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

✍ Scribed by Vincenzo Cunsolo; Salvatore Foti; Carmelo La Rosa; Rosaria Saletti; G. W. Canters; M. Ph. Verbeet

• Publisher: John Wiley and Sons
• Year: 2003
• Tongue: English
• Weight: 182 KB
• Volume: 38
• Category: Article
• ISSN: 1076-5174
• DOI: 10.1002/jms.460

✦ Synopsis

Abstract

An electrospray ionisation (ESI) mass spectrometric method for the determination of the equilibrium constant and free energy (Δ__G__) 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 Δ__G__° derivation was attempted. The Δ__G__° 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. Copyright © 2003 John Wiley & Sons, Ltd.