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 standardized variant of native 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.
Electrophoresis buffer and device
The QPNC-PAGE procedure is accomplished in a commercially available 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.
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 (see Figure Electropherogram). Most protein molecules are stable in aqueous solution, at pH values from 3 to 10 if the temperature is below 50 °C. The separation system, including the electrophoresis chamber and a fraction collector, is cooled in a refrigerator at 4 °C (see Figure Equipment).
Gel and gel properties
The time of polymerization of the gel may directly 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 insure maximum reproducibility in gel pore size and to obtain a fully polymerized and non-restrictive large pore gel for a PAGE run, the polyacrylamide gel is polymerized for a time period of 69 hr at room temperature. The exothermic heat generated by the polymerization processes is dissipated constantly. The lowest energy state of the gel occurs after 69 hr of polymerization. As a result, the prepared gel is homogeneous, inherently stable and free of monomers or radicals. Fresh polyacrylamide gels are further hydrophilic, electrically neutral and do not bind proteins.
The 4% T, 2.67% C gel is pre-run to equilibriate it. It is essentially non-sieving and optimal for electrophoresis of proteins that are smaller and larger than 200 ku. Proteins migrate in it more or less on the basis of their free mobility. For these reasons interactions of the gel with the biomolecules are negligibly low and the proteins separate cleanly and predictably. The separated metalloproteins (e.g., metal chaperones, prions, metal transport proteins, amyloids, metalloenzymes, metallopeptides (< 6 ku)) are not dissociated into apoproteins and metal cofactors.
Reproducibility and recovery
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 fractions after a PAGE run (see Figure Electropherogram). A shifting peak in the respective electropherogram may either indicate that a denatured metalloprotein is available in the original protein mixture to be separated or the standardized time of gel polymerization (69 hr, RT) is not implemented in a PAGE experiment. A lower deviation of this standardized polymerization time stands for imcomplete polymerization, whereas exceeding this time limit is an indicator of gel aging (see Figure Electropherogram). Under standard conditions metalloproteins with different molecular mass ranges and isoelectric points have been recovered in biologically active form at a quantitative yield of more than 95%.
Quantification and identification
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. In the process the structural information of the associated molecules is irreversibly lost. 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 a few specific PAGE fractions, the related structures of these analytes can be elucidated quantitatively by using solution NMR spectroscopy under non-denaturing conditions.
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 (AD) or Amyotrophic Lateral Sclerosis (ALS). 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. Currently, quantitative preparative native continuous PAGE is applied in the field of molecular biology to purify enzymes and recombinant proteins of microbial strains.
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