Thermally induced denaturation has been measured for both oxidised and reduced forms of the tryptic fragment or bovine microsomal cytochrome b 5 using spectrophotometric methods. In the oxidised state, the tryptic fragment of cytochrome b 5 (Ala7-Lys90) denatures in a single cooperative transition with a midpoint temperature (T m) of ∼ 67°C (pH 7.0). The reduced form of the tryptic fragment of cytochrome b 5 shows a higher transition temperature of ∼ 73°C at pH 7.0 and this is reflected in the values of ΔH m, ΔS m, and Δ(ΔG) of ∼ 310kJ · mol−1, 900J · mol−1 · K−1 and 5 kJ · mol−1. Increased thermal stability is demonstrated for a variant protein that contains the first 90 amino acid residues of cytochrome b 5. These novel increases in stability are observed in both redox states and result from the presence of six additional residues at the amino-terminus. The two forms of cytochrome b 5 do not differ significantly in structure with the results suggesting that the reorganisation energy (λ) of the variant protein, as measured indirectly from redox-linked differences in conformational stability, is small. Consequently the reported subtle differences in reactivity between variants of cytochrome b 5 may result from the presence of additional N-terminal residues on the surface of the protein.
