Jun 10, 2003

Monitoring of unfolding of metallo-proteins by electrospray ionization mass spectrometry

Journal of Mass Spectrometry : JMS
V CunsoloM P Verbeet

Abstract

An electrospray ionisation (ESI) mass spectrometric method for the determination of the equilibrium constant and free energy (DeltaG) of protein unfolding was used to monitor the denaturation process at different pH of three metallo-proteins, i.e. wild-type copper azurin, zinc azurin and wild-type amicyanin. The time course of the unfolding process was followed by dissolving the proteins under denaturing conditions (methanol-water (1 : 1, v/v)) at different pH (2.5, 3.0, 3.5) and recording ESI spectra at time intervals. The spectra showed two series of peaks, corresponding to the native holo-protein and the unfolded apo-protein. From the intensity ratio of these two series of peaks at increasing time and at equilibrium, the equilibrium constants for the unfolding process for the three proteins could be determined. From these equilibrium constants a DeltaG degrees derivation was attempted. The DeltaG degrees values obtained decrease with decrease in pH, in agreement with the expected reduction of conformational stability of proteins at lower pH. The results obtained confirm that ESI-MS can be used for monitoring of unfolding process and to derive quantitative thermodynamic data.

  • References27
  • Citations7

Citations

Mentioned in this Paper

Thermodynamics
Bacterial Proteins
Amicyanin Activity
Azurin
Dicom Derivation
Azurin Activity
Metalloproteins
Copper
Blue copper protein, Alcaligenes faecalis
Zinc

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