J. Biochem, 1968, Vol. 64, No. 2 147-159
© 1968 Japanese Biochemical Society
research-article |
Spectral Interactions of Protoheme with Ethyl Isocyanide and Some Other Ligands in Neutral pH Region
From the Division of Physiology, Institute for Protein Research, Osaka University Kita-ku, Osaka
1. At neutral pH values, dithionite-reduced protoheme interacted with ethyl isocyanide to form three spectrally different compounds. From the dependence of the spectrum on the ethyl isocyanide concentration, the products having Soret peaks at 414 and 428 mµ were assigned to the mono-isocyanide compound and the normal di-isocyanide compound ("n" form), respectively. The third species ("a" form) was anomalous in that it showed a Soret peak at 455 mµ.
2. Various experimental conditions affected profoundly the intensities of the peaks at 428 and 455 mµ in the presence of a saturating concentration of ethyl isocyanide. Thus, decrease in pH, increase in protoheme concentration, and increase in ionic strength all caused increased formation of the a form, accompanied by corresponding decrease in the amount of the n form. On the other hand, addition of organic solvents, such as phenol and aliphatic alcohols, and of deoxycholate shifted the equilibrium in favor of the formation of the n form. The maximum extent of the mono-isocyanide compound formation was also affected by pH; its formation was increased as the medium became more alkaline.
3. Circular dichroism measurements indicated that the a form, but not the n form, was optically asymmetric.
4. Pyridine, another lipophilic ligand, also showed anomalous behavior, similar to that of ethyl isocyanide, on interaction with protoheme in the neutral pH region. However, no such unusual interactions were observed between protoheme and hydrophilic ligands such as cyanide and imidazole.
5. From these observations, it was suggested that the a form of the protoheme compound with ethyl isocyanide or any other lipophilic ligand represented aggregated states of the protoheme compound carrying two moles of ligand attached to the ferrous iron. The formation of such aggregates seemed to be favored by the high hydrophobicity of the protoheme compound.
6. The spectral behavior of the protoheme-ethyl isocyanide system was similar in many respects to that of the ethyl isocyanide compound of the reduced form of P-450. The state of protoheme in this micro-somal hemoprotein is discussed based on these similarities.