Urea Denaturation of Taka-amylase A
II. Kinetic Aspects
1961 Volume 49 Issue 4 Pages 267-277
Details
1961 Volume 49 Issue 4 Pages 267-277
1. Effects of Urea concentration and temperature on denaturation of taka-amylase A were examined by the measure ment of viscosity. The experiment was car-ried out for taka-amylase A solution prepared by dialysis against water. When this solution was treated with urea in an acetate buffer of pH 5.1 and ionic strength 0.1, the viscosity reached a steady value after it increased with time.
2. The final steady value of viscosity increased with urea concentration and changed abruptly in the region of 6M to 7 M urea.
3. The steady value of viscosity was de-termined only by the temperature at which the viscosity was measured, irrespective of the temperature at which the protein had been denatured. It was higher as the tem-perature was lower.
4. If the taka-amylase A solution de-natured by concentrated urea solutions was diluted with or dialysed against the buffer solution, precipitation occurred and filtrate showed no absorbance in an ultraviolet region at 280 mμ.
5. The denaturation obeyed the first order reaction with respect to the protein concentration in solutions in which the urea concentration was not higher than 6.43M, whereas it deviated from the first order kinetics at higher urea concentrations.
6. A possible reaction mechanism of denaturation was deduced to fit all these ex-perimental results. It may be expressed by the scheme
Native protein k1→Denatured component 1 k2 _??_ k3Denatured component 2
7. The rate constant of the first step of denaturation, k1, was dependent on urea con-centration.
8. The denaturation rate had a positive temperature coefficient over the region of 12°C to 50°C. The Arrhenius activation energy, and the heat, the free energy and the entropy of activation at different urea concentrations and temperatures were estimated.
9. The unfolding of taka-amylase Amolecule by urea treatment proceeded with a relatively slow rate, but the protein was readily denatured even in urea solution of low concentration. Consequently, taka-amylase A has a high “kinetic stability” but a low “thermodynamic stability”.
The author wishes to express her gratitude to Prof. T. Isemura for kind guidance throughout the present work.
