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Title: Plastocyanin microheterogeneity in Scenedesmus acutus MT8
Authors: Dimitrov, Mitko I
Donchev, Anthony A
Toromanov, Georgi R
Getov, Vladimir I
Toncheva-Panova, Tonka G
Keywords: Plastocyanin
Isoelectric focusing
Issue Date: Oct-2008
Publisher: CSIR
Abstract: 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 lPC into a smaller oxidized [lPC (II)] and a larger reduced [lPC(I)] fractions, in contrast to sPC, where sPC(II) greatly dominated over sPC(I). Analytical isoelectric focusing (IEF) separated lPC(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 lPC(II) into two blue fractions – a more alkaline lPC'(II) and a more acidic lPC''(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 nonalkylated protein. Additionally, a simple procedure for simultaneous separation of both primary structure and stereoisomers of PC was developed.
Page(s): 310-316
ISSN: 0301-1208
Source:IJBB Vol.45(5) [October 2008]

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