Critical Temperature of Secondary Structural Change of Myoglobin in Thermal Denaturation up to 130 °C and Effect of Sodium Dodecyl Sulfate on the Change

Abstract

The secondary structural change of horse heart myoglobin was examined in the thermal denaturation up to 130 degrees C. The original helicity of 82% gradually decreased to 67% with rise of temperature until 75 degrees C. Thereafter, it suddenly decreased to 24% at 90 degrees C and then slightly decreased to 14% at 130 degrees C. The helices of this protein were mostly destroyed between 75 and 100 degrees C. On the other hand, upon cooling to 25 degrees C from temperatures below 75 degrees C, the helicity completely recovered to the original value, but it did not after heating to temperatures above 80 degrees C. Thus, myoglobin maintains the reversibility of the structural change up to a temperature as high as 75 degrees C. This protein had another critical temperature around 90-100 degrees C in addition to 75 degrees C in the present thermal denaturation. Upon cooling to 25 degrees C after heating to temperatures above 80 degrees C, the extent of recovered helicity decreased with rise of temperature before cooling. The additive effect of sodium dodecyl sulfate (SDS) on the structural change of myoglobin differed below and above the critical temperature at 75 degrees C. In the temperature range below 75 degrees C where the structural change was reversible, the presence of SDS cooperated with the thermal denaturation to disrupt the structure. On the contrary, the presence of the surfactant more or less restrained the decrement of helicity at high temperatures above 85 degrees C. The helicity decreased and increased with an increase of SDS concentration upon cooling to 25 degrees C after heating to temperatures below 75 degrees C and after heating to temperatures above 85 degrees C, respectively. Then, upon cooling to 25 degrees C from any temperature, the helicity settled to a magnitude around 60% in the presence of the surfactant above 0.6 mM.

Quantitative Protein Topography Measurements by High Resolution Hydroxyl Radical Protein Footprinting Enable Accurate Molecular Model Selection

“…The FPOP labeled products were collected in quench solution as previously reported to eliminate excess hydrogen peroxide and any long-lived secondary oxidants 18, 20, 30 . Protein denaturation was performed right before FPOP irradiation by heating the protein sample to 90 °C for 30 min for myoglobin sample as previously reported 31 . For denaturation of lysozyme sample, TCEP was added to a final concentration of 500 μM with heating at 90 °C for 30 min before FPOP experiments.…”

Kinetic Aspects of Surfactant-Induced Structural Changes of Proteins－Unsolved Problems of Two-State Model for Protein Denaturation－

“…Moreover, the structural recoveries of the proteins, except lysozyme, are seen upon cooling after heating in restricted temperature ranges. Myoglobin maintains the recovery of the structural change up to a temperature as high as 75 24 Here, the helicity, that is, the secondary structure of each protein changes beyond a critical temperature and ○: helicity upon keeping at each temperature of abscissa; •: helicity upon cooling to 25℃ from each temperature of abscissa.…”

Quantitative Protein Topography Measurements by High Resolution Hydroxyl Radical Protein Footprinting Enable Accurate Molecular Model Selection

“…18,20,30 Protein denaturation was performed right before FPOP irradiation by heating the protein sample to 90°C for 30 min for myoglobin sample as previously reported. 31 For denaturation of lysozyme sample, TCEP was added to a final concentration of 500μM with heating at 90°C for 30 min before FPOP experiments. Adenine dosimeter UV absorbance at 260 nm was measured by a Thermo NanoDrop 2000c UV spectrophotometer (Thermo Fisher Scientific) to measure the effective radical dose delivered.…”