Thermal denaturation of human gamma-interferon. A calorimetric and spectroscopic study.
The thermal denaturation of a recombinant human gamma-interferon has been studied as a function of pH in the range from 2 to 10 and buffer concentration in the range from 5 to 100 mM by differential scanning calorimetry, circular dichroism, fluorescence, 1H NMR, and biological activity measurements. The thermal transitions are irreversible at high buffer concentrations at all pH values studied, although they are reversible between pH 3.5 and 5.4 at low buffer concentrations. The denaturation enthalpy, DeltaH(Tm), at denaturation temperature Tm was a function of both Tm and the buffer concentration, and this resulted in heat capacity changes decreasing with buffer concentration. When the denaturation enthalpies were corrected for Tm dependence, they did not appear to change versus pH. The denaturation entropies, however, appeared to decrease with pH, leading to a small but appreciable increase in the stability of the protein with pH. The difference between the number of moles of protons stoichiometrically bound to a mole of protein in the native and thermally denatured state, was calculated from the variation of Tm versus pH at each buffer concentration. The values obtained appear to depend on pH alone rather than upon temperature or buffer concentration, a result which agrees with the invariance of the denaturation enthalpies with pH. This dependence was fitted to the titration curve of a group with a pK of 5.4.
Beldarrain
A
A
Centro de Ingeniería Genética y Biotecnología, La Habana, Cuba.
López-Lacomba
J L
JL
Furrazola
G
G
Barberia
D
D
Cortijo
M
M
D002152:Calorimetry, Differential Scanning / Q000379:methods; D002942:Circular Dichroism; D003903:Deuterium; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007371:Interferon-gamma / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D011994:Recombinant Proteins; D007371:Interferon-gamma; D003903:Deuterium