Development of methods for separation and characterization of proteins from gorgonian sources

Research into the chemical constituents of marine organisms is a relatively new and growing field, given that much of the marine environment has only in the last half century become accessible. Despite the strides that have been made in ocean exploration, the marine biome has still been only barely explored. Much of the impetus for the isolation and identification of chemical entities from marine organisms has been related to their potential as medicines. For a number of reasons, including synthetic efficiency, this has meant that many marine compounds isolated and studied have been small molecules. In recent years, large strides have been made in techniques for the isolation and identification of biological macromolecules, especially proteins. The understanding of these molecules, and their relationship to the biochemical processes of the marine organisms from which they have been isolated, is important not only because of the potential for this information to help in the synthesis of medicines, but because it may help in the recognition of processes that affect the very viability of marine organisms increasingly exposed to anthropogenic threats to their environment. This work consists of four related studies involving the development of methods for the separation and identification of proteins from a number of gorgonian species. Chromatographic and gel based methods were used to isolate an elisabethatriene synthase (ELS) which shows promise as a biotechnological tool for the production of pseudopterosins. A number of gorgonians were screened for the presence of proteins that might have antimicrobial properties, and several organisms were identified that might be of interest in this context. Two-dimensional gel electrophoresis methods were then developed to allow the construction of gel maps for the azooxanthellate gorgonian Leptogorgia minimata and the xoozanthellae gorgonian Pseudopterogorgia elisabethae, the separated proteins were digested and analyzed by LC/MSMS, and the information generated was used to examine the proteome of the organism for functional and phylogenetic relationships. Finally, the gorgonian Eunicea fusca was exposed to several environmental stressors in the laboratory, and the two-dimensional proteomic methods developed were used to examine the effect of the stressors on the organism.