Thermal denaturation of human gamma-interferon. A calorimetric and spectroscopic study.


Abstract

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. Study holds ProTherm entries: 5779, 5780, 5781, 5782, 5783, 5784, 5785, 5786, 5787, 5788, 5789, 5790, 5791, 5792, 5793, 5794, 5795, 5796, 5797, 5798, 5799, 5800, 5801, 5802, 5803, 5804, 5805, 5806, 5807, 5808, 5809, 5810, 5811, 5812, 5813, 5814, 5815, 5816 Extra Details: recombinant human gamma-interferon; buffer concentration;,enthalpy; heat capacity change

Submission Details

ID: w9s6gBAM3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:30 p.m.

Version: 1

Publication Details
Beldarrain A;López-Lacomba JL;Furrazola G;Barberia D;Cortijo M,Biochemistry (1999) Thermal denaturation of human gamma-interferon. A calorimetric and spectroscopic study. PMID:10387027
Additional Information

Number of data points 76
Proteins Interferon beta ; Gamma-interferon-inducible lysosomal thiol reductase
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: dHcal pH:7.6, buffers:Sodium phosphate: 100 mM ; Experimental Assay: Tm pH:7.6, buffers:Sodium phosphate: 100 mM ; Experimental Assay: dHcal buffers:Sodium phosphate: 100 mM, pH:6.6 ; Experimental Assay: Tm buffers:Sodium phosphate: 100 mM, pH:6.6 ; Experimental Assay: dHcal pH:5.7, buffers:Acetic acid-Sodium acetate: 100 mM ; Experimental Assay: Tm pH:5.7, buffers:Acetic acid-Sodium acetate: 100 mM ; Experimental Assay: dHcal pH:4.3, buffers:Acetic acid-Sodium acetate: 100 mM ; Experimental Assay: Tm pH:4.3, buffers:Acetic acid-Sodium acetate: 100 mM ; Experimental Assay: dHcal pH:4.0, buffers:Acetic acid-Sodium acetate: 100 mM ; Experimental Assay: Tm pH:4.0, buffers:Acetic acid-Sodium acetate: 100 mM ; Experimental Assay: dHcal buffers:Sodium phosphate: 50 mM, pH:6.6 ; Experimental Assay: Tm buffers:Sodium phosphate: 50 mM, pH:6.6 ; Experimental Assay: dHcal pH:4.9, buffers:Acetic acid-Sodium acetate: 50 mM ; Experimental Assay: Tm pH:4.9, buffers:Acetic acid-Sodium acetate: 50 mM ; Experimental Assay: dHcal pH:4.3, buffers:Acetic acid-Sodium acetate: 50 mM ; Experimental Assay: Tm pH:4.3, buffers:Acetic acid-Sodium acetate: 50 mM ; Experimental Assay: dHcal pH:4.1, buffers:Acetic acid-Sodium acetate: 50 mM ; Experimental Assay: Tm pH:4.1, buffers:Acetic acid-Sodium acetate: 50 mM ; Experimental Assay: dHcal pH:3.9, buffers:Acetic acid-Sodium acetate: 50 mM ; Experimental Assay: Tm pH:3.9, buffers:Acetic acid-Sodium acetate: 50 mM ; Experimental Assay: dHcal buffers:Sodium phosphate: 20 mM, pH:8.8 ; Experimental Assay: Tm buffers:Sodium phosphate: 20 mM, pH:8.8 ; Experimental Assay: dHcal buffers:Sodium phosphate: 20 mM, pH:7.8 ; Experimental Assay: Tm buffers:Sodium phosphate: 20 mM, pH:7.8 ; Experimental Assay: dHcal buffers:Sodium phosphate: 20 mM, pH:6.6 ; Experimental Assay: Tm buffers:Sodium phosphate: 20 mM, pH:6.6 ; Experimental Assay: dHcal pH:5.8, buffers:Acetic acid-Sodium acetate: 20 mM ; Experimental Assay: Tm pH:5.8, buffers:Acetic acid-Sodium acetate: 20 mM ; Experimental Assay: dHcal buffers:Acetic acid-Sodium acetate: 20 mM, pH:4.9 ; Experimental Assay: Tm buffers:Acetic acid-Sodium acetate: 20 mM, pH:4.9 ; Experimental Assay: dHcal buffers:Acetic acid-Sodium acetate: 20 mM, pH:4.7 ; Experimental Assay: Tm buffers:Acetic acid-Sodium acetate: 20 mM, pH:4.7 ; Experimental Assay: dHcal buffers:Acetic