QPNC-PAGE, or quantitative preparative native continuous polyacrylamide gel electrophoresis, is a high-resolution technique applied in biochemistry and bioinorganic chemistry to separate proteins by isoelectric point. This variant of gel electrophoresis is used by biologists to isolate active or native metalloproteins in biological samples and to resolve properly and improperly folded metal cofactor-containing proteins or protein isoforms in complex protein mixtures.[1]
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The QPNC-PAGE procedure is accomplished in a special electrophoresis chamber for separating charged biomolecules. Due to the specific properties of the prepared gel and electrophoresis buffer solution (which is basic and contains Tris-HCl and NaN3), most proteins of a biological system are charged negatively in the solution, and will migrate from the cathode to the anode due to the electric field (see Figure "Electrophoresis chamber").[2]
Although the pH value (10.00) of the electrophoresis buffer does not correspond to a physiological pH value within a cell or tissue type, the separated ring-shaped protein bands are eluted continuously into a physiological buffer solution (pH 8.00) and isolated in different fractions. The separation system, including the electrophoresis chamber and a fraction collector, is cooled in a refrigerator.[3]
The time of polymerization of the gel may affect the peak-elution times of separated metalloproteins in the electropherogram due to the compression of the gels and their pores with the longer incubation times (see Figure "Electropherogram"). In order to obtain a fully polymerized gel for a PAGE run, the polyacrylamide gel is polymerized for a time period of 69 hr at room temperature. As a result, the prepared gel is homogeneous, mechanically stable and free of monomers or radicals. The pore sizes of the gel are very large and therefore, sieving effects become minimized during the electrophoretic separations. For these reasons interactions of the gel with the biomolecules can be neglected. The separated metalloproteins (e.g., metal chaperones, prions, metal transport proteins, amyloids, metalloenzymes, metallopeptides) are not dissociated into apoproteins and metal cofactors.[4]
The bioactive structures (native or 3D conformation) of the isolated protein molecules do not undergo any significant conformational changes. Active metal cofactor-containing proteins can be isolated reproducibly in the same fraction after a PAGE run (see Figure "Electropherogram"). A shifting peak in the respective electropherogram may indicate that a denatured metalloprotein is available in the original protein mixture to be separated. Metalloproteins with different molecular mass ranges have been recovered in biologically active form at a yield of more than 95 %.[5]
Low concentrations (ppb-range) of Fe, Cu, Zn, Ni, Mo, Pd, Co, Mn, Pt, Cr, Cd and other metal cofactors can be identified and absolutely quantified by inductively coupled plasma mass spectrometry (ICP-MS), for example. Because of high purity and optimized concentration of the separated metalloproteins, for example, therapeutic recombinant plant-made pharmaceuticals such as copper chaperone for superoxide dismutase (CCS) from medicinal plants, in specific PAGE fractions, the related structures of these analytes can be elucidated by using solution NMR spectroscopy under non-denaturing conditions.[6]
Improperly folded metal proteins, for example, CCS or superoxide dismutase (SOD1) present in brain, blood or other clinical samples, are indicative of neurodegenerative diseases like Alzheimer’s disease or Amyotrophic Lateral Sclerosis. Active CCS or SOD molecules contribute to intracellular homeostatic control of essential metal ions (e.g., Cu1+/2+, Zn2+, Fe2+/3+) in organisms and thus, these biomolecules can balance pro-oxidative and antioxidative processes in the cytoplasm.[7]
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