Abstract
The molecular properties and stability of a laccase from the white‐rot fungus Trametes hirsuta (ThL) were studied to exploit the unique capability of electrospray ionization mass spectrometry (ESI‐MS) to monitor conformational and molecular‐based heterogeneities and metal ion binding simultaneously. Acid and organic solvents were applied as denaturing agents. In aqueous acidic solution, ThL existed in two major forms, distinguished by their mass difference; in addition to these, two other forms were detected. This molecular heterogeneity was due to the variable glycan content of ThL. Additionally, copper‐depleted forms of laccase were observed in mass spectra measured from aqueous acidic solution. A small amount of organic solvent (acetonitrile, CH~3~CN) increased the loss of one Cu atom from folded states and led to unfolding. In the unfolded state, ThL was depleted of all four copper atoms, and the charge state distribution was shifted to lower mass‐to‐charge region. Thus, denaturation took place in two stages: first, the loss of one Cu resulting in an inactive form; second, complete denaturation with the loss of the three remaining Cu atoms. After all coppers were lost, ThL was unfolded, as was clearly seen in the increased number of charge states in the mass spectra. Different stabilities of the glycoforms were observed in the denaturation triggered in acid and organic solvents. Copyright © 2005 John Wiley & Sons, Ltd.