Kinetic analysis of the acid and the alkaline unfolded states of staphylococcal nuclease

  • Chen H
  • You J
  • Markin V
 et al. 
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Thermodynamic analysis by differential scanning calorimetry shows that the folding/ unfolding transition of staphylococcal nuclease is consistent with the two-state process. Stopped-flow kinetic measurements, monitoring the Trp140 fluorescence and covering five decades in time (2 ms to 300 s), indicate that the unfolding from pH 7-0 to 3-1 is monophasic (time constant 1.15 s) and from pH 7.0 to 12.2 is biphasic (time constants: one < 2 ms and the other 0.6 s). However, the folding, either from pH 3.1 to 7.0 or from pH 12.2 to 7-0, is triphasic (time constants 150 ms, 850 ms and 30 s from acid, 90 ms, 565 ms and 33 s from alkaline). A simple sequential model, which agrees with the above observations for acidic folding/unfolding is, D3ag D2ag D1ag N. The three Ds denote three sub-states of the unfolded state and N denotes the native state. These sub-states of D have similar enthalpy and tryptophan fluorescence, and their equilibrium cannot be shifted by temperature changes. However, they are kinetically distinctive. Data do not favor alternative mechanisms assuming parallel transitions of the three Ds to N, or complexity of the N state, or parallel transitions of sub-states of N1, N2and N3to D. Other more complex, branched or cyclic, kinetics are not considered because of the lack of evidence. pH dependence of the unfolding kinetics suggests that the unfolding is triggered by protonation of 0-8(±0.3) ionizable groups, with a pKaof 3.9 or by deprotonation of 1.6(±0.4) ionizable groups with pKavalues near 10.5. Circular dichroisms indicate that these three D states retain non-random chain conformation. Possible role of these "chain conformation" in the protein folding is discussed. © 1991.

Author-supplied keywords

  • circular dichroism
  • intermediates
  • kinetics
  • protein folding
  • stopped-flow

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  • Hueih M. Chen

  • J. L. You

  • V. S. Markin

  • Tian Yow Tsong

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