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Subunit dissociations in natural and recombinant hemoglobins
Author(s) -
Manning Lois R.,
Jenkins W. Terry,
Hess John R.,
Vandegriff Kim,
Winslow Robert M.,
Manning James M.
Publication year - 1996
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560050423
Subject(s) - tetramer , recombinant dna , chemistry , dissociation constant , dimer , hemoglobin , protein subunit , allosteric regulation , dissociation (chemistry) , size exclusion chromatography , biochemistry , stereochemistry , chromatography , receptor , enzyme , organic chemistry , gene
A precise and rapid procedure employing gel filtration on Superose‐12 to measure the tetramer‐dimer dissociation constants of some natural and recombinant hemoglobins in the oxy conformation is described. Natural sickle hemoglobin was chosen to verify the validity of the results by comparing the values with those reported using an independent method not based on gel filtration. Recombinant sickle hemoglobin, as well as a sickle double mutant with a substitution at the Val‐6( β ) receptor site, had approximately the same dissociation constant as natural sickle hemoglobin. Of the two recombinant hemoglobins with amino acid replacements in the α 1 β 2 subunit interface, one was found to be extensively dissociated and the other completely dissociated. In addition, the absence of an effect of the allosteric regulators DPG and IHP on the dissociation constant was demonstrated. Thus, a tetramer dissociation constant can now be determined readily and used together with other criteria for characterization of hemoglobins and their interaction with small regulatory molecules.