Quantitation of interaction between cytochrome P-450scc and adrenodoxin--analysis in the median UV-region by second derivative spectroscopy.
Interaction between the essential protein components of the bovine adrenal mitochondrial enzyme system (cytochrome P-450scc, adrenodoxin and adrenodoxin reductase) were studied in the median UV-region utilizing second derivative difference spectroscopy. Complex formation of cytochrome P-450scc with adrenodoxin induces a signal in the second derivative difference spectrum which can be attributed to tyrosine due to its minimum at 283 nm. Based on this signal cytochrome P-450scc was titrated with adrenodoxin in dependence on different effectors (reductase, phospholipid, cholesterol). The dissociation constants (Kd) of the P-450scc/adrenodoxin complexes derived therefrom revealed an increasing affinity between both components starting from titrations in buffer solution without additional components up to the completely reconstituted system. A high affinity between P-450scc and adrenodoxin corresponds to a high turnover rate of cholesterol. Dissociation constants of the P-450scc/adrenodoxin complex were also derived from spectral changes in the Soret region. But these data do not correlate with the substrate turnover.
Shkumatov
V M
VM
Institute of Bioorganic Chemistry, Academy of Sciences of the BSSR, Minsk, U.S.S.R.
Smettan
G
G
Ristau
O
O
Rein
H
H
Ruckpaul
K
K
Chaschin
V L
VL
Akhrem
A A
AA
D000325:Adrenodoxin; D000818:Animals; D002417:Cattle; D002784:Cholesterol; D002786:Cholesterol Side-Chain Cleavage Enzyme; D004347:Drug Interactions; D005287:Ferredoxin-NADP Reductase; D007700:Kinetics; D010649:Phenylalanine; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan; D014443:Tyrosine
D000325:Adrenodoxin; D014443:Tyrosine; D010649:Phenylalanine; D014364:Tryptophan; D002784:Cholesterol; D002786:Cholesterol Side-Chain Cleavage Enzyme; D005287:Ferredoxin-NADP Reductase