Discrimination between Conformational States of Mitochondrial Cytochrome P-450scc by Selective Modification with Pyridoxal 5-Phosphate

A. G. Lapko^1* and K. Ruckpaul²

¹Sakharov International Institute of Radioecology, ul. Dolgobrodskaya 23, Minsk, 220009 Belarus; fax: (0172) 30-6897

²Institute of Biochemistry, Humboldt University, Charite, Max-Delbrueck-Centrum of Molecular Medicine, Robert-Roessle-Str. 10, 13122 Berlin-Buch, Germany; fax: (49-30) 940-63-794

^* To whom correspondence should be addressed.

Received September 25, 1997; Revision received November 22, 1997

---

Electrophoretically homogeneous cytochrome P-450scc preparation isolated by the standard method from adrenal cortex mitochondria comprises two protein forms differing in the accessibility of their amino groups to specific chemical modification with pyridoxal 5-phosphate. The protein form whose lysine amino groups are accessible to the modifier constitutes about 60-70% of the preparation. Being covalently bound to pyridoxal 5-phosphate, this protein form loses enzymatic activity and affinity for adrenodoxin. This protein form can be separated by affinity chromatography on adrenodoxin-Sepharose. The cytochrome P-450scc form whose amino groups are not accessible to the modifier is retained on the affinity matrix, and after elution from adrenodoxin-Sepharose has the absorption spectrum typical of the high-spin protein with a spectral homogeneity index A₃₉₂/A₂₇₈ = 1.0. The enzymatic activity of the hemoprotein form whose lysine amino groups are inaccessible to the modification is identical to that of the initial unmodified protein.

---

KEY WORDS: cytochrome P-450, terminal monooxygenase, chemical modification, affinity chromatography, dynamic equilibrium of conformers

---