ISSN:
1075-4261
Keywords:
myoglobin
;
protein folding
;
pH-jump
;
molten globule
;
ionic strength
;
Chemistry
;
Analytical Chemistry and Spectroscopy
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Physics
Notes:
The acid unfolding of deoxymyoglobin (deoxyMb) from the native (N) form to the unfolded (U) form proceeds through at least two spectroscopically distinct heme intermediates. The 426-nm absorbing heme intermediate (I′-form) occurs in the pH ∼ 3.5-4.5 range. In the I′-form, the iron-proximal histidine bond is broken; however, the heme is five-coordinate due to binding of a water molecule. The I′-form was first observed in pH-jump (neutral to acid conditions) experiments, where it was characterized as a transient species which rapidly forms (10 ms) and dissipates. Recently, however, it was shown that the I′-intermediate also forms under equilibrium conditions. To elucidate the factors which control the formation of the I′-intermediate, a detailed series of equilibrium and slow kinetic (〉2-s) experiments were performed. Equilibrium pH titrations reveal that the I′-intermediate forms at successively higher pH as the ionic strength increases. pH-jump experiments (pH 6.9 to 3.2 and pH 4.4 to 3.2) indicate that the rate of formation of the intermediate is dramatically affected by the ionic strength conditions. If the ionic strength is held constant during the pH-jump, the I′-intermediate forms slowly (∼ 35 s) and the formation rate is independent of ionic strength. If the ionic strength is jumped from low to high values during the pH-jump, the formation rate of the I′-intermediate monotonically increases. Conversely, if the ionic strength is jumped from high to low values during the pH-jump, the rate monotonically decreases. The former result explains the finding of early pH-jump experiments wherein the I′-intermediate was found to form very rapidly. In these experiments, the ionic strength was also jumped from low to very high values during the pH-jump. In both types experiments where the pH and ionic strength are simultaneously jumped, the rate of formation of the I′-intermediate is independent of the initial and final ionic strength and depends only on the difference. The kinetic and equilibrium data are well accounted for with a simple three-state model in which the N-form is transformed into the I′-form via a single transition (T) state, and the free energy of the various forms depends linearly on the ionic strength. The model predicts that both the N-form and the T-state are stabilized with increasing ionic strength and that the extent of stabilization is approximately the same for both (-4.84 cal/mol per mM). The I′-form is also stabilized with increasing ionic strength; however, the extent of stabilization is greater than for the N-form. This picture is qualitatively consistent with a simple Born model which predicts that a medium with higher dielectric constant should impart greater stabilization to a species with higher overall charge. The I′-form is stabilized relative to the N-form at higher ionic strength (higher dielectric constant) because it is formed in a pH region where several of the histidine residues in the protein titrate, thus increasing the net positive charge on the protein relative to the N-form at neutral pH. Collectively, the studies provide a self-consistent picture of the factors which control the acid-induced transformation of deoxyMb from the N- to I′-forms. © 1997 John Wiley & Sons, Inc. Biospect 3: 17-29, 1997
Additional Material:
8 Ill.
Type of Medium:
Electronic Resource
Permalink
