih je različna: strukturna, funkcijska in regulatorna. Na
stabilnost danega proteina v raztopini vplivajo njegova koncentracija, vrednost pH in
vrsta pufra, vrsta in koncentracija dodatkov (npr. soli, sladkorji, polimeri), temperatura,
tlak itd. Študij pogojev fazne stabilnosti proteinov v raztopinah (denaturacija, agregacija,
fazna separacija) je eden izmed ključnih dejavnikov pri razvoju zdravil, saj sta od nje
odvisna učinkovitost in rok uporabnosti biološkega zdravila. Agregacija proteinov ima za
posledico vrsto bolezenskih stanj (Alzheimerjeva, Parkinsonova, siva mrena), med tem
pa je lahko fazna separacija tudi zaželen pojav, denimo pri čiščenju proteinov
(kristalizacija).
V okviru magistrskega dela sem proučeval vpliv polietilenglikola (PEG) in soli na fazno
stabilnost govejega serumskega albumina (BSA) v acetatnem pufru pri dveh pHvrednostih (blizu in pod izoionsko točko). Uporabil sem PEG z molsko maso 6000, 10000
in 20000 g/mol ter različne natrijeve soli (NaF, NaCl, NaBr, NaI, NaNO3, NaOAc) ter
alkalijske kloride (LiCl, NaCl, KCl, RbCl, CsCl). Koncentracija BSA je bila v vseh
primerih 90 mg/mL. Raztopine sem pripravil v 0,1 M acetatnem pufru s pH = 4,6 in 4,0.
Dodatek PEG destabilizira raztopino (pomakne temperaturo točke zmotnitve k višjim
vrednostim) tem bolj čim večja je njegova molska masa ter višja kot je njegova
koncentracija. Efekt dodanega PEG je v raztopinah s pH = 4,6 močnejši kot pri 4,0. Ob
naraščajoči koncentraciji soli je efekt soli pri pH = 4,6 stabilizacijski, pri pH = 4,0 pa
destabilizacijski. Vidni so ionospecifični efekti tako pri anionih kot pri kationih. Med
Tc
in standardno prosto entalpijo hidratacije anionov/kationov dodane soli obstaja
korelacija. Pri pH ≈ pI velja, da ion z bolj negativno standardno prosto entalpijo
hidratacije zviša Tc
(destabilizira raztopino), medtem ko pri pH < pI velja, da jo tak ion
zniža (stabilizira raztopino).
Ključne besede
proteini;goveji serumski albumin;BSA;polietilen glikol;(1-1) elektroliti;fazna stabilnost;topnostna krivulja;fazna separacija tekoče-tekoče;Hofmeistrova vrsta;magistrska dela;
Podatki
Jezik: |
Slovenski jezik |
Leto izida: |
2020 |
Tipologija: |
2.09 - Magistrsko delo |
Organizacija: |
UL FKKT - Fakulteta za kemijo in kemijsko tehnologijo |
Založnik: |
[M. Belak Vivod] |
UDK: |
577.322(043.2) |
COBISS: |
34951683
|
Št. ogledov: |
320 |
Št. prenosov: |
52 |
Ocena: |
0 (0 glasov) |
Metapodatki: |
|
Ostali podatki
Sekundarni jezik: |
Angleški jezik |
Sekundarni naslov: |
Influence of salts on the phase stability of BSA-polyethylene glycol mixtures |
Sekundarni povzetek: |
Proteins are biological macromolecules build of amino acids. They differ in number and
sequence of amino acids that are linked together (primary structure). This affects protein
coagulation in its native form (secondary, tertiary and quaternary structure). Proteins have
many different roles in living organism: structural, functional and regulatory. The
stability of a given protein in solution is affected by its concentration, pH value and type
of buffer, type and concentration of additives (salt, sugars, polymers), temperature,
pressure etc. The study of the conditions of phase stability of proteins in solutions
(denaturation, aggregation, phase separation) is one of the key factors in the development
of drugs, as the effectiveness and shelf life of a biological drug depend on it. Protein
aggregation results in a number of disease states (Alzheimer's, Parkinson's, cataracts),
while phase separation can also be a desirable phenomenon, for example in protein
purification (crystallization).
As part of my master's thesis, I studied the effect of polyethylene glycol (PEG) and salt
on the phase stability of bovine serum albumin (BSA) in acetate buffer at two pH values
(near and below the isoionic point). I used PEG with molar masses of 6000, 10000 and
20000 g/mol and various sodium salts (NaF, NaCl, NaBr, NaI, NaNO3, NaOAc) and
alkali chlorides (LiCl, NaCl, KCl, RbCl, CsCl). The BSA concentration was 90 mg/mL
in all cases. The solutions were prepared in 0.1 M acetate buffer with pH = 4.6 and 4.0.
PEG destabilizes the solution (shifts the cloud point temperature to higher values) the
higher its molar mass and the higher its concentration. The effect of added PEG is
observed earlier in solutions with pH = 4.6 than in 4.0. With increasing salt concentration,
the effect of salt is stabilizing at pH = 4.6 and destabilizing at pH = 4.0. Ionospecific
effects are seen in both anions and cations. There is a correlation between Tc and the
standard free enthalpy of hydration of anions / cations of the added salt. At pH ≈ pI, an
ion with a more negative standard free hydration enthalpy is considered to increase Tc
(destabilize the solution), while at pH < pI, such ion is considered to decrease Tc (stabilize
the solution). |
Sekundarne ključne besede: |
phase stability;bovine serum albumin;polyethylene glycol;(1-1) electrolyte;Hofmeister series; |
Vrsta dela (COBISS): |
Magistrsko delo/naloga |
Študijski program: |
1000375 |
Komentar na gradivo: |
Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, smer Kemija |
Strani: |
63 str. |
ID: |
12977414 |