Scarpa M, Viglino P, Momo F, Bracco F, Battistin L, Rigo A
Department of Biological Chemistry, University of Padua, Italy.
J Biochem Biophys Methods. 1991 Feb-Mar;22(2):135-44. doi: 10.1016/0165-022x(91)90026-s.
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.
基于对F -纵向核弛豫时间（T1）的测量，已开发出一种用于测定肝脏和大脑等组织匀浆中含铜和锰的超氧化物歧化酶（Cu - 和MnSOD）的方法，作为体内测量这些酶的初步方法。F -的弛豫速率随SOD浓度呈线性增加，它还取决于酶活性位点中存在的金属离子的氧化态。发现氧化型、还原型和周转型CuSOD的弛豫率值分别为9.6×10⁶、远小于1×10²和5.2×10⁶ M⁻¹ s⁻¹，而对于MnSOD，相应的值分别为2.9×10⁶、4.2×10⁶和3.6×10⁶ M⁻¹ s⁻¹。这些高弛豫率值使得能够在脑和肝匀浆中检测到SOD，在有氧条件下，这些酶以稳态形式存在。通过添加二乙基二硫代氨基甲酸盐或氰化物来测量两种类型的SOD对匀浆中F -弛豫速率的贡献，这两种物质均选择性抑制CuSOD。NMR和活性数据之间的比较证实了通过NMR对匀浆中的SOD进行准确和精确测量的可能性。
