The principal objectives are twofold: to provide experimental data to test a proposed model of the quaternary structure of Lumbricus terrestris hemoglobin in which two of the four subunits (D1 and D2) are assumed to form a scaffolding or serve as linkers for complexes made up of the remaining two subunits (M and T) and to relate the cooperativity of oxygen binding of the earthworm hemoglobin to the various levels of its quaternary structure.
The specific aims i n the first part are to characterize using electrophoresis and gel filtration the various reassociated hexagonal bilayer structures obtained from several different fragments obtained by dissociation at extremes of pH or at neutral pH in the presence of various dissociating agents as well as from combinations of the isolated subunits M, D1, D2 and T, to use mass mapping by scanning transmission electron microscopy to determine the molecular size and dimensions of the different reassociated hexagonal bilayer structures, to use monoclonal antibodies specific to the individual subunits of Lumbricus hemoglobin to locate their positions in the hexagonal bilayer structure by electron microscopy and to examine using dissociation and reassociation whether the quaternary structures of other annelid hemoglobins such as those of Arenicola, Amphitrite, Tubifex and Macrobdella and of chlorocruorins such as those of Myxicola and Eudistylia, fit the model proposed for Lumbricus hemoglobin. In the second part the specific aims are to determine the oxygen binding curves of the four subunits of Lumbricus hemoglobin, of the two fragments, consisting of subunits M and D1+D2+T and of putative one-twelfth fragments obtained by dissociation at neutral pH and of the reassociated hexagonal bilayer structures obtained from combinations of subunits and fragments.
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