|
|
CN-PAGE as a tool for separating pigment–protein complexes and studying their thermal stability in spruce and barley thylakoid membranes
Kurasová, Irena ; Svrčinová, K. ; Karlický, Václav ; Špunda, Vladimír
The central aim of our study was to develop a method for solubilization and native electrophoretic (colourless native polyacrylamide gel electrophoresis; CN-PAGE) separation of pigment–protein complexes (PPCs) embedded in thylakoid membranes (tBMs) isolated from spruce. Subsequently, we focused on studying the effect of temperature on the composition and PPC stability of two different species: barley and spruce. We found that the mild detergent n-dodecyl β-D-maltoside (β-DM) is suitable for PPC solubilization of spruce tBMs, but longer solubilization and a higher ratio of detergent to total chlorophyll are needed for spruce than are needed for barley. We also unified CN-PAGE protocols to optimize the separation of spruce and barley PPCs that resulted in the separation of photosystem I (PSI) and photosystem II (PSII) supercomplexes (SCs), PSI and PSII core dimers, PSII core monomers, trimeric and monomeric light-harvesting complexes of PSII, and bands with free pigments. Studying the effect of elevated temperature on PPCs using CN-PAGE revealed different thermal stability of PPCs in spruce and barley tBMs. Pronounced PPCs changes were observed at temperatures at or above 40°C. We observed partial disappearance of PSII SCs bands at 44°C in barley and at 52°C in spruce. In addition, spruce PSI SCs exhibited slightly higher thermal stability than did barley PSI SCs. The increased thermal stability of spruce tBMS in comparison to that of barley tBM was also confirmed by the circular dichroism spectra of isolated tBMs at different temperatures (Karlický et al. 2015).
|
host ::
RSS.