5 research outputs found
Application of electron paramagnetic resonance spectroscopy in the study of albumin conformational changes by spin-labeling method
Π‘Π΅ΡΡΠΌΡΠΊΠΈ Π°Π»Π±ΡΠΌΠΈΠ½ ΡΠ΅ Π½Π°ΡΠ·Π°ΡΡΡΠΏΡΠ΅Π½ΠΈΡΠΈ ΠΏΡΠΎΡΠ΅ΠΈΠ½ Ρ ΠΊΡΠ²Π½ΠΎΡ ΠΏΠ»Π°Π·ΠΌΠΈ, ΠΊΠΎΡΠΈ ΠΈΠΌΠ°
Π²ΠΈΡΠ΅ Π²Π°ΠΆΠ½ΠΈΡ
ΡΠΈΠ·ΠΈΠΎΠ»ΠΎΡΠΊΠΈΡ
ΡΡΠ½ΠΊΡΠΈΡΠ°, Π·Π±ΠΎΠ³ ΡΠ΅Π³Π° ΡΠ΅ Π΄Π΅ΡΠ°ΡΠ½ΠΎ ΠΏΡΠΎΡΡΠ°Π²Π°Π½ ΡΠ°Π·Π½ΠΈΠΌ
ΡΠ΅Ρ
Π½ΠΈΠΊΠ°ΠΌΠ°. ΠΠ΅Π΄Π½Π° ΠΎΠ΄ ΡΠ΅Ρ
Π½ΠΈΠΊΠ° ΠΊΠΎΡΠΈΡΡΠ΅Π½ΠΈΡ
Π·Π° ΠΏΡΠΎΡΡΠ°Π²Π°ΡΠ΅ ΠΊΠΎΠ½ΡΠΎΡΠΌΠ°ΡΠΈΠΎΠ½ΠΈΡ
ΠΏΡΠΎΠΌΠ΅Π½Π° Π°Π»Π±ΡΠΌΠΈΠ½Π° ΠΈ ΡΠ΅Π³ΠΎΠ²ΠΎΠ³ ΠΊΠ°ΠΏΠ°ΡΠΈΡΠ΅ΡΠ° Π·Π° Π²Π΅Π·ΠΈΠ²Π°ΡΠ΅ ΡΠ°Π·Π½ΠΈΡ
ΡΡΠΏΡΡΠ°Π½ΡΠΈΡΠ° ΡΠ΅
Π΅Π»Π΅ΠΊΡΡΠΎΠ½ΡΠΊΠ° ΠΏΠ°ΡΠ°ΠΌΠ°Π³Π½Π΅ΡΠ½Π° ΡΠ΅Π·ΠΎΠ½Π°Π½ΡΠ½Π° (ΠΠΠ ) ΡΠΏΠ΅ΠΊΡΡΠΎΡΠΊΠΎΠΏΠΈΡΠ° Ρ ΡΠΏΡΠ΅Π·ΠΈ ΡΠ° ΡΠΏΠΈΠ½ΡΠΊΠΈΠΌ
ΠΎΠ±Π΅Π»Π΅ΠΆΠ°Π²Π°ΡΠ΅ΠΌ. Π£ ΡΡ ΡΠ²ΡΡ
Ρ ΡΠ΅ ΠΊΠΎΡΠΈΡΡΠ΅Π½ΠΎ Π½Π΅ΠΊΠΎΠ»ΠΈΠΊΠΎ ΡΠ°Π·Π»ΠΈΡΠΈΡΠΈΡ
ΡΠΏΠΈΠ½ΡΠΊΠΈΡ
ΠΎΠ±Π΅Π»Π΅ΠΆΠΈΠ²Π°ΡΠ°, ΠΌΠ΅ΡΡ ΠΊΠΎΡΠΈΠΌΠ° ΡΡ ΡΠΏΠΈΠ½ΡΠΊΠΈ ΠΎΠ±Π΅Π»Π΅ΠΆΠ΅Π½Π΅ ΠΌΠ°ΡΠ½Π΅ ΠΊΠΈΡΠ΅Π»ΠΈΠ½Π΅, Ρ ΠΎΠ±Π·ΠΈΡΠΎΠΌ Π½Π° ΡΠΎ
Π΄Π° ΡΠ΅ Π°Π»Π±ΡΠΌΠΈΠ½ Π³Π»Π°Π²Π½ΠΈ ΡΡΠ°Π½ΡΠΏΠΎΡΡΠ΅Ρ ΠΌΠ°ΡΠ½ΠΈΡ
ΠΊΠΈΡΠ΅Π»ΠΈΠ½Π°. ΠΡΡΠ³ΠΈ ΡΠ΅ΡΡΠΎ ΠΊΠΎΡΠΈΡΡΠ΅Π½ΠΈ
ΠΎΠ±Π΅Π»Π΅ΠΆΠΈΠ²Π°Ρ ΡΠ΅ 3-ΠΌΠ°Π»Π΅ΠΈΠΌΠΈΠ΄ΠΎ ΠΏΡΠΎΠΊΡΠΈΠ» (5-ΠΠ‘Π) ΠΊΠΎΡΠΈ ΡΠ΅ ΠΊΠΎΠ²Π°Π»Π΅Π½ΡΠ½ΠΎ Π²Π΅Π·ΡΡΠ΅ Π·Π°
ΡΠ»ΠΎΠ±ΠΎΠ΄Π°Π½ ΡΠΈΡΡΠ΅ΠΈΠ½.
