D. magna Hemoglobin (Hb) was discovered with the use of a spectroscope by Lankester more than one hundred years ago [1]. This Hb is noted for its ability to perform the extraordinarily large variation in concentration. The body color of Daphnia, therefore, changes from bright red to pale [2,3]. The molecular weight of D. pulex Hb appears to be six times that of mammalian Hb by the sedimentation coefficient, and is composed of subunits containing two heme groups per 35,000 mol. wt polypeptide chain [4,5].

The large variation in the Hb concentration has attracted much attention in the searching for a key to solve its functional significance. The physiological function of the Hb in extremely small invertebrate animals has been estimated from the depression of the activity of animals whose Hb had been inactivated by exposure to CO. However, in Daphnia, the activity in red animals can be comparatively examined by using Hb-poor (pale) animals. In general, the Hb-rich (red) animals have been mainly used to investigate the physiological properties of this Hb, because large amounts of Hb can be obtained from red animals. On the other hand, only trace amounts of Hb existed in pale animals, and only a few studies have been done on this Hb. We have been studying this Hb, as well as red animals, to better understand the physiological properties of D. magna Hb.

As described above, D. magna contains different kinds of Hb molecules of different O₂ affinities, where as the Hb was proposed to be composed of 16 identical polypeptide chains each carrying two heme-binding domains [6]. These suggest that D. magna contains several very similar subunit chains. To determine whether D. magna contains heterogeneous Hb chains or not and to elucidate the properties of each subunit chains, we recently separated and chemically determined their N-terminal amino acid sequences. We also isolated and analyzed genes encoding these subunit chains [7,8]. The results obtained from such analyses are reviewed in this paper.