acid-Sodium acetate: 20 mM, pH:4.3 ; Experimental Assay: Tm buffers:Acetic acid-Sodium acetate: 20 mM, pH:4.3 ; Experimental Assay: dHcal buffers:Acetic acid-Sodium acetate: 20 mM, pH:4.1 ; Experimental Assay: Tm buffers:Acetic acid-Sodium acetate: 20 mM, pH:4.1 ; Experimental Assay: dHcal pH:3.9, buffers:Acetic acid-Sodium acetate: 20 mM ; Experimental Assay: Tm pH:3.9, buffers:Acetic acid-Sodium acetate: 20 mM ; Experimental Assay: dHcal pH:3.8, buffers:Acetic acid-Sodium acetate: 20 mM ; Experimental Assay: Tm pH:3.8, buffers:Acetic acid-Sodium acetate: 20 mM ; Experimental Assay: dHcal buffers:Sodium phosphate: 10 mM, pH:7.8 ; Experimental Assay: Tm buffers:Sodium phosphate: 10 mM, pH:7.8 ; Experimental Assay: dHcal buffers:Sodium phosphate: 10 mM, pH:6.6 ; Experimental Assay: Tm buffers:Sodium phosphate: 10 mM, pH:6.6 ; Experimental Assay: dHcal pH:5.8, buffers:Acetic acid-Sodium acetate: 10 mM ; Experimental Assay: Tm pH:5.8, buffers:Acetic acid-Sodium acetate: 10 mM ; Experimental Assay: dHcal buffers:Acetic acid-Sodium acetate: 10 mM, pH:5.3 ; Experimental Assay: Tm buffers:Acetic acid-Sodium acetate: 10 mM, pH:5.3 ; Experimental Assay: dHcal buffers:Acetic acid-Sodium acetate: 10 mM, pH:5.1 ; Experimental Assay: Tm buffers:Acetic acid-Sodium acetate: 10 mM, pH:5.1 ; Experimental Assay: dHcal buffers:Acetic acid-Sodium acetate: 10 mM, pH:4.9 ; Experimental Assay: Tm buffers:Acetic acid-Sodium acetate: 10 mM, pH:4.9 ; Experimental Assay: dHcal buffers:Acetic acid-Sodium acetate: 10 mM, pH:4.7 ; Experimental Assay: Tm buffers:Acetic acid-Sodium acetate: 10 mM, pH:4.7 ; Experimental Assay: dHcal pH:4.5, buffers:Acetic acid-Sodium acetate: 10 mM ; Experimental Assay: Tm pH:4.5, buffers:Acetic acid-Sodium acetate: 10 mM ; Experimental Assay: dHcal buffers:Acetic acid-Sodium acetate: 10 mM, pH:4.3 ; Experimental Assay: Tm buffers:Acetic acid-Sodium acetate: 10 mM, pH:4.3 ; Experimental Assay: dHcal buffers:Acetic acid-Sodium acetate: 10 mM, pH:4.1 ; Experimental Assay: Tm buffers:Acetic acid-Sodium acetate: 10 mM, pH:4.1 ; Experimental Assay: dHcal pH:3.9, buffers:Acetic acid-Sodium acetate: 10 mM ; Experimental Assay: Tm pH:3.9, buffers:Acetic acid-Sodium acetate: 10 mM ; Experimental Assay: dHcal buffers:Acetic acid-Sodium acetate: 10 mM, pH:3.8 ; Experimental Assay: Tm buffers:Acetic acid-Sodium acetate: 10 mM, pH:3.8 ; Experimental Assay: dHcal buffers:Acetic acid-Sodium acetate: 10 mM, pH:3.5 ; Experimental Assay: Tm buffers:Acetic acid-Sodium acetate: 10 mM, pH:3.5 ; Experimental Assay: dHcal buffers:Acetic acid-Sodium acetate: 10 mM, pH:3.0 ; Experimental Assay: Tm buffers:Acetic acid-Sodium acetate: 10 mM, pH:3.0 ; Experimental Assay: dHcal buffers:Sodium phosphate: 5 mM, pH:6.6 ; Experimental Assay: Tm buffers:Sodium phosphate: 5 mM, pH:6.6 ; Experimental Assay: dHcal buffers:Acetic acid-Sodium acetate: 5 mM, pH:4.9 ; Experimental Assay: Tm buffers:Acetic acid-Sodium acetate: 5 mM, pH:4.9 ; Experimental Assay: dHcal pH:4.3, buffers:Acetic acid-Sodium acetate: 5 mM ; Experimental Assay: Tm pH:4.3, buffers:Acetic acid-Sodium acetate: 5 mM ; Experimental Assay: dHcal buffers:Acetic acid-Sodium acetate: 5 mM, pH:4.1 ; Experimental Assay: Tm buffers:Acetic acid-Sodium acetate: 5 mM, pH:4.1
Libraries Mutations for sequence MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN
Sequence Assay Result Units