The second peak had a very low R of 1.7, consistent with a mixture of apo- and holo-NP4 and perhaps other impurities.
Therefore, we conclude that the fortuitous presence of an approximately 50 50 mixture of apo- and metallo-enzyme, as isolated from E. coli, allowed the apoenzyme to cocrystallize with the metallo-enzyme and thereby be trapped for crystallographic analysis.
Consistent with this, most of these proteins elute as dimers from a gel filtration column when loaded with 0.5 equiv of Zn II), whereas Atx1ZiaAN elutes as a monomer, most probably as a mixture of apo- and Zn II -protein.
Insoluble material was removed by centrifugation; the soluble lysate contained a mixture of apo- and holo-His6-IsdB-N2, and apoprotein could be separated at 4 °C using a HisTrap nickel affinity column (GE Healthcare) by elution with an imidazole gradient.
Cytb562 was appended to the N-terminus of EGFP via a Gly-Ser linker and the resultant fusion protein was successfully expressed in Escherichia coli as a mixture of the apo- and the holo-forms as to the cytb562 moiety.
Zn2+ and Mn2+ were added to the mixture of 5 µM apo-SsTroA and 500 µM ANS to 10 µM final concentration, and the mixtures were incubated at 25°C for 15 minutes prior to collecting the excitation spectrum.
Aminotransferases were reported to occur as mixtures of their apo- and holo-forms also when isolated from their natural environment (Moss 1976).
Furthermore, most often, lactoferrin preparation is a mixture of various Lf species (apo, holo, monoferric N-domain, and monoferric C-domain forms), and using just one coefficient does not reflect their complexity.
Sommerburg et al. applied an Ultrasphere C18 column with 5 μm particles using a mixture of ACN/THF/methanol/ammonium acetate for separation of β-apo-4´-, β-apo-8´-, β-apo-12´-carotenal, and retinal.
A mixture of oxidative CPs and β-apo-8´-carotenal have further been shown to be genotoxic in primary rat hepatocytes [ 4, 32 34], indicating that these CPs may be responsible for the increased risk of lung cancer.
