Energetics of oxygenation-linked subunit interactions in human hemoglobin.
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Binding Sites↗
Biomedical subjects
F C Mills
Publications and source records attributed to F C Mills.
The linkage between oxygenation and subunit dissociation in human hemoglobin. Consequences for the analysis of oxygenation curves.
For human hemoglobin, a pronounced dependence of oxygenation curves upon protein concentration can be demonstrated experimentally in the range between 10(-4) and 2 X 10(-6) M heme. The effects of such protein concentration dependence upon analysis of saturation curves have been explored using a model-independent linkage analysis which incorporates the dissociation of tetramers to dimers. We have carried out stimulations of oxygenation curves representing a variety of energy distributions designed to cover a wide range of values which are relevant to known hemoglobin systems and experimental conditions. The resulting simulated oxygenation curves were analyzed by least-squares minimization procedures in terms of the tetramer binding isotherm to yield the four apparent Adair constants. These derived constants were compared with the originally assumed values used in the simulation in order to assess the extent to which their values may be altered by the presence of dimer. For each energy distribution the analysis has been carried out over a wide range of protein concentration. We have found that the presence of even small amounts of dimer that are necessarily present at the low protein concentrations commonly employed may have a devastating effect upon the reliability of Adair constant determinations. In addition to these simulated cases, we have analyzed two sets of highly precise experimental data from the literature in order to assess the degree to which constants obtained may have been influenced by the presence of dimer.
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Selective unfolding of erythroid chromatin in the region of the active beta-globin gene.
Globin gene expression, which occurs exclusively in the erythroid cell lineage, is controlled at the level of transcription. It is thus of some considerable interest to compare the chromatin structure of this gene with that of inactive genes in erythroid cell nuclei and to compare the chromatin structure of the globin gene in its active and inactive states in nuclei of different cell types. Other workers have observed that globin genes in erythroid cell nuclei exhibit the enhanced overall sensitivity to nucleases and the hypersensitive site in the 5'-flanking sequence typical of many active genes. The nature of the structural changes giving rise to nuclease sensitivity are however obscure. We have investigated the local higher order structure of chromatin in the region of unique genes in chicken by sucrose gradient sedimentation of chromatin restriction fragments. We find that ovalbumin and alpha 2-collagen gene fragments in erythrocyte chromatin and an adult beta-globin gene fragment in spleen chromatin sediment with bulk chromatin fragments of the same DNA size, whereas the beta-globin gene fragment in erythrocyte chromatin sediments more slowly than bulk fragments of equivalent size. The simplest interpretation of the results is that the solenoid structure in the region of the globin gene is selectively and permanently unfolded on gene activation.
Animals↗
DNase I hypersensitive sites in the chromatin of human mu immunoglobulin heavy-chain genes.
An immunoglobulin polypeptide chain is encoded by multiple gene segments that lie far apart in germ-line DNA and must be brought together to allow expression of an immunoglobulin gene active in B lymphocytes. For the immunoglobulin heavy chain genes, one of many variable (V) region genes becomes joined to one of several diversity (D) segments which are fused to one of several joining (J) segments lying 5' of the constant region (C) genes. Here we show that the rearranged mu genes of an IgM-producing human B-lymphocyte cell line exhibit pancreatic deoxyribonuclease (DNase I) hypersensitive sites in the JH-C mu intron that are absent in naked DNA or the chromatin of other differentiated cell types. DNA sequence analysis reveals that the major hypersensitive site maps to a conserved region of the JH-C mu intron recently shown to function as a tissue-specific enhancer of heavy-chain gene expression. A similar association of an enhancer-like element with a DNase I hypersensitive site has been reported for the mouse immunoglobulin light-chain J kappa-C kappa intron. These results implicate disruption of local chromatin structure in the mechanism of immunoglobulin enhancer function.
B-Lymphocytes↗