NMR method for superoxide dismutase assay in brain and liver homogenates.

ABSTRACT

A method for copper- and manganese-containing superoxide dismutase (Cu- and MnSOD) assay in tissue homogenates such as liver and brain, based on the measurement of the longitudinal nuclear relaxation time (T1) of F-, has been developed as a preliminary approach to in vivo measurement of these enzymes. The relaxation rate of F-, which increases linearly with the SOD concentration, also depends on the oxidation state of the metal ion present in the active site of the enzyme. The relaxivity values of the oxidized, reduced and turnovering CuSOD were found to be 9.6 x 10(6), much less than 1 x 10(2) and 5.2 x 10(6) M-1 s-1, respectively, while for MnSOD the corresponding values were 2.9 x 10(6), 4.2 x 10(6) and 3.6 x 10(6) M-1 s-1, respectively. These high relaxivity values allow the detection of SODs in brain and liver homogenates where, under aerobic conditions, these enzymes appear in the steady-state. The contribution of the two types of SOD to the F- relaxation rate in the homogenates was measured by addition of either diethyldithiocarbamate or cyanide, both of which selectively inhibit the CuSOD. The comparison between NMR and activity data confirmed the possibility of carrying out accurate and precise measurements of SODs in homogenates by NMR.