TWEETEN, KA*; CURTIS, J; DERBY, R; SEEFELD, M; VANG, L; College of St. Catherine, St. Paul, MN; College of St. Catherine, St. Paul, MN; College of St. Catherine, St. Paul, MN; College of St. Catherine, St. Paul, MN; College of St. Catherine, St. Paul, MN: Analysis of the Extracellular Hemoglobin from Lumbriculus variegatus

The major carrier of oxygen in the California blackworm, Lumbriculus variegatus, is an extracellular, high molecular weight molecule. The objective of this project was to characterize this molecule to determine how similar it is to annelid hemoglobins that have already been studied. The hemoglobin was isolated from homogenates of worm tissue by ultracentrifugation and size exclusion chromatography. The morphology, subunit composition, and size of the hemoglobin were determined by electron microscopy and showed that the structure consisted of two hexagonal-shaped rings that were 265 � in width and comprised of six subunits each. Western blot analysis showed that antibodies against human hemoglobin bound to four of the proteins with molecular weights of 14,900, 15,300, 16,100, and 16,500 daltons, suggesting these are the oxygen-binding proteins. Five to six potential linker proteins with molecular weights ranging from 26,200 to 36,000 daltons were also observed. The glycoprotein composition of L. variegatus hemoglobin appeared to be more complex than that of earthworm hemoglobin with all four oxygen-binding proteins and the predominant linker protein being glycosylated. Hemoglobins from L. variegatus and Lumbricus terrestris were compared by two-dimensional gel electrophoresis to further evaluate the similarities and differences in the proteins comprising these assemblages.