Biochemical and Stability Properties of Recombinant Human MnSOD

Abstract

The light absorption spectral properties of recornbinant human MnSOD. which contains an N-terminal additional methionyl residue, were investigated as a function of pH in the range 4.5–10.5. Whereas the extinction coefficient, ϵ_M at the UV maximum (282 nm) was essentially independent of pH, the ϵ_M values of the visible spectrum maximum (482 nm) displayed a bell-shaped dependence with a plateau between pH 6.5 and B. Those spectral changes were reversible and the enzymatic activity was not affected by exposure to buffered solutions at 25°C in the pH range 5–10.5. The stability of MnSOD was determined between 25 and 60°C at two different pH: 6.5 and 8.2. The enzyme was found to be considerably more stable at pH 6.5 than at pH 8.2, both toward aggregation and degradation. The gel permeation properties of MnSOD were investigated: the enzyme is a tetramer, with a subunit of 22.2 kD; however. it elutes from a Superose 12 column (Pharmacia) with an apparent molecular weight of ∼60kD. Under dissociative conditions (such as guanidine-HCI). molecular weights corresponding to the dimer and monomer could also be demonstrated. It thus appears that the tetramer adopts a non-globular shape. which causes the deviation from the Stokes radius corresponding to its molecular weight.

Key Words:

• Biochemistry of human liver manganese-SOD
• stability of recombinant MnSOD
• light absorption characteristics of MnSOD
• pH-dependence of spectral properties of MnSOD
• subunit composition of MnSOD under dissociative conditions
• aggregation of MnSOD upon “aging”
• abnormal Stokes radius of human MnSOD.