Acid-induced denaturation of myoglobin studied by time-resolved electrospray ionization mass spectrometry.
The acid-induced denaturation of holo-myoglobin (hMb) following a pH-jump from 6.5 to 3.2 has been studied by electrospray ionization (ESI) mass spectrometry in combination with a continuous flow mixing technique (time-resolved ESI MS). Different protein conformations are detected by the different charge state distributions that they generate during ESI. The changes in intensity of the peaks in the mass spectrum as a function of time can be described by two exponential lifetimes of 0.38 +/- 0.06 s and 6.1 +/- 0.5 s, respectively. The acid-induced denaturation of hMb was also studied in stopped-flow experiments by monitoring changes in the Soret absorption. The lifetimes measured by this method are in good agreement with those obtained by time-resolved ESI MS. The shorter lifetime is associated with the formation of a transient intermediate which shows the mass of the intact heme-protein complex but leads to the formation of much higher charge states during ESI than native hMb at pH 6.5. This form of hMb has an absorption spectrum similar to that of the native protein, indicating a relatively unperturbed chromophore environment inside the heme binding pocket. The intermediate can thus be characterized as an unfolded form of hMb with essentially intact heme-protein interactions. The longer of the two lifetimes is associated with the formation of a product which has a blue-shifted absorption spectrum with a much lower maximum absorption coefficient than observed for native hMb. In the ESI mass spectrum, this product appears as the apoprotein with high charge states which indicates the disruption of the native heme-protein interactions and a considerable degree of unfolding compared to native apo-myoglobin. The mechanism of acid-induced denaturation of hMb, therefore, appears to follow the sequence (heme-protein)native --> (heme-protein)unfolded --> heme + (protein)unfolded.
Konermann
L
L
Department of Chemistry, University of British Columbia, Vancouver, Canada.
Rosell
F I
FI
Mauk
A G
AG
Douglas
D J
DJ
D019342:Acetic Acid; D000818:Animals; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013058:Mass Spectrometry; D009211:Myoglobin / Q000737:chemistry* / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding
D009211:Myoglobin; D006418:Heme; D019342:Acetic Acid