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. The presented technique is based on a new principle and a new physical constant of gel electrophoresis implying the accurate control of gel pore size and stability by the time of polymerization of acrylamide (tpolAA=69 hr, RT).
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 is reached 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 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 physical constant of the time of gel polymerization (tpolAA=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. 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.
History and principle
In the 20th century it was generally accepted that ammonium persulfate/TEMED-initiated reactions should be allowed to proceed for 2 hours to insure maximum reproducibility in gel pore size of PAGE gels. Thus, longer incubation times may not have any essential effects on the protein separation process although residual monomer still remains beyond this time period. These views, however, have changed fundamentally at the beginning of the 21st century.
In 2001 a new basic principle of gel electrophoresis was discovered at the Forschungszentrum Jülich. This indicates that the time of polymerization of a polyacrylamide gel directly affects the result of a protein purification because the separation properties implying the mechanical and chemical stability of a gel and its pores are determined by this parameter. As a consequence, the peak-elution times implying the reproducibility and recovery of the separated proteins in the electropherogram may vary considerably. Accordingly, such procedures result in a loss of quality of analytical measurements. On the other hand the results of protein electrophoresis can be optimized in terms of reliability by strict adherence to a standardized time of polymerization (physical constant, tpolAA=69 hr, RT) for PAGE gels as postulated by Kastenholz and Garfin.
Said principle and the associated physical constant as an integral part of a new technique were applied for patent in 2003, subsequently issued in 2014 (German Patent DE 10336540) and realized by QPNC-PAGE. Several publications concerning the applications of this technique were edited or coauthored by the well-known American scientist and internationally recognized expert in the field of protein electrophoresis David E. Garfin. In the 1970s and the early 1980s Dr. Garfin became one of the main investigators and pioneers in the field of prion research in the team of Stanley B. Prusiner. For significant contributions to the history of electrophoresis Dave Garfin received the 2013 AES Electrophoresis Society Career Award in San Francisco.
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- Youn HD, Kim EJ, Roe JH, Hah YC, Kang SO (1996). "A novel nickel-containing superoxide dismutase from Streptomyces spp". Biochemical Journal 318 (Pt 3): 889–96. PMC 1217701. PMID 8836134.
- McLellan T (1982). "Electrophoresis buffers for polyacrylamide gels at various pH". Analytical Biochemistry 126 (1): 94–99. doi:10.1016/0003-2697(82)90113-0. ISSN 0003-2697. PMID 7181120.
- Garfin DE (2009). "25th Annual Meeting of the American Electrophoresis Society". Expert Review of Proteomics 6 (3): 239–241. doi:10.1586/epr.09.18. PMID 19489696.
- Garfin DE (2009) . "Chapter 29 one-dimensional gel electrophoresis". Methods in Enzymology 463: 497–513. doi:10.1016/S0076-6879(09)63029-9. ISBN 978-0-12-374536-1. PMID 19892189.
- Fitri N, Kastenholz B, Buchari B, Amran MB, Warganegara FM (2008). "Molybdenum speciation in raw phloem sap of castor bean". Analytical Letters 41 (10): 1773–84. doi:10.1080/00032710802162442. ISSN 0003-2719.
- Kastenholz B (2006). "Important contributions of a new quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE) procedure for elucidating metal cofactor metabolisms in protein-misfolding diseases – a theory". Protein and Peptide Letters 13 (5): 503–8. doi:10.2174/092986606776819637. PMID 16800806.
- Kastenholz B, Garfin DE (2009). "Medicinal plants: a natural chaperones source for treating neurological disorders". Protein and Peptide Letters 16 (2): 116–120. doi:10.2174/092986609787316234. PMID 19200033.
- Robinson NJ, Winge DR (2010). "Copper metallochaperones". Annual Review of Biochemistry 79: 537–562. doi:10.1146/annurev-biochem-030409-143539. PMID 20205585.
- Research at BIOMIG
- Wittig I, Braun H-P, Schägger H (2006). "Blue native PAGE". Nature Protocols 1 (1): 418–428. doi:10.1038/nprot.2006.62. PMID 17406264.
- Isolation of Acidic, Basic and Neutral Metalloproteins by QPNC-PAGE
- German Patent DE 10336540
- Prusiner SB, Garfin DE, Cochran SP, McKinley MP, Groth DF, Hadlow WJ, Race RE, Eklund CM (1980). "Experimental scrapie in the mouse: electrophoretic and sedimentation properties of the partially purified agent". Journal of Neurochemistry 35 (3): 574–582. doi:10.1111/j.1471-4159.1980.tb03693.x. PMID 6778963.
- AES 2013: Annual Meeting of the AES Electrophoresis Society
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- Kastenholz B. "Author Profile". Researchgate