Π£ ΠΎΠ²ΠΎΡ Π΄ΠΎΠΊΡΠΎΡΡΠΊΠΎΡ Π΄ΠΈΡΠ΅ΡΡΠ°ΡΠΈΡΠΈ Π΄Π²Π΅ ΡΠΏΠΈΠ½ΡΠΊΠΈ ΠΎΠ±Π΅Π»Π΅ΠΆΠ΅Π½Π΅ ΡΡΠ΅Π°ΡΠΈΠ½ΡΠΊΠ΅ ΠΊΠΈΡΠ΅Π»ΠΈΠ½Π΅
ΠΊΠΎΡΠ΅ ΡΠ°Π΄ΡΠΆΠ΅ ΠΏΠ°ΡΠ°ΠΌΠ°Π³Π½Π΅ΡΠ½Ρ Π΄ΠΎΠΊΡΠΈΠ» Π³ΡΡΠΏΡ (Π³ΡΡΠΏΠ° ΠΎΠ΄ ΠΊΠΎΡΠ΅ ΠΏΠΎΡΠΈΡΠ΅ ΠΠΠ ΡΠΈΠ³Π½Π°Π») Π½Π°
ΠΏΠ΅ΡΠΎΠΌ (5-Π΄ΠΎΠΊΡΠΈΠ» ΡΡΠ΅Π°ΡΠΈΠ½ΡΠΊΠ° ΠΊΠΈΡΠ΅Π»ΠΈΠ½Π°, 5-ΠΠ‘) ΠΈ Π½Π° ΡΠ΅ΡΠ½Π°Π΅ΡΡΠΎΠΌ (16-Π΄ΠΎΠΊΡΠΈΠ»
ΡΡΠ΅Π°ΡΠΈΠ½ΡΠΊΠ° ΠΊΠΈΡΠ΅Π»ΠΈΠ½Π°, 16-ΠΠ‘) ΡΠ³ΡΠ΅Π½ΠΈΠΊΠΎΠ²ΠΎΠΌ Π°ΡΠΎΠΌΡ ΠΌΠ΅ΡΠΈΠ»Π΅Π½ΡΠΊΠΎΠ³ Π»Π°Π½ΡΠ° ΡΡ
ΠΊΠΎΡΠΈΡΡΠ΅Π½Π΅ Π·Π° ΠΏΡΠΎΡΡΠ°Π²Π°ΡΠ΅ ΠΊΠΎΠ½ΡΠΎΡΠΌΠ°ΡΠΈΠΎΠ½ΠΈΡ
ΠΏΡΠΎΠΌΠ΅Π½Π° Ρ Ρ
ΡΠΌΠ°Π½ΠΎΠΌ ΡΠ΅ΡΡΠΌΡΠΊΠΎΠΌ
Π°Π»Π±ΡΠΌΠΈΠ½Ρ (Π₯Π‘Π). ΠΡΠΈΠΌ ΡΠΎΠ³Π°, ΠΊΠΎΠ½ΡΠΎΠΌΠ°ΡΠΈΠΎΠ½Π΅ ΠΏΡΠΎΠΌΠ΅Π½Π΅ Ρ Π³ΠΎΠ²Π΅ΡΠ΅ΠΌ ΡΠ΅ΡΡΠΌΡΠΊΠΎΠΌ
Π°Π»Π±ΡΠΌΠΈΠ½Ρ (ΠΠ‘Π) ΡΡ ΠΏΡΠΎΡΡΠ°Π²Π°Π½Π΅ ΠΎΠ±Π΅Π»Π΅ΠΆΠ°Π²Π°ΡΠ΅ΠΌ ΠΠ‘Π ΡΠ° 5-ΠΠ‘Π. ΠΠΎΠ½ΡΠΎΡΠΌΠ°ΡΠΈΠΎΠ½Π΅
ΠΏΡΠΎΠΌΠ΅Π½Π΅ ΡΡ Π±ΠΈΠ»Π΅ ΠΈΠ½Π΄ΡΠΊΠΎΠ²Π°Π½Π΅ Π²Π°ΡΠΈΡΠ°ΡΠ΅ΠΌ ΡΠ΅ΠΌΠΏΠ΅ΡΠ°ΡΡΡΠ΅ ΠΈ pH, Π΄ΠΎΠ΄Π°Π²Π°ΡΠ΅ΠΌ Π΅ΡΠ°Π½ΠΎΠ»Π°,
Π²Π΅Π·ΠΈΠ²Π°ΡΠ΅ΠΌ Π½Π΅ΠΊΠΎΠ»ΠΈΠΊΠΎ Π»ΠΈΠ³Π°Π½Π°Π΄Π° ΡΠΈΠΏΠΈΡΠ½ΠΈΡ
Π·Π° Π°Π»Π±ΡΠΌΠΈΠ½ (ΠΌΠ°ΡΠ½Π΅ ΠΊΠΈΡΠ΅Π»ΠΈΠ½Π΅ ΠΈ Π»Π΅ΠΊΠΎΠ²ΠΈ) ΠΈ
ΠΈΠ·Π»Π°Π³Π°ΡΠ΅ΠΌ Π΄Π΅ΡΡΡΠ²Ρ ΡΠ°ΠΊΠΈΡ
ΠΎΠΊΡΠΈΠ΄ΡΡΡΡΠΈΡ
Π°Π³Π΅Π½Π°ΡΠ°, Π²ΠΎΠ΄ΠΎΠ½ΠΈΠΊ-ΠΏΠ΅ΡΠΎΠΊΡΠΈΠ΄Ρ ΠΈ
ΡΡΠΏΠ΅ΡΠΎΠΊΡΠΈΠ΄Π½ΠΎΠΌ Π°Π½ΡΠΎΠ½ΡΠΊΠΎΠΌ ΡΠ°Π΄ΠΈΠΊΠ°Π»Ρ. ΠΠΎΠ΄Π°ΡΠ½ΠΈ ΡΠΈΡ ΠΎΠ²Π΅ Π΄ΠΈΡΠ΅ΡΡΠ°ΡΠΈΡΠ΅ ΡΠ΅ Π±ΠΈΠΎ Π΄Π° ΡΠ΅
ΠΈΡΠΏΠΈΡΠ° Π΄Π° Π»ΠΈ 5-ΠΠ‘Π ΠΌΠΎΠΆΠ΅ Π΄Π° ΡΠ΅ ΠΊΠΎΡΠΈΡΡΠΈ Π·Π° Π΄Π΅ΡΠ΅ΠΊΡΠΈΡΡ ΡΡΠΏΡΠΈΠ»Π½ΠΈΡ
ΠΊΠΎΠ½ΡΠΎΡΠΌΠ°ΡΠΈΠΎΠ½ΠΈΡ
ΠΏΡΠΎΠΌΠ΅Π½Π° ΡΠ·ΡΠΎΠΊΠΎΠ²Π°Π½ΠΈΡ
Π²Π΅Π·ΠΈΠ²Π°ΡΠ΅ΠΌ Π»ΠΈΠ³Π°Π½Π°Π΄Π°, Ρ ΠΎΠ±Π·ΠΈΡΠΎΠΌ Π½Π° ΡΠΎ Π΄Π° ΡΠ΅ 5-ΠΠ‘Π Π½Π°Π»Π°Π·ΠΈ Ρ
ΡΠΈΠ³ΠΈΠ΄Π½ΠΎΡ ΡΡΠ΅Π΄ΠΈΠ½ΠΈ ΠΊΠ°Π΄Π° ΡΠ΅ Π²Π΅ΠΆΠ΅ Π·Π° ΡΠ΅Π΄ΠΈΠ½ΠΈ ΡΠ»ΠΎΠ±ΠΎΠ΄Π°Π½ ΡΠΈΡΡΠ΅ΠΈΠ½ Ρ ΠΠ‘A ΠΊΠΎΡΠΈ ΡΠ΅ Π½Π°Π»Π°Π·ΠΈ
Π½Π° ΠΏΠΎΠ·ΠΈΡΠΈΡΠΈ 34 Ρ Π°ΠΌΠΈΠ½ΠΎΠΊΠΈΡΠ΅Π»ΠΈΠ½ΡΠΊΠΎΡ ΡΠ΅ΠΊΠ²Π΅Π½ΡΠΈΡΠΈ (Cys-34).
Π Π΅Π·ΡΠ»ΡΠ°ΡΠΈ ΠΎΠ²Π΅ Π΄ΠΈΡΠ΅ΡΡΠ°ΡΠΈΡΠ΅ ΠΏΠΎΠΊΠ°Π·ΡΡΡ Π΄Π° ΡΡ Π΄ΠΎΠΊΡΠΈΠ» Π³ΡΡΠΏΠ΅ ΠΎΠ±Π΅Π»Π΅ΠΆΠΈΠ²Π°ΡΠ° 5-ΠΠ‘ ΠΈ
