| Summary: | 博士 === 國立臺灣大學 === 海洋研究所 === 87 === Phycoerythrin, the major water-soluble-protein in red algae, together with phycocyanin and allophycocyanin as known as the accessory light-harvesting pigments in photosynthesis of Rhodophyta. R-phycoerythrin of Bangia atropurpurea conchocelis, purified from the gel filtration chromatography of its phosphate extract, showed the existence of various charge isomers through an anion exchange chromatographic separation. To further describe the biochemical nature of these R-PE isomers, we used a Bio-Rad MA7P (7.8×50mm) column for their purification. Systematically variables of the chromatographic parameters have been applied to observe the different resolutions obtained in separations. A linear gradient elution of NaCl under a phosphate buffer of pH 6 showed the best resolution. Changes of flow rate of the same gradient elution did not affect the resolution of these R-PE isomers. However, the resolution was improved by reducing the steepness of NaCl gradient in elution level that was needed to elute the respected protein molecule was found to raise with the steepness of the NaCl gradient applied. A linear correlation was also found between the logarithm of the relative retention and the logarithm of the inserted salt concentration that eluted the proteins under different gradient elutions in different slopes. Three lines of such correlation were obtained for the isolated PE isomers. According to the stoichiometric displacement model (logk’=const.－(Zp/Zs)log[S] in the ion exchange chromatography of high molecular weight molecules, the different slopes of the line for different isomers gave evidences to show the difference of their specific surface charges.
Filaments of various species of red algae grown under the same environmental condition gave their specific pattern of chromatograms upon the ion exchange separation of their PE molecules. The similarities and differences of the chromatograms given by their compositions of PE isomers coincided with the relationships of these red algal species in their phylogenic position. Further studies on the composition of PE isomers of Bangia atropurpurea and Porphyra angusta conchocelis showed that the species of PE isomers in these algae were not affected by the change of experimental temperature, light quality and light intensity, but the relative abundance of different PE isomers were altered by the light intensity and different light color.
There are evidences to show that the genes of some PE subunits are located in nuclear chromosome and some are in the chloroplast DNA. B. atropurpurea has two types of independently-grown plant bodies containing different chromosome numbers, one of is the conchocelis (2N) phase and the other, the thallus (1N) phase. We compared the composition of PE isomers in plants of these two phases. It was found that the major components of PE isomer in conchocelis were different from those in thallus. According to their retention time in ion exchange chromatography, PE isomers of conchocelis were denoted as PE-I, -II and -III. Both field-collected and laboratory-cultured thalli of B. atropurpurea showed the same major composition of PE-II and PE-III as in conchocelis, but with less amount of PE-I. Thalli of B. atropurpurea also contained significant amount of PE-IV, that is the same component also found in conchocelis but in relatively trace amount. It was concluded that there were four components of PE isomers in B. atropurpurea. Among them, both PE-II and PE-III were common in both phases. Meanwhile PE-IV in conchocelis and PE-I in thallus were much less than those in the other phase.
PE charge isomers were further isolated either from thallus or conchocelis by column gel filtration and the following ion-exchange column separation. Protein subunits of these PE isomers were analyzed by methods, such as SDS PAGE, reverse phase HPLC, and MALDI-TOF analysis. N-terminal amino acid sequence and amino acid composition of the protein subunits were also studies individually. The result showed that PE-II or PE-III molecules in either conchocelis or thallus were identical in each other due to the same compositions of α, β andγsubunits. All the αsubunits in different PE isomers were found identical, so were the β subunits. However γ subunits were found different from each other in different isomers. They were separated by HPLC and their molecular weights determined by MALDI-TOF as~27kDa in PE-I, ~30kDa in PE-II, ~27kDa in PE-III, and ~30kDa PE-IV. This result offered further evidences to the statement given in the past literatures that PE charge isomers were different in their γsubunit.
|
|---|
