Portland State University. Department of Chemistry
John H. Golbeck
Date of Award
Master of Science (M.S.) in Chemistry
1 online resource (48 p.)
Iron proteins -- Analysis, Iron -- Metabolism, Anabaena, Trichodesmium, High performance liquid chromatography
Iron is an essential nutrient for growth of photosynthetic microorganisms such as cyanobacteria and algae. Iron is required for proteins involved in the important processes of carbon and nitrogen assimilation. Low concentrations of iron in cultures or natural waters can lead to iron limitation which affects many aspects of algal metabolism. In natural waters, iron limitation can have effects on the patterns and rates of primary productivity.
The cellular content of certain proteins can be affected by media iron concentrations. Methods have been used that assay components of the cell as an indirect measure of iron nutritional status. For example, spectroscopy can be performed to determine the cellular concentration of iron-containing proteins involved in photosynthesis. Organisms grown in media that imitate natural conditions, or organisms collected from their natural habitat are usually dilute. Methods that assay iron nutritional status such as spectroscopy and column chromatography require large sample sizes which are difficult to obtain from natural samples. In addition, methods that utilize techniques such as immunology or radioactive labelling are complex and time-consuming. These considerations led to the necessity of developing a technique that would be simple, rapid and effective on dilute samples. The method developed here utilized fast protein liquid chromatography (FPLC), which fulfilled these requirements. A complete analysis could be done within two to three hours with minimal sample treatment. The FPLC was simple to operate and was effective on a sample containing less than 100 μg of protein.
Some photosynthetic organisms, when iron-depleted, can produce the flavin-containing protein flavodoxin (Flv). This protein substitutes for the iron-containing protein ferredoxin (Fd) in Fd-dependent reactions such as the light-induced reduction of NADP. The FPLC technique identified and quantified, in relative terms, Fd and Flv in the cell. Optical spectroscopy was used to verify FPLC retention time assignments. The results illustrated how the FPLC could be used to observe the changes in relative Fd and Flv content as a function of media iron concentration in cultures of the cyanobacterium Anabaena grown in the laboratory. It was found that Fd content decreased and Flv content increased with decreasing media iron concentration. In addition, samples of the cyanobacterium Trichodesmium collected from the ocean near Barbados were analyzed using FPLC to assay relative Fd and Flv content. By analogy with Anabaena, Fd and Flv retention times were identified. Using this technique conclusions could be drawn regarding the changing iron nutritional status of Trichodesmium in its natural habitat .
Jones, Karen Lorraine, "Analysis of ferredoxin and flavodoxin in Anabaena and Trichodesmium using fast protein liquid chromatography" (1988). Dissertations and Theses. Paper 3812.