title: Molecular symmetry and arrangement of subunits in extracellular hemoglobin from Caenestheria inopinata.
abstract: The subunit structure of extracellular hemoglobin from the clam shrimp Caenestheria inopinata was studied using the method of cross-linking by bifunctional reagents followed by SDS/PAGE. Two phases were distinguished in the cross-linking by glutardialdehyde: a fast phase characterized by the appearance of two bands in electrophoresis corresponding to single polypeptide chains and cross-linked chain pairs, and a slow phase where five bands corresponding to even numbers, 2-10, of cross-linked polypeptide chains are observed. Theoretical curves for the distribution of protein among the various cross-linked species were calculated assuming allowed arrangements of ten identical subunits. Equally good descriptions of the cross-linking were provided by two models with dihedral symmetry: a ring arrangement with two types of alternating interactions and a two-layered eclipsed arrangement with one type of interaction between subunits from different layers and another between subunits within the same layer. A way out of the ambiguity was found by carrying out the fast phase of the cross-linking reaction with dimethyl-3,3'-dithiobispropionimidate.2HCl, a bifunctional reagent containing an S-S bond that can be cleaved by 2-mercaptoethanol, following up with glutardialdehyde in the slow phase. The observation in the presence of 2-mercaptoethanol of electrophoretic bands corresponding to trimeric and higher cross-linked polypeptide chain species rules out the alternating ring and confirms the two-layered eclipsed model. The arrangement of subunits found in this work from consecutive cross-linking can account satisfactorily for molecular profiles previously obtained from electron microscopy of Caenestheria hemoglobin.
authors.affiliation: Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Israel.
authors.forename: Y
authors.initials: Y
authors.lastname: Tsfadia
authors.forename: I
authors.initials: I
authors.lastname: Shaked
authors.forename: E
authors.initials: E
authors.lastname: Daniel
mesh_terms: D000818:Animals; D003432:Cross-Linking Reagents; D003445:Crustacea / Q000502:physiology*; D005976:Glutaral / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry / Q000648:ultrastructure*; D046911:Macromolecular Substances; D015394:Molecular Structure; D008970:Molecular Weight
chemical_list: D003432:Cross-Linking Reagents; D006454:Hemoglobins; D046911:Macromolecular Substances; D005976:Glutaral