THERMAL DENATURATION OF MYOGLOBIN .I. KINETIC RESOLUTION OF REACTION MECHANISM

The thermal denaturation of sperm whale myoglobin was investigated kinetically at pH 6.85 and ionic strength 0.096 in sodium phosphate buffer. Progress curves of the absorbance changes at 290, 409, and 540 mμ were obtained for temperatures between 40 and 90°. Three reaction phases in the denaturation process were resolved experimentally. The first phase was a rapid equilibrium. In the second phase, the reaction rate showed a very sharp dependence on temperature at 80°. This effect was reflected in the Arrhenius plot as a deviation from linearity. The limiting slope of the Arrhenius plot at the higher temperatures gave a value of ΔH≠ = + 109 kcal/mole. Assuming ΔF≠ = +30 kcal/mole, the value ΔS≠ = +224 eu was calculated. The third phase was the formation of precipitate. An interpretation of mechanism is suggested, involving a conformational disturbance in the region of the heme group in the first phase, unfolding of the helical regions in the molecule in the second phase, followed by a sequence of polymerization steps, leading to precipitation in the third phase. © 1968, American Chemical Society. All rights reserved.