Origin of the anomalous circular dichroism spectra of many apomyoglobin mutants.
Several authors have reported that many sperm whale apomyoglobin mutants show anomalous circular dichroism spectra. These mutants have a low molar ellipticity compared to the wild-type protein but in several cases have the same stability of unfolding. A model in which native apomyoglobin is not folded in the same manner as that in other proteins and in which mutants show progressive reductions in their degree of folding has been suggested to explain this phenomenon. However, nuclear magnetic resonance of the native apomyoglobin conformation has shown that this state is folded and compact, raising the possibility that the anomalous circular dichroism spectra could have another explanation. We studied several mutants with anomalous circular dichroism spectra and found that these proteins were all contaminated with nucleic acid that contributed to the ultraviolet absorption and caused uncertainty in the determination of protein concentration. The resulting overestimation of the concentration of apomyoglobin explains the phenomenon of anomalous circular dichroism spectra. We describe a procedure to remove the contaminant nucleic acid which yields accurate protein concentration measurements and provides the normal circular dichroism spectra. Our findings support a well-structured native conformation for apomyoglobin and may also be of the interest to scientists working with the purification of recombinant proteins.
Ribeiro
Euripedes A
EA
Laboratório Nacional de Luz Síncrotron, Centro de Biologia Molecular Estrutural, PO Box 6192, 13084-971, Campinas, SP, Brazil.
Ramos
Carlos H I
CH
D000818:Animals; D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002942:Circular Dichroism / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D009211:Myoglobin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D014907:Whales / Q000378:metabolism
D001059:Apoproteins; D009211:Myoglobin; C010857:apomyoglobin