Naslov
Jetreni citokrom P-450. Istraživanje mikrosomalnog, solubiliziranog i djelomično rekonstituiranog sistema protonskom magnetskom relaksacijom
(Hepatic Cytochrome P-450. A Proton Magnetic Relaxation Study of Microsomal, Solubilized and Partilally Reconstituted Enzyme System)

Autori
Ruckpaul, K. ; Maričić, Siniša ; Jänig, G.-R. ; Benko, Bojan ; Vuk-Pavlović, Stanimir ; Rein, H.

Izvornik
Croatica chemica acta (0011-1643) 48 (1976), 1; 69-86

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
Cytochrom P-450; Microsom; NADPH-dependent reductase; Phenobarbital; Proton magnetic relaxation

Sažetak
The longitudinal proton magnetic relaxation times, T1, were measured from - 5 to 40 °C for microsomal, solubilized and reconstituted cytochrome P-450 obtained from phenobarbital-induced rat livers. The paramagnetic contribution to the rates was derived by subtraction of the rates measured on dithionite-CO-reduced samples. The same values were obtained for microsomal P-450 on reduction with NADPH. PMR titration by KCN yielded a dissociation constant of about 30 mM. This is three order of magnitude larger than for metmyoglobin. It is concluded that the measured PMR rates are most likely due to the P-450 (and P-420) haem-iron while the 30% non-haem iron found in both the microsomal and solubilized P-450 is ineffective for the PMR rates. These rates increase several times on isotopic dilution (D2O for H2O) with the microsomes and diminish for the solubilized samples. Microsomes show 17% residual, encaged, H2O. Most of their paramagnetic PMR rate is due to the paramagnetic iron located on the outside of microsomes. This is demonstrated by measurements with deuterated samples to which 19% H2O had been added. Hence, the solubilized P-450 is homogeneous regarding PMR, but the microsomes are not. The paramagnetic molar relaxation rates are very high (103 to 2x103 s-1M-1) for the predominately low-spin iron in these samples. If the only relaxation mechanism is the magnetic dipole-dipole interactionof electron and nuclear spin, then the correlation time ought to be of the order of 10-10 s. Lack of the knowledge of the actual relaxation mechanism prevents quantitative evaluation of the results. The comparison of relaxation rates measured by water protons, or (in deuterated solution) by aliphatic protons of deuterated glycerol indicates a very accesible haem-iron. The Arrhenius plots show clearly two temperature regions with the kink between 13 and 20 °C, both for the original and the CO-reduced samples. The molar paramagnetic rates derived as differences from the former pairs of data (original v. CO) have a PMR discontinuity around 18 °C. The PMR rates for the microsomes are twice as large as those for the solubilized P-450. When the latter is combined with the solubilized NADPH-dependent reductase, the PMR rates almost coincide with those of the original microsomes. It thus appears that the missing phospholipid in the reconstituted sample is not determining the conformation around the haem-iron: Rather, interaction of the first-neighbour protein molecules separated possibly byone lipid molecule bring about a functional (?) conformation sensitive to structural changes in the membrane around the "transition" temperature.

Izvorni jezik
Engleski

Znanstvena područja
Fizika, Kemija, Biologija

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