The thermal stability of the tryptic fragment of bovine microsomal cytochrome b5 and a variant containing six additional residues.
Thermally induced denaturation has been measured for both oxidised and reduced forms of the tryptic fragment of bovine microsomal cytochrome b5 using spectrophotometric methods. In the oxidised state, the tryptic fragment of cytochrome b5 (Ala7-Lys90) denatures in a single cooperative transition with a midpoint temperature (Tm) of approximately 67 degrees C (pH 7.0). The reduced form of the tryptic fragment of cytochrome b5 shows a higher transition temperature of approximately 73 degrees C at pH 7.0 and this is reflected in the values of delta Hm, delta Sm and delta(delta G) of approximately 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 b5. 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 b5 do not differ significantly in structure with the results suggesting that the reorganisation energy (lambda) 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 b5 may result from the presence of additional N-terminal residues on the surface of the protein.
Newbold
R J
RJ
Department of Biochemistry, Queen Mary and Westfield College, London, UK.
Hewson
R
R
Whitford
D
D
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D015786:Cytochromes b5 / Q000737:chemistry* / Q000378:metabolism; D004795:Enzyme Stability; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D013057:Spectrum Analysis; D013696:Temperature; D013816:Thermodynamics; D014357:Trypsin
D010446:Peptide Fragments; D015786:Cytochromes b5; D014357:Trypsin