Analytical and Semipreparative HPLC Analysis and Isolation of Hemocyanin from the American LobsterHomarus americanus

Abstract

Hemocyanin, which has numerous research and commercial biotechnology applications, is currently available from just two sources: the keyhole limpet and the horseshoe crab. Obtaining and purifying hemocyanin from these sources is a complex but necessary process because this compound cannot be synthesized in the laboratory. Lobster hemolymph, an abundant by-product of the lobster meat processing industry, could be developed as a new source of hemocyanin. A reliable and inexpensive method of extracting hemocyanin from lobster hemolymph would increase hemocyanin supplies through development of a value-added product from processing waste. Currently, ultracentrifugation is the most common commercial method of purifying hemocyanin. This technique, which requires several days of processing time, is not a reliable method of separating hemocyanin oligomers. We report new separation and purification techniques based on size-exclusion high-performance liquid chromatography, and methods of determining the quantitative and qualitative properties of hemocyanin derived from lobster hemolymph. In addition, an analytical and semipreparative method was developed to separate and purify hexamers and dodecamers from American lobster hemolymph. The shelf-life of hemocyanin hexamers and dodecamers extracted using the newly developed methods was determined to be more than 30 days with no observed degradation of the hemocyanin oligomers.