16-ΠΠ‘ (ΠΊΠΎΡΠ΅ Π΄Π°ΡΡ ΠΈΠ½ΡΠΎΡΠΌΠ°ΡΠΈΡΠ΅ ΠΎ ΠΏΡΠΎΠΌΠ΅Π½Π°ΠΌa Ρ ΡΠ²ΠΎΡΠΎΡ ΠΎΠΊΠΎΠ»ΠΈΠ½ΠΈ), Π»ΠΎΡΠΈΡΠ°Π½Π΅ Π½Π°
ΡΠ°Π·Π»ΠΈΡΠΈΡΠΈΠΌ ΠΌΠ΅ΡΡΠΈΠΌΠ° Ρ Π₯Π‘Π. ΠΠ°ΠΈΠΌΠ΅, Π΄ΠΎΠΊΡΠΈΠ» Π³ΡΡΠΏΠ° 5-ΠΠ‘ ΡΠ΅ Π½Π°Π»Π°Π·ΠΈ Ρ Ρ
ΠΈΠ΄ΡΠΎΡΠΎΠ±Π½ΠΎΡ
ΡΠ½ΡΡΡΠ°ΡΡΠΎΡΡΠΈ Π₯Π‘Π, Π΄ΠΎΠΊ ΡΠ΅ Π·Π° 16-ΠΠ‘ Π»ΠΎΡΠΈΡΠ°Π½Π° Π±Π»ΠΈΠ·Ρ ΠΏΠΎΠ²ΡΡΠΈΠ½Π΅ Π₯Π‘Π ΠΈΠ»ΠΈ ΡΠ°ΠΊ
ΠΏΡΠΎΠ»Π°Π·ΠΈ ΠΊΡΠΎΠ· ΡΡ. Π‘ΡΠΎΠ³Π°, ΠΎΠ²Π° Π΄Π²Π° ΡΠ΅Π΄ΠΈΡΠ΅ΡΠ° Π΄Π°ΡΡ ΠΈΠ½ΡΠΎΡΠΌΠ°ΡΠΈΡΠ΅ ΡΠ° ΡΠ°Π·Π»ΠΈΡΠΈΡΠΈΡ
ΠΌΠ΅ΡΡΠ°
Π½Π° ΠΌΠΎΠ»Π΅ΠΊΡΠ»Ρ Π₯Π‘Π. Π’Π°ΠΊΠΎΡΠ΅ ΡΠ΅ ΡΠΎΡΠ΅Π½ΠΎ Π΄Π° ΠΠΠ ΡΠΏΠ΅ΠΊΡΡΠΈ ΠΎΠ±Π° ΠΠ‘ ΠΎΠ±Π΅Π»Π΅ΠΆΠΈΠ²Π°ΡΠ° Π²Π΅Π·Π°Π½ΠΈΡ
Π·Π° Π₯Π‘Π ΠΌΠΎΠ³Ρ Π΄Π° ΡΠ΅ ΡΠ°Π·Π»ΠΎΠΆΠ΅ Π½Π° ΠΊΠΎΠΌΠΏΠΎΠ½Π΅Π½ΡΠ΅ ΠΊΠΎΡΠ΅ ΠΎΠ΄Π³ΠΎΠ²Π°ΡΠ°ΡΡ ΡΠ°ΠΊΠΎ (ΠΠ), ΡΠ»Π°Π±ΠΎ Π²Π΅Π·Π°Π½ΠΎΠΌ
(Π‘Π) ΠΈ Π½Π΅Π²Π΅Π·Π°Π½ΠΎΠΌ (ΠΠ) ΠΎΠ±Π΅Π»Π΅ΠΆΠΈΠ²Π°ΡΡ. ΠΠ° 5-ΠΠ‘Π Π²Π΅Π·Π°Π½ Π·Π° ΠΠ‘Π, ΠΏΠΎΠΊΠ°Π·Π°Π½ΠΎ ΡΠ΅ Π΄Π° ΡΠ΅
ΠΠΠ ΡΠΏΠ΅ΠΊΡΡΠΈ ΡΠ°ΡΡΠΎΡΠ΅ ΠΈΠ· Π΄Π²Π΅ ΠΊΠΎΠΌΠΏΠΎΠ½Π΅Π½ΡΠ΅, ΠΠ ΠΈ Π‘Π, ΠΊΠΎΡΠ΅ ΠΏΠΎΡΠΈΡΡ ΠΎΠ΄ 5-ΠΠ‘Π Π²Π΅Π·Π°Π½ΠΎΠ³ Π·Π°
Cys-34 ΠΈ Π°ΠΌΠΈΠ½ΠΎ Π³ΡΡΠΏΠ΅, ΡΠ΅ΡΠΏΠ΅ΠΊΡΠΈΠ²Π½ΠΎ...Serum albumin is the most abundant blood plasma protein, which has been
thoroughly studied using a plethora of techniques, due to its multiple important
physiological roles. One of the methods used to monitor albumin conformational
changes and binding capacity is electron paramagnetic resonance (EPR) spin-labeling
methodology. For this purpose, albumin has been studied using various spin-labels,
among which spin-labeled fatty acids have been the most commonly employed, since
albumin is the main carrier of fatty acids. The other frequently used spin-label is 3-
maleimido-proxyl (5-MSL) which binds covalently to the free cysteine residues.
In this doctoral dissertation, two spin-labeled stearic acids containing a
