Modification of trypsin-solubilized cytochrome b5, apocytochrome b5, and liposome-bound cytochrome b5 by diethylpyrocarbonate.
The interactions of diethylpyrocarbonate (DEP) with the various forms of cytochrome b5 were studied to gain a better understanding of the factors that influence the extent of modification of the axial histidines of cytochrome b5. Very low concentrations of DEP were able to decrease the heme binding capacity of apocytochrome b5. 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 b5. Trypsin-solubilized bovine cytochrome b5 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 b5 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 b5 are monomeric in their respective environments while detergent-solubilized cytochrome b5 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 b5. Examination of the cytochrome b5 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.
Konopka
K
K
Department of Anesthesia, Veterans Administration Medical Center, San Francisco, California 94121.
Waskell
L
L
D000818:Animals; D001059:Apoproteins / Q000378:metabolism*; D003573:Cytochrome b Group / Q000302:isolation & purification / Q000378:metabolism*; D045303:Cytochromes b; D015786:Cytochromes b5; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D004134:Dimyristoylphosphatidylcholine; D005561:Formates / Q000494:pharmacology*; D006418:Heme / Q000378:metabolism; D007700:Kinetics; D008081:Liposomes; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D008958:Models, Molecular; D010713:Phosphatidylcholines; D011487:Protein Conformation; D011817:Rabbits; D012995:Solubility; D014357:Trypsin
D001059:Apoproteins; D003573:Cytochrome b Group; D005561:Formates; D008081:Liposomes; D010713:Phosphatidylcholines; D006418:Heme; D045303:Cytochromes b; D015786:Cytochromes b5; D014357:Trypsin; D004047:Diethyl Pyrocarbonate; D004134:Dimyristoylphosphatidylcholine