Abstract
The interactions of diethylpyrocarbonate (DEP) with the various forms of cytochrome b 5 were studied to gain a better understanding of the factors that influence the extent of modification of the axial histidines of cytochrome b 5. Very low concentrations of DEP were able to decrease the heme binding capacity of apocytochrome b 5. Moreover, it was shown that two additional histidines, presumed to be the axial ligands (His 39 and 63), were modified in the apo but not the holo form of a given preparation of cytochrome b 5. Trypsin-solubilized bovine cytochrome b 5 was resistant to the effects of DEP. A 200-fold molar excess of DEP displaced only 15% of the heme in the trypsin-solubilized protein in contrast to an 84% displacement of the heme in the detergent-solubilized protein. However, detergent-solubilized cytochrome b 5 which had been incorporated into phospholipid vesicles exhibited the same reactivity with DEP as did the trypsin-solubilized protein. This is attributed to the fact that the two resistant preparations of cytochrome b 5 are monomeric in their respective environments while detergent-solubilized cytochrome b 5 is known to exist as an octamer in aqueous solutions. Our studies suggest that dissociation of the octamer to the monomer results in a conformational change that decreases the reactivity of the axial ligands of the hydrophilic heme-containing domain of cytochrome b 5. Examination of the cytochrome b 5 molecule by computer graphics indicates that a tunnel leads from the surface of the molecule to axial histidine 63 and that axial histidine 39 is buried.
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