Plastocyanin microheterogeneity in Scenedesmus acutus MT8.
Two total plastocyanin (PC) fractions - loosely bound (lPC) and strongly bound (sPC) were extracted (84% and 16%, respectively) from the homogenate of Scenedesmus acutus MT8. Two-fold isolation-purification procedure including DE-52 chromatography separated IPC into a smaller oxidized [IPC (II)] and a larger reduced [IPC(I)] fractions, in contrast to sPC, where sPC(ll) greatly dominated over sPC(I). Analytical isoelectric focusing (IEF) separated IPC(II) into two main fractions only in the presence of 8 M urea, implying microheterogeneity. Preparative IEF in immobiline pH-gradient of 3.2-4.1 separated IPC(II) into two blue fractions - a more alkaline IPC(II) and a more acidic IPC"(II), which were probably stereoisomers. Their UV-Vis spectra exhibited rarely observed tryptophane (291.5 nm) and some differences at 270 and 287 nm. The exact molecular masses of apo-/holo-lPC (10131 Da/10194 Da) were determined by mass spectrometry. The number of -SH groups was determined from the mass difference between alkylated with 4-vinylpyridine (4-VP) and non-alkylated protein. Additionally, a simple procedure for simultaneous separation of both primary structure and stereoisomers of PC was developed.
Dimitrov
Mitko I
MI
Insitute of Biophysics, Bulgarian Academy of Sciences, Sofia, Bulgaria. mitkod@bio21.bas.bg
Donchev
Anthony A
AA
Toromanov
Georgi R
GR
Getov
Vladimir I
VI
Toncheva-Panova
Tonka G
TG
D002852:Chromatography, Ion Exchange; D007525:Isoelectric Focusing; D010970:Plastocyanin / Q000737:chemistry*; D044444:Scenedesmus / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D013056:Spectrophotometry, Ultraviolet
D010970:Plastocyanin