The effects of temperature on hemoglobin in Capitella teleta

Abstract

Annelid hemoglobin represents an impressive diversity in structure and function that has been highly in uential in developing the current understanding of how this large class of protein works. Unique to the annelids are giant extracellular pigments of two main varieties: the 3600 kDa erythrocruorins and the 400 kDa hexagonal- bilayer hemoglobins. The marine polychaete Capitella teleta has served as a model organism for numerous biological studies, yet very little work has been dedicated to understanding the types and properties of respiratory pigments utilized by this worm. As a temperate opportunist, this species is ideal for studying the effects laboratory induced changes in temperature and oxygen saturation on the structure and functionality of hemoglobin. The goal of this work was to determine what type of extracellular hemoglobin is normally present in C. teleta, and to determine the effects of warm versus cold acclimation on the concentration and subunit composition of this hemoprotein. It was observed that cold-acclimation induces two major changes in the morphology of C. teleta: an increase in body size and a change in red pigmentation. Using the pyridine hemochrome assay in conjunction with a heme activity assay, it was determined that the difference in pigmentation was due to a combination of effects. The 21°C worms had a greater concentration of hemoglobin than the 4°C worms, but less heme b. It has been suggested that this was due to a difference in the functional status of the heme b, as well as a minor difference in hemoglobin stability. Using size exclusion chromatography and native gel electrophoresis, the major extracellular hemoglobin in both warm- and cold-acclimated C. teleta was determined to be a 333 kDa hexagonal-bilayer hemoglobin composed of three major and up to three minor polypeptide chains that covalently bind to form disulfide-linked dimers and trimers. A major difference in the expression of these subunits between the experimental cultures was not detected. This study represents the first attempt at identifying and characterizing extracellular hemoglobins from C. teleta. It has established a strong foundation for future work on the purifcation and analysis of this important respiratory protein in a model annelid.