The thermal stability of the tryptic fragment of bovine microsomal cytochrome b5 and a variant containing six additional residues

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Abstract
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.