Molecular symmetry and arrangement of subunits in extracellular hemoglobin from Caenestheria inopinata.
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.
Tsfadia
Y
Y
Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Israel.
Shaked
I
I
Daniel
E
E
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
D003432:Cross-Linking Reagents; D006454:Hemoglobins; D046911:Macromolecular Substances; D005976:Glutaral