paramagnetic doxyl group (the EPR-active group) attached at fifth (5-doxyl-stearic acid,
5-DS) and sixteenth (16-doxyl stearic acid, 16-DS) carbon atom of the methylene chain,
were used to study the conformational changes of human serum albumin (HSA).
Furthermore, conformational changes of bovine serum albumin (BSA) were studied by
labeling BSA with 5-MSL. The conformational changes were induced by varying
temperature and pH, addition of ethanol, binding several typical ligands (fatty acids and
drugs) and exposure to strong oxidizing agents, hydrogen peroxide and superoxide
anion radical. The additional goal of this dissertation was to reveal whether 5-MSL can
be used to track subtle conformational changes arising from ligand binding, since 5-
MSL is located in the fairly rigid environment when bound to the single free cysteine
residue in BSA, located at the position 34 in amino acid sequence (Cys-34).
The results from this study show that the doxyl groups (group which reports the
changes in its environment) of 5-DS and 16-DS, are located at different sites in HSA.
Namely, doxyl group of 5-DS is located in the hydrophobic interior, while for 16-DS it
is close to, or even protrudes the surface of HSA. Hence, these two compounds give
information from the different locations in HSA molecule. It was also observed that
EPR spectra of both DS spin-labels bound to HSA could be decomposed into
components corresponding to the strongly (SB), weakly bound (WB) and unbound (UB)
label. In case of 5-MSL bound to BSA, it was shown that the corresponding EPR
spectra consist of SB and WB component which originate from 5-MSL bound to Cys-34
and amino groups, respectively..
European contribution to the study of ROS: A summary of the findings and prospects for the future from the COST action BM1203 (EU-ROS).
