Thermal dissociation and unfolding of insulin
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Thermal dissociation and unfolding of insulin. / Huus, Kasper; Havelund, Svend; Olsen, Helle B; van de Weert, Marco; Frokjaer, Sven.
In: Biochemistry, Vol. 44, No. 33, 23.08.2005, p. 11171-7.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Thermal dissociation and unfolding of insulin
AU - Huus, Kasper
AU - Havelund, Svend
AU - Olsen, Helle B
AU - van de Weert, Marco
AU - Frokjaer, Sven
PY - 2005/8/23
Y1 - 2005/8/23
N2 - The thermal stability of human insulin was studied by differential scanning microcalorimetry and near-UV circular dichroism as a function of zinc/protein ratio, to elucidate the dissociation and unfolding processes of insulin in different association states. Zinc-free insulin, which is primarily dimeric at room temperature, unfolded at approximately 70 degrees C. The two monomeric insulin mutants Asp(B28) and Asp(B9),Glu(B27) unfolded at higher temperatures, but with enthalpies of unfolding that were approximately 30% smaller. Small amounts of zinc caused a biphasic thermal denaturation pattern of insulin. The biphasic denaturation is caused by a redistribution of zinc ions during the heating process and results in two distinct transitions with T(m)'s of approximately 70 and approximately 87 degrees C corresponding to monomer/dimer and hexamer, respectively. At high zinc concentrations (>or=5 Zn(2+) ions/hexamer), only the hexamer transition is observed. The results of this study show that the thermal stability of insulin is closely linked to the association state and that the zinc hexamer remains stable at much higher temperatures than the monomer. This is in contrast to studies with chemical denaturants where it has been shown that monomer unfolding takes place at much higher denaturant concentrations than the dissociation of higher oligomers [Ahmad, A., et al. (2004) J. Biol. Chem. 279, 14999-15013].
AB - The thermal stability of human insulin was studied by differential scanning microcalorimetry and near-UV circular dichroism as a function of zinc/protein ratio, to elucidate the dissociation and unfolding processes of insulin in different association states. Zinc-free insulin, which is primarily dimeric at room temperature, unfolded at approximately 70 degrees C. The two monomeric insulin mutants Asp(B28) and Asp(B9),Glu(B27) unfolded at higher temperatures, but with enthalpies of unfolding that were approximately 30% smaller. Small amounts of zinc caused a biphasic thermal denaturation pattern of insulin. The biphasic denaturation is caused by a redistribution of zinc ions during the heating process and results in two distinct transitions with T(m)'s of approximately 70 and approximately 87 degrees C corresponding to monomer/dimer and hexamer, respectively. At high zinc concentrations (>or=5 Zn(2+) ions/hexamer), only the hexamer transition is observed. The results of this study show that the thermal stability of insulin is closely linked to the association state and that the zinc hexamer remains stable at much higher temperatures than the monomer. This is in contrast to studies with chemical denaturants where it has been shown that monomer unfolding takes place at much higher denaturant concentrations than the dissociation of higher oligomers [Ahmad, A., et al. (2004) J. Biol. Chem. 279, 14999-15013].
KW - Amino Acid Substitution
KW - Calorimetry, Differential Scanning
KW - Circular Dichroism
KW - Hot Temperature
KW - Humans
KW - Insulin
KW - Point Mutation
KW - Protein Denaturation
KW - Protein Folding
KW - Zinc
U2 - 10.1021/bi0507940
DO - 10.1021/bi0507940
M3 - Journal article
C2 - 16101301
VL - 44
SP - 11171
EP - 11177
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 33
ER -
ID: 44640563