Regeneration of native ovalbumin conformation disrupted by citraconylation.
Ovalbumin was reacted with a 960-fold molar excess of citraconic anhydride, and 91% of the epsilon-amino groups, representing 18 of the 20 lysine residues, were citraconylated. As detected by fluorescence and far-ultraviolet circular dichroic (CD) measurements, the modified protein displayed significant disruption of the native conformation. Treatment at pH 2.2 for 5 h resulted in the hydrolysis of 10 of the 18 citraconyl groups, but when subjected to the acid conditions for 12 h, all 18 modifying groups were removed. Electrophoretically, the 5-h and the 12-h acid-treated proteins were homogeneous and showed decreasing anodic mobility at pH 8.3; indeed, the anodic mobility of the 12-h acid-treated protein was identical to that of the native protein. Similarly, the 12-h acid-treated protein possessed conformational properties almost indistinguishable from the native protein. These properties included similar emission fluorescence spectra and far-ultraviolet CD spectra, similar resistance to undergoing helix-to-coil transition at pH 12.2, and identical urea unfolding curves, and thus identical urea transition mid-point of about 8.0 M. These observations indicate that the protein with all the epsilon-amino groups regenerated by acid treatment has the same conformational stability as the native ovalbumin.
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Department of Biochemistry, School of Medicine and College of Science and Mathematics, Dayton, Ohio 45435.
D000596:Amino Acids / Q000737:chemistry; D002942:Circular Dichroism; D002949:Citraconic Anhydrides / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D010047:Ovalbumin / Q000737:chemistry*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014508:Urea / Q000737:chemistry
D000596:Amino Acids; D002949:Citraconic Anhydrides; D014508:Urea; D010047:Ovalbumin