The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed.The EU-ROS consortium (COST Action BM1203) was supported by the European Cooperation in Science and Technology (COST). The present overview represents the final Action dissemination summarizing the major achievements of COST Action BM1203 (EU-ROS) as well as research news and personal views of its members. Some authors were also supported by COST Actions BM1005 (ENOG) and BM1307 (PROTEOSTASIS), as well as funding from the European Commission FP7 and H2020 programmes, and several national funding agencies
Application of electron paramagnetic resonance spectroscopy in the study of albumin conformational changes by spin-labeling method
Π‘Π΅ΡΡΠΌΡΠΊΠΈ Π°Π»Π±ΡΠΌΠΈΠ½ ΡΠ΅ Π½Π°ΡΠ·Π°ΡΡΡΠΏΡΠ΅Π½ΠΈΡΠΈ ΠΏΡΠΎΡΠ΅ΠΈΠ½ Ρ ΠΊΡΠ²Π½ΠΎΡ ΠΏΠ»Π°Π·ΠΌΠΈ, ΠΊΠΎΡΠΈ ΠΈΠΌΠ°
Π²ΠΈΡΠ΅ Π²Π°ΠΆΠ½ΠΈΡ
ΡΠΈΠ·ΠΈΠΎΠ»ΠΎΡΠΊΠΈΡ
ΡΡΠ½ΠΊΡΠΈΡΠ°, Π·Π±ΠΎΠ³ ΡΠ΅Π³Π° ΡΠ΅ Π΄Π΅ΡΠ°ΡΠ½ΠΎ ΠΏΡΠΎΡΡΠ°Π²Π°Π½ ΡΠ°Π·Π½ΠΈΠΌ
ΡΠ΅Ρ
Π½ΠΈΠΊΠ°ΠΌΠ°. ΠΠ΅Π΄Π½Π° ΠΎΠ΄ ΡΠ΅Ρ
Π½ΠΈΠΊΠ° ΠΊΠΎΡΠΈΡΡΠ΅Π½ΠΈΡ
Π·Π° ΠΏΡΠΎΡΡΠ°Π²Π°ΡΠ΅ ΠΊΠΎΠ½ΡΠΎΡΠΌΠ°ΡΠΈΠΎΠ½ΠΈΡ
ΠΏΡΠΎΠΌΠ΅Π½Π° Π°Π»Π±ΡΠΌΠΈΠ½Π° ΠΈ ΡΠ΅Π³ΠΎΠ²ΠΎΠ³ ΠΊΠ°ΠΏΠ°ΡΠΈΡΠ΅ΡΠ° Π·Π° Π²Π΅Π·ΠΈΠ²Π°ΡΠ΅ ΡΠ°Π·Π½ΠΈΡ
ΡΡΠΏΡΡΠ°Π½ΡΠΈΡΠ° ΡΠ΅
Π΅Π»Π΅ΠΊΡΡΠΎΠ½ΡΠΊΠ° ΠΏΠ°ΡΠ°ΠΌΠ°Π³Π½Π΅ΡΠ½Π° ΡΠ΅Π·ΠΎΠ½Π°Π½ΡΠ½Π° (ΠΠΠ ) ΡΠΏΠ΅ΠΊΡΡΠΎΡΠΊΠΎΠΏΠΈΡΠ° Ρ ΡΠΏΡΠ΅Π·ΠΈ ΡΠ° ΡΠΏΠΈΠ½ΡΠΊΠΈΠΌ
ΠΎΠ±Π΅Π»Π΅ΠΆΠ°Π²Π°ΡΠ΅ΠΌ. Π£ ΡΡ ΡΠ²ΡΡ
Ρ ΡΠ΅ ΠΊΠΎΡΠΈΡΡΠ΅Π½ΠΎ Π½Π΅ΠΊΠΎΠ»ΠΈΠΊΠΎ ΡΠ°Π·Π»ΠΈΡΠΈΡΠΈΡ
ΡΠΏΠΈΠ½ΡΠΊΠΈΡ
ΠΎΠ±Π΅Π»Π΅ΠΆΠΈΠ²Π°ΡΠ°, ΠΌΠ΅ΡΡ ΠΊΠΎΡΠΈΠΌΠ° ΡΡ ΡΠΏΠΈΠ½ΡΠΊΠΈ ΠΎΠ±Π΅Π»Π΅ΠΆΠ΅Π½Π΅ ΠΌΠ°ΡΠ½Π΅ ΠΊΠΈΡΠ΅Π»ΠΈΠ½Π΅, Ρ ΠΎΠ±Π·ΠΈΡΠΎΠΌ Π½Π° ΡΠΎ
Π΄Π° ΡΠ΅ Π°Π»Π±ΡΠΌΠΈΠ½ Π³Π»Π°Π²Π½ΠΈ ΡΡΠ°Π½ΡΠΏΠΎΡΡΠ΅Ρ ΠΌΠ°ΡΠ½ΠΈΡ
ΠΊΠΈΡΠ΅Π»ΠΈΠ½Π°. ΠΡΡΠ³ΠΈ ΡΠ΅ΡΡΠΎ ΠΊΠΎΡΠΈΡΡΠ΅Π½ΠΈ
ΠΎΠ±Π΅Π»Π΅ΠΆΠΈΠ²Π°Ρ ΡΠ΅ 3-ΠΌΠ°Π»Π΅ΠΈΠΌΠΈΠ΄ΠΎ ΠΏΡΠΎΠΊΡΠΈΠ» (5-ΠΠ‘Π) ΠΊΠΎΡΠΈ ΡΠ΅ ΠΊΠΎΠ²Π°Π»Π΅Π½ΡΠ½ΠΎ Π²Π΅Π·ΡΡΠ΅ Π·Π°
ΡΠ»ΠΎΠ±ΠΎΠ΄Π°Π½ ΡΠΈΡΡΠ΅ΠΈΠ½.
