Characterization of L-DOPA Containing Organic Matrix Proteins in the Eastern Oyster Crassostrea virginica
Anna Kathleen Benton
The eastern oyster, Crassostrea virginica, forms shell through the process of layer by layer construction. There are two components of shell: the hard layer made of calcium carbonate and the soft layer made of organic matrix. This project focuses on a specific class of proteins found in the organic layer that contain a unique amino acid, L-3,4-dihydroxyphenylalanine (L-DOPA). This class of proteins is of interest because of the crosslinking capabilities of L-DOPA. L-DOPA can adhesively bind to calcium carbonate and cohesively bind to other amino acids as confirmed by the study of L-DOPA-containing Mytilus edulis foot proteins. L-DOPA can be used as a biomarker for these proteins and can be assayed or tracked using the ARNOW assay, a catechol stain, on adductor muscle tissue. Market quality oysters were shucked, and tissue samples were collected from the adductor muscle. Proteins were extracted by homogenizing the tissue, soaking the tissue in 0.7% perchloric acid solution, precipitating proteins with cold acetone, and resuspension in 5% acetic acid. Once the initial extraction was complete, the protein solution was concentrated using ultrafiltration under inert gas to prepare the solution to be run on a high-pressure liquid chromatography (HPLC). After HPLC separation, proteins are further characterized using acid-urea polyacrylamide gel electrophoresis. The AAA-Direct Amino Acid Analysis System was then used to determine the percentage concentrations of the amino acids present in the purified proteins. Purified protein with high L-DOPA content will be sequenced using a commercial vendor. Once the sequence is obtained, proteins will be modeled using ChemDraw, Chem3D, and Gaussian-9 software. Modeling using published sequences of putative shell formation proteins has revealed the occurrence of specific folding domains that may play a role in adhesive and cohesive binding of those proteins. We anticipate finding similar tertiary structure in proteins isolated and characterized from Crassostrea virginica.
Douglas C. Hansen, Karolyn M. Hansen
Primary Advisor's Department
Stander Symposium project
"Characterization of L-DOPA Containing Organic Matrix Proteins in the Eastern Oyster Crassostrea virginica" (2019). Stander Symposium Projects. 1464.