Probing the unfolding and refolding processes of carbonic anhydrase 2 using electrospray ionization mass spectrometry combined with pH jump.
A novel method for proving the time course of the unfolding and refolding processes of metalloprotein bovine carbonic anhydrase 2 (CA2) is demonstrated using electrospray ionization mass spectrometry (ESI MS) combined with pH jumps between 3.6 and 4.4. The shift in mass accompanied by the release or coordination of a zinc ion and the change in the charge state distribution were measured to evaluate the folding process. The time course of the ESI mass spectra revealed the existence of four types of ions in the experimental system, i.e., lower charged apo-CA2 and holo-CA2 ions and higher charged apo-CA2 and holo-CA2 ions. The deconvolution spectrum of the ion peak ensemble for each type of ion was processed and time course plots of the relative intensities of the four ions were prepared in order to analyze the folding processes. These analyses revealed the coexistence of two folding states of the lower and higher charged apo-CA2 under the condition of pH 3.6. The lower and higher charged apoproteins spontaneously refolded to the lower charged holoprotein by a pH jump from 3.6 to 4.4 without the addition of an extra zinc ion. The higher charged holoprotein observed during both the unfolding and refolding processes was considered to be an intermediate of the change in folding. The present study indicates that ESI MS combined with pH jump would be a powerful method to probe the unfolding and refolding of proteins. This method simultaneously measures mass spectra and analyzes the folding processes as a function of time using deconvolution spectra constructed by selecting a suitable m/z range for the analysis from the peaks of charge state distributions.
Nabuchi
Yoshiaki
Y
Fuji Gotemba Research Laboratories, Chugai Pharmaceutical Co., Ltd., Gotemba, Shizuoka 412-8513, Japan. nabuchiysa@chugai-pharm.co.jp
Murao
Naoaki
N
Asoh
Yoshinori
Y
Takayama
Mitsuo
M
D000818:Animals; D024402:Carbonic Anhydrase II / Q000032:analysis*; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D017510:Protein Folding; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013997:Time Factors
D024402:Carbonic Anhydrase II