Π£ ΠΎΠ²ΠΎΡ Π΄ΠΎΠΊΡΠΎΡΡΠΊΠΎΡ Π΄ΠΈΡΠ΅ΡΡΠ°ΡΠΈΡΠΈ Π΄Π²Π΅ ΡΠΏΠΈΠ½ΡΠΊΠΈ ΠΎΠ±Π΅Π»Π΅ΠΆΠ΅Π½Π΅ ΡΡΠ΅Π°ΡΠΈΠ½ΡΠΊΠ΅ ΠΊΠΈΡΠ΅Π»ΠΈΠ½Π΅
ΠΊΠΎΡΠ΅ ΡΠ°Π΄ΡΠΆΠ΅ ΠΏΠ°ΡΠ°ΠΌΠ°Π³Π½Π΅ΡΠ½Ρ Π΄ΠΎΠΊΡΠΈΠ» Π³ΡΡΠΏΡ (Π³ΡΡΠΏΠ° ΠΎΠ΄ ΠΊΠΎΡΠ΅ ΠΏΠΎΡΠΈΡΠ΅ ΠΠΠ ΡΠΈΠ³Π½Π°Π») Π½Π°
ΠΏΠ΅ΡΠΎΠΌ (5-Π΄ΠΎΠΊΡΠΈΠ» ΡΡΠ΅Π°ΡΠΈΠ½ΡΠΊΠ° ΠΊΠΈΡΠ΅Π»ΠΈΠ½Π°, 5-ΠΠ‘) ΠΈ Π½Π° ΡΠ΅ΡΠ½Π°Π΅ΡΡΠΎΠΌ (16-Π΄ΠΎΠΊΡΠΈΠ»
ΡΡΠ΅Π°ΡΠΈΠ½ΡΠΊΠ° ΠΊΠΈΡΠ΅Π»ΠΈΠ½Π°, 16-ΠΠ‘) ΡΠ³ΡΠ΅Π½ΠΈΠΊΠΎΠ²ΠΎΠΌ Π°ΡΠΎΠΌΡ ΠΌΠ΅ΡΠΈΠ»Π΅Π½ΡΠΊΠΎΠ³ Π»Π°Π½ΡΠ° ΡΡ
ΠΊΠΎΡΠΈΡΡΠ΅Π½Π΅ Π·Π° ΠΏΡΠΎΡΡΠ°Π²Π°ΡΠ΅ ΠΊΠΎΠ½ΡΠΎΡΠΌΠ°ΡΠΈΠΎΠ½ΠΈΡ
ΠΏΡΠΎΠΌΠ΅Π½Π° Ρ Ρ
ΡΠΌΠ°Π½ΠΎΠΌ ΡΠ΅ΡΡΠΌΡΠΊΠΎΠΌ
Π°Π»Π±ΡΠΌΠΈΠ½Ρ (Π₯Π‘Π). ΠΡΠΈΠΌ ΡΠΎΠ³Π°, ΠΊΠΎΠ½ΡΠΎΠΌΠ°ΡΠΈΠΎΠ½Π΅ ΠΏΡΠΎΠΌΠ΅Π½Π΅ Ρ Π³ΠΎΠ²Π΅ΡΠ΅ΠΌ ΡΠ΅ΡΡΠΌΡΠΊΠΎΠΌ
Π°Π»Π±ΡΠΌΠΈΠ½Ρ (ΠΠ‘Π) ΡΡ ΠΏΡΠΎΡΡΠ°Π²Π°Π½Π΅ ΠΎΠ±Π΅Π»Π΅ΠΆΠ°Π²Π°ΡΠ΅ΠΌ ΠΠ‘Π ΡΠ° 5-ΠΠ‘Π. ΠΠΎΠ½ΡΠΎΡΠΌΠ°ΡΠΈΠΎΠ½Π΅
ΠΏΡΠΎΠΌΠ΅Π½Π΅ ΡΡ Π±ΠΈΠ»Π΅ ΠΈΠ½Π΄ΡΠΊΠΎΠ²Π°Π½Π΅ Π²Π°ΡΠΈΡΠ°ΡΠ΅ΠΌ ΡΠ΅ΠΌΠΏΠ΅ΡΠ°ΡΡΡΠ΅ ΠΈ pH, Π΄ΠΎΠ΄Π°Π²Π°ΡΠ΅ΠΌ Π΅ΡΠ°Π½ΠΎΠ»Π°,
Π²Π΅Π·ΠΈΠ²Π°ΡΠ΅ΠΌ Π½Π΅ΠΊΠΎΠ»ΠΈΠΊΠΎ Π»ΠΈΠ³Π°Π½Π°Π΄Π° ΡΠΈΠΏΠΈΡΠ½ΠΈΡ
Π·Π° Π°Π»Π±ΡΠΌΠΈΠ½ (ΠΌΠ°ΡΠ½Π΅ ΠΊΠΈΡΠ΅Π»ΠΈΠ½Π΅ ΠΈ Π»Π΅ΠΊΠΎΠ²ΠΈ) ΠΈ
ΠΈΠ·Π»Π°Π³Π°ΡΠ΅ΠΌ Π΄Π΅ΡΡΡΠ²Ρ ΡΠ°ΠΊΠΈΡ
ΠΎΠΊΡΠΈΠ΄ΡΡΡΡΠΈΡ
Π°Π³Π΅Π½Π°ΡΠ°, Π²ΠΎΠ΄ΠΎΠ½ΠΈΠΊ-ΠΏΠ΅ΡΠΎΠΊΡΠΈΠ΄Ρ ΠΈ
ΡΡΠΏΠ΅ΡΠΎΠΊΡΠΈΠ΄Π½ΠΎΠΌ Π°Π½ΡΠΎΠ½ΡΠΊΠΎΠΌ ΡΠ°Π΄ΠΈΠΊΠ°Π»Ρ. ΠΠΎΠ΄Π°ΡΠ½ΠΈ ΡΠΈΡ ΠΎΠ²Π΅ Π΄ΠΈΡΠ΅ΡΡΠ°ΡΠΈΡΠ΅ ΡΠ΅ Π±ΠΈΠΎ Π΄Π° ΡΠ΅
ΠΈΡΠΏΠΈΡΠ° Π΄Π° Π»ΠΈ 5-ΠΠ‘Π ΠΌΠΎΠΆΠ΅ Π΄Π° ΡΠ΅ ΠΊΠΎΡΠΈΡΡΠΈ Π·Π° Π΄Π΅ΡΠ΅ΠΊΡΠΈΡΡ ΡΡΠΏΡΠΈΠ»Π½ΠΈΡ
ΠΊΠΎΠ½ΡΠΎΡΠΌΠ°ΡΠΈΠΎΠ½ΠΈΡ
ΠΏΡΠΎΠΌΠ΅Π½Π° ΡΠ·ΡΠΎΠΊΠΎΠ²Π°Π½ΠΈΡ
Π²Π΅Π·ΠΈΠ²Π°ΡΠ΅ΠΌ Π»ΠΈΠ³Π°Π½Π°Π΄Π°, Ρ ΠΎΠ±Π·ΠΈΡΠΎΠΌ Π½Π° ΡΠΎ Π΄Π° ΡΠ΅ 5-ΠΠ‘Π Π½Π°Π»Π°Π·ΠΈ Ρ
ΡΠΈΠ³ΠΈΠ΄Π½ΠΎΡ ΡΡΠ΅Π΄ΠΈΠ½ΠΈ ΠΊΠ°Π΄Π° ΡΠ΅ Π²Π΅ΠΆΠ΅ Π·Π° ΡΠ΅Π΄ΠΈΠ½ΠΈ ΡΠ»ΠΎΠ±ΠΎΠ΄Π°Π½ ΡΠΈΡΡΠ΅ΠΈΠ½ Ρ ΠΠ‘A ΠΊΠΎΡΠΈ ΡΠ΅ Π½Π°Π»Π°Π·ΠΈ
Π½Π° ΠΏΠΎΠ·ΠΈΡΠΈΡΠΈ 34 Ρ Π°ΠΌΠΈΠ½ΠΎΠΊΠΈΡΠ΅Π»ΠΈΠ½ΡΠΊΠΎΡ ΡΠ΅ΠΊΠ²Π΅Π½ΡΠΈΡΠΈ (Cys-34).
Π Π΅Π·ΡΠ»ΡΠ°ΡΠΈ ΠΎΠ²Π΅ Π΄ΠΈΡΠ΅ΡΡΠ°ΡΠΈΡΠ΅ ΠΏΠΎΠΊΠ°Π·ΡΡΡ Π΄Π° ΡΡ Π΄ΠΎΠΊΡΠΈΠ» Π³ΡΡΠΏΠ΅ ΠΎΠ±Π΅Π»Π΅ΠΆΠΈΠ²Π°ΡΠ° 5-ΠΠ‘ ΠΈ
