A quantitative understanding of the stability and expression of hemoglobins and myoglobins must take into account all of the factors which govern heme affinity and the folding of the globin portions of the molecules. In order to evaluate these factors, a new assay for measuring the rate of hemin loss was developed, and fluorescence techniques were adapted to monitor apoglobin unfolding.
Native apomyoglobin has roughly the same size and shape as the holoprotein, including a hydrophobic crevice for heme binding, but its overall structure is less helical and more solvated. Addition of small amounts of denaturant to apomyoglobin produces an intermediate which lacks a well-defined heme pocket but still contains a hydrophobic core made up of three $\alpha$ helices. Further addition of denaturants produces a completely unfolded state. This three state unfolding reaction has traditionally been monitored by changes in circular dichroism but is more easily visualized by changes in tryptophan fluorescence. The heme pocket in native apomyoglobin is stabilized by the presence of hydrophobic amino acids which exclude solvent from the binding site. Introduction of polar amino acids into this region greatly destabilizes native apomyoglobin causing formation of the molten globular intermediate due to solvation of the heme pocket.
The binding of hemin to apomyoglobin is very favorable and results in the release of $\sim{-}$77 kJ/mol of Gibbs' free energy under physiological conditions. The association rate constant is $\sim1 \times 10\sp8$ M$\sp{{-}1}$s$\sp{{-}1}$ and little affected by the structure of the apoglobin or reaction conditions. As a result, the equilibrium constant for hemin binding is determined primarily by its dissociation rate constant which is markedly dependent on apoprotein structure and ranges from $$100 h$\sp{{-}1}$ at pH 7, 37$\sp\circ$C. The principle factors determining hemin affinity are: (1) non-specific hydrophobic interactions, which account for $\sim{-}$36 kJ/mol of the free energy change produced by the formation of holomyoglobin from hemin and apoglobin; (2) specific van der Waals and electrostatic interactions between the porphyrin and heme-pocket amino acid residues, which account for another $-$18 kJ/mol; and (3) the covalent bond between His$\sp{93}$(F8) and the iron atom, which accounts for the remaining $\sim{-}$23 kJ/mol.
| Identifer | oai:union.ndltd.org:RICE/oai:scholarship.rice.edu:1911/16910 |
| Date | January 1996 |
| Creators | Hargrove, Mark Scott |
| Source Sets | Rice University |
| Language | English |
| Detected Language | English |
| Type | Thesis, Text |
| Format | application/pdf |
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