Denaturation of oxyhemoglobulin S by mechanical shaking.

The oxy form of sickle hemoglobin precipitates rapidly during mechanical shaking. The rate of precipitation depends on shaking conditions such as stroke amplitude, frequency, angle between the vial axis and the shaking motion, volume and viscosity of hemoglobin solution and temperature. The rate increases linearly with either stroke (S) or frequency (n) above a certain value (S = 5 mm or n = 10Hz). The rate constant was maximum when the angle 0 between the vial axis and the shaking direction was 30 degrees. The rate of precipitation of sickle oxyhemoglobin was faster at high temperatures and always about 10 times greater than that of normal hemoglobin. The activation energies of precipitation of hemoglobin S and A are 20.3 and 21.3 kcal/mol, respectively. The thermogram of hemoglobin was measured by a differential scanning calorimeter. The denaturation temperatures determined from the peak of denaturation curves were 83.5 degrees C for oxyhemoglobin S and 85 degrees C for oxyhemoglobin A. The precipitated hemoglobin after mechanical shaking did not show any denaturation peak indicating that the protein molecules are irreversibly denatured by shaking.