16-ΠΠ‘ (ΠΊΠΎΡΠ΅ Π΄Π°ΡΡ ΠΈΠ½ΡΠΎΡΠΌΠ°ΡΠΈΡΠ΅ ΠΎ ΠΏΡΠΎΠΌΠ΅Π½Π°ΠΌa Ρ ΡΠ²ΠΎΡΠΎΡ ΠΎΠΊΠΎΠ»ΠΈΠ½ΠΈ), Π»ΠΎΡΠΈΡΠ°Π½Π΅ Π½Π°
ΡΠ°Π·Π»ΠΈΡΠΈΡΠΈΠΌ ΠΌΠ΅ΡΡΠΈΠΌΠ° Ρ Π₯Π‘Π. ΠΠ°ΠΈΠΌΠ΅, Π΄ΠΎΠΊΡΠΈΠ» Π³ΡΡΠΏΠ° 5-ΠΠ‘ ΡΠ΅ Π½Π°Π»Π°Π·ΠΈ Ρ Ρ
ΠΈΠ΄ΡΠΎΡΠΎΠ±Π½ΠΎΡ
ΡΠ½ΡΡΡΠ°ΡΡΠΎΡΡΠΈ Π₯Π‘Π, Π΄ΠΎΠΊ ΡΠ΅ Π·Π° 16-ΠΠ‘ Π»ΠΎΡΠΈΡΠ°Π½Π° Π±Π»ΠΈΠ·Ρ ΠΏΠΎΠ²ΡΡΠΈΠ½Π΅ Π₯Π‘Π ΠΈΠ»ΠΈ ΡΠ°ΠΊ
ΠΏΡΠΎΠ»Π°Π·ΠΈ ΠΊΡΠΎΠ· ΡΡ. Π‘ΡΠΎΠ³Π°, ΠΎΠ²Π° Π΄Π²Π° ΡΠ΅Π΄ΠΈΡΠ΅ΡΠ° Π΄Π°ΡΡ ΠΈΠ½ΡΠΎΡΠΌΠ°ΡΠΈΡΠ΅ ΡΠ° ΡΠ°Π·Π»ΠΈΡΠΈΡΠΈΡ
ΠΌΠ΅ΡΡΠ°
Π½Π° ΠΌΠΎΠ»Π΅ΠΊΡΠ»Ρ Π₯Π‘Π. Π’Π°ΠΊΠΎΡΠ΅ ΡΠ΅ ΡΠΎΡΠ΅Π½ΠΎ Π΄Π° ΠΠΠ ΡΠΏΠ΅ΠΊΡΡΠΈ ΠΎΠ±Π° ΠΠ‘ ΠΎΠ±Π΅Π»Π΅ΠΆΠΈΠ²Π°ΡΠ° Π²Π΅Π·Π°Π½ΠΈΡ
Π·Π° Π₯Π‘Π ΠΌΠΎΠ³Ρ Π΄Π° ΡΠ΅ ΡΠ°Π·Π»ΠΎΠΆΠ΅ Π½Π° ΠΊΠΎΠΌΠΏΠΎΠ½Π΅Π½ΡΠ΅ ΠΊΠΎΡΠ΅ ΠΎΠ΄Π³ΠΎΠ²Π°ΡΠ°ΡΡ ΡΠ°ΠΊΠΎ (ΠΠ), ΡΠ»Π°Π±ΠΎ Π²Π΅Π·Π°Π½ΠΎΠΌ
(Π‘Π) ΠΈ Π½Π΅Π²Π΅Π·Π°Π½ΠΎΠΌ (ΠΠ) ΠΎΠ±Π΅Π»Π΅ΠΆΠΈΠ²Π°ΡΡ. ΠΠ° 5-ΠΠ‘Π Π²Π΅Π·Π°Π½ Π·Π° ΠΠ‘Π, ΠΏΠΎΠΊΠ°Π·Π°Π½ΠΎ ΡΠ΅ Π΄Π° ΡΠ΅
ΠΠΠ ΡΠΏΠ΅ΠΊΡΡΠΈ ΡΠ°ΡΡΠΎΡΠ΅ ΠΈΠ· Π΄Π²Π΅ ΠΊΠΎΠΌΠΏΠΎΠ½Π΅Π½ΡΠ΅, ΠΠ ΠΈ Π‘Π, ΠΊΠΎΡΠ΅ ΠΏΠΎΡΠΈΡΡ ΠΎΠ΄ 5-ΠΠ‘Π Π²Π΅Π·Π°Π½ΠΎΠ³ Π·Π°
Cys-34 ΠΈ Π°ΠΌΠΈΠ½ΠΎ Π³ΡΡΠΏΠ΅, ΡΠ΅ΡΠΏΠ΅ΠΊΡΠΈΠ²Π½ΠΎ...Serum albumin is the most abundant blood plasma protein, which has been
thoroughly studied using a plethora of techniques, due to its multiple important
physiological roles. One of the methods used to monitor albumin conformational
changes and binding capacity is electron paramagnetic resonance (EPR) spin-labeling
methodology. For this purpose, albumin has been studied using various spin-labels,
among which spin-labeled fatty acids have been the most commonly employed, since
albumin is the main carrier of fatty acids. The other frequently used spin-label is 3-
maleimido-proxyl (5-MSL) which binds covalently to the free cysteine residues.
In this doctoral dissertation, two spin-labeled stearic acids containing a
paramagnetic doxyl group (the EPR-active group) attached at fifth (5-doxyl-stearic acid,
