Effects of pH on the kinetic reaction mechanism of myoglobin unfolding studied by time-resolved electrospray ionization mass spectrometry

In most cases, kinetic unfolding reactions of proteins follow a simple one- step mechanism that does not involve any detectable intermediates. One exam ple for a more complicated unfolding reaction is the acid-induced denaturat ion of holo-myoglobin (hMb). This reaction proceeds through a transient int ermediate and can be described by a sequential two-step mechanism (Konerman n et al. Biochemistry 1997, 36, 6448-6454). Time-resolved electrospray ioni zation mass spectrometry (ESI MS) is a new technique for monitoring the kin etics of protein folding and unfolding in solution. Different protein confo rmations can be distinguished by the different charge state distributions t hat they generate during ESI. At the same time this technique allows monito ring the loss or binding of noncovalent protein ligands. In this work, time -resolved ESI MS is used to study the dependence of the kinetic unfolding m echanism of hMb on the specific solvent conditions used in the experiment I t is shown that hMb, unfolds through a short-lived intermediate only at aci dic pH. Under basic conditions no intermediate is observed. These findings are confirmed by the results of optical stopped-flow absorption experiments . This appears to be the first time that a dependence of the kinetic mechan ism for protein unfolding on external conditions such as pH has been observ ed. (C) (J Am Soc Mass Spectrom 2000, 11, 312-319) (C) 2000 American Societ y for Mass Spectrometry.