QPNC-PAGE

QPNC-PAGE (Abbr.: quantitative preparative native continuous polyacrylamide gel electrophoresis) is a high-resolution technique applied in biochemistry and bioinorganic chemistry to separate proteins according to their isoelectric point. Furthermore, this method is well-known as a specific type of gel electrophoresis used by scientists to isolate active or native metalloproteins in biological samples and to resolve properly and improperly folded metal cofactor-containing proteins in complex protein mixtures.

Electrophoresis buffer
The QPNC-PAGE procedure is accomplished in a special electrophoresis chamber for separating bioactive molecules. Due to the specific properties of the prepared gel and electrophoresis buffer solution (20 mM Tris-HCl, 1 mM NaN3, pH 10.00), most proteins (MW 6 - ≥ 200 kDa) of a biological system are charged negatively and migrate from the cathode to the anode in the electric field. Although the pH value (pH 10.00) of the electrophoresis buffer does not correspond to a physiological pH value within a cell or tissue type, the proteins are eluted continuously by 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 at 4° C.

Gel properties
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 prepared 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. The bioactive structures (native or 3D conformation) of the isolated protein molecules do not undergo any conformational changes by using QPNC-PAGE. Consequently, quantitative amounts of highly purified metalloproteins and protein isomers are isolated in the different PAGE fractions. In this context, QPNC-PAGE can be regarded as a 'breakthrough method' compared with other electrophoretic separation techniques such as SDS-PAGE, 2-DE, isotachophoresis or colorless native polyacrylamide gel electrophoresis (CN-PAGE) which are well-known as semiquantitative methods.

Additional information concerning other important parameters of the QPNC-PAGE procedure are available in the mentioned references.

Quantification and identification
Fe, Cu, Zn, Ni, Mo, Pd, Co, Mn, Pt, Cr, Cd and other metal cofactors can be identified and quantified by ICP-MS (Abbr.: inductively coupled plasma mass spectrometry), for example. Because of high purity and optimized concentration of the separated metalloproteins in specific PAGE fractions, the related structures of these analytes can be elucidated by using solution NMR spectroscopy under non-denaturing conditions.

Applications
It is of enormous importance to determine the structure-function relationships of isolated metalloproteins in brain, blood or other clinical samples because improperly folded metallochaperones, for example, copper chaperone for superoxide dismutase (CCS), present in these biomatrices may be responsible for neurodegenerative diseases like Alzheimer’s disease or Amyotrophic Lateral Sclerosis. A combined procedure consisting of QPNC-PAGE, SEC, ICP-MS and NMR is a groundbreaking approach for elucidating the structures of physiologically relevant metalloproteins in biofluids of patients and probands. It is anticipated that the results obtained by this analytical process will contribute to an improved diagnosis and therapy of several protein-misfolding diseases.