The molecular size of Myxicola infundibulum chlorocruorin and its subunits

The molecular shape and size of the extracellular chlorocruorin of Myxicola infundibulum was determined using scanning transmission electron microscopy and its dissociation in the presence of sodium dodecyl sulfate (SDS) was investigated using polyacrylamide gel electrophoresis. The shape of the chlorocruorin is that of a two-tiered hexagon with a vertex-to-vertex diameter of 29.0-29.5 nm and a height of 19.0-19.7 nm: it appears to be smaller by 5-10% relative to several annelid extracellular hemoglobins examined by scanning transmission electron microscopy. The quaternary structure of the chlorocruorin appears to be sensitive to Ca(II) concentration; dissociation fragments of the whole molecule were observed, consisting of octamers an dimers of one-twelfth subunits. The unreduced chlorocruorin dissociated into two subunits with estimated molecular masses of 23 000 (1) and 60 000 (2); the reduced chlorocruorin dissociated into subunits with estimated molecular masses of 13 000 (I), 14 000 (II) and 30 000 (III). SDS-polyacrylamide gel electrophoresis of reduced subunits 1 and 2 showed that subunit 1 corresponded to subunit III and that subunit 2 dissociated to subunits I and II. Densitometry of the polyacrylamide gels indicates that 85-90% of the Myxicola chlorocruorin consists of disulfide-bonded tetramers of polypeptide chains of about 15 000. Such a pattern of subunit aggregation has not been observed previously in annelid extracellular hemoglobins and chlorocruorins.