5-DS) and sixteenth (16-doxyl stearic acid, 16-DS) carbon atom of the methylene chain,
were used to study the conformational changes of human serum albumin (HSA).
Furthermore, conformational changes of bovine serum albumin (BSA) were studied by
labeling BSA with 5-MSL. The conformational changes were induced by varying
temperature and pH, addition of ethanol, binding several typical ligands (fatty acids and
drugs) and exposure to strong oxidizing agents, hydrogen peroxide and superoxide
anion radical. The additional goal of this dissertation was to reveal whether 5-MSL can
be used to track subtle conformational changes arising from ligand binding, since 5-
MSL is located in the fairly rigid environment when bound to the single free cysteine
residue in BSA, located at the position 34 in amino acid sequence (Cys-34).
The results from this study show that the doxyl groups (group which reports the
changes in its environment) of 5-DS and 16-DS, are located at different sites in HSA.
Namely, doxyl group of 5-DS is located in the hydrophobic interior, while for 16-DS it
is close to, or even protrudes the surface of HSA. Hence, these two compounds give
information from the different locations in HSA molecule. It was also observed that
EPR spectra of both DS spin-labels bound to HSA could be decomposed into
components corresponding to the strongly (SB), weakly bound (WB) and unbound (UB)
label. In case of 5-MSL bound to BSA, it was shown that the corresponding EPR
spectra consist of SB and WB component which originate from 5-MSL bound to Cys-34
and amino groups, respectively..
European contribution to the study of ROS:a summary of the findings and prospects for the future from the COST action BM1203 (EU-ROS)
Abstract
The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed