Molecular Pharmacology, Vol 13, 15-30, Copyright © 1977 by the American Society for Pharmacology and Experimental Therapeutics

Nuclear Magnetic Resonance Studies of Substrate-Hemoprotein Complexes in Solution

I. The Interaction of Xylidine with Myoglobin, Hemoglobin, and Cytochrome P450

¹ Department of Pharmacology, Northwestern University Medical and Dental Schools, Chicago, Illinois 60611

¹H nuclear magnetic resonance longitudinal relaxation time (T₁) measurements were used to study the interaction of xylidine (2,6-dimethylaniline) with the hemoproteins myoglobin, hemoglobin, and solubilized rat liver microsomal cytochrome P450. Upon addition of various amounts of ferrimyoglobin, ferrihemoglobin, or ferricytochrome P450 to solutions of xylidine, the T₁ values for the methyl and phenyl protons of the xylidine molecule decreased markedly. The observed changes showed that xylidine was much more sensitive to addition of cytochrome P450 than to myoglobin or hemoglobin. The relative effects upon the specific moieties of the substrate also differed; whereas myoglobin produced essentially the same effect upon the relaxation rates (T_11p^-1) of the phenyl and methyl protons of xylidine, hemoglobin and cytochrome P450 produced differential changes in the (T_11p^-1) values, phenyl > methyl. These results were shown to reflect specific xylidine-ferrihemoprotein complexes. A nonsubstrate, noninteracting internal reference (tetramethylammonium phosphate) was added to each sample as a control both for experimental variation and for effects due to changes in solution viscosity; in no case was a substantial change in the T₁ value of the reference observed. Variable-temperature studies confirmed that the residence times for the xylidineferrimyoglobin and xylidine-cytochrome P450 interactions were in the region of fast exchange with respect to the NMR time scale (i.e., _M [unknown] T_11M). Formation of the cyano derivative of ferrimyoglobin or ferrihemoglobin changes the paramagnetic spin state from S = 5/2 to S = 1/2, and conversion to the carbonmonoxyferrous derivative results in a diamagnetic species, S = 0. When the ferrihemoproteins were so converted in situ in the presence of xylidine, the T₁ values for the xylidine moieties increased. Since the carbonmonoxyferrous derivatives of all three hemoproteins are diamagnetic, the latter type of experiment was performed in all cases to give the control values (1/T₁⁰), which allowed calculation of the paramagnetic relaxation rate values, 1/T_11p. The ¹H T₁ results, in conjunction with the value obtained for the dissociation constant of the xylidine-cytochrome P450 complex (K_D) = 4.1 x 10^-4 M) and the estimated correlation time for the complex, allowed calculation of distances between the heme iron atom and specific portions of the substrate molecule.

Note:
ACKNOWLEDGMENTS The authors thank Drs. T. J. Swift and A. F. Boyne for critical review of the manuscript.

This article has been cited by other articles:

				P. Hlavica, I. Golly, M. Lehnerer, and J. Schulze Primary aromatic amines: their N-oxidative bioactivation Human and Experimental Toxicology, August 1, 1997; 16(8): 441 - 448. [Abstract] [PDF]