QPNC-PAGE

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Folding funnel: By minimizing its free energy an unfolded polypeptide chain assumes its native structure. To protect the native state from unfolding, the conditions of native GE must be precisely adjusted for subsequent 3-D structure determination. Folding funnel schematic.svg
Folding funnel: By minimizing its free energy an unfolded polypeptide chain assumes its native structure. To protect the native state from unfolding, the conditions of native GE must be precisely adjusted for subsequent 3-D structure determination.

QPNC-PAGE, or QuantitativePreparativeNativeContinuousPolyacrylamideGel Electrophoresis, is an advanced bioanalytical, high-resolution and high-precision zone electrophoresis technique applied in biochemistry and bioinorganic chemistry to separate proteins or protein isoforms by isoelectric point and by continuous elution from a gel column for further characterization. [1] [2]

Contents

This standardized 1-D hybrid variant of native gel electrophoresis and preparative polyacrylamide gel electrophoresis is used to quantitatively resolve physiological concentrations of macromolecules with high recovery, for example, into active or native metalloproteins in biological samples or into properly and improperly folded metal cofactor-containing proteins in complex protein mixtures. [1] [3]

QPNC-PAGE reduces the concentration of contaminating metal ions and dipolar compounds, thus preventing the misidentification of apo-metalloproteins (without a cofactor) as holo-metalloproteins (with a cofactor) and metal exchange reactions. This is critical for studying diseases related to protein misfolding, such as Alzheimer's disease, which are influenced by metal cofactors. [1] [3]

Introduction

Proteins perform several functions in living organisms, including catalytic reactions and transport of molecules or ions within the cells, the organs or the whole body. The understanding of the metabolic processes in human organisms, which are mainly driven by biochemical reactions and protein-protein interactions, depends to a great extent on the ability to isolate active proteins in biological extracts or fluids for more detailed examination of chemical structure and physiological function. This essential information can imply an important indication of a patient's state of health. [4]

As about 30–40% of all known proteins contain one or more metal ion cofactors (e.g., ceruloplasmin, ferritin, amyloid-beta precursor protein, matrix metalloproteinase, or metallochaperones), especially native and denatured metalloproteins have to be isolated, identified and quantified after liquid biopsy or tissue extraction. Many of these cofactors (e.g., iron, copper, or zinc) play a key role in vital enzymatic catalytic processes, such as electron transport and redox transformations, or stabilize globular protein molecules driven by protein-cofactor interactions. [5] Therefore, the high-precision gel electrophoresis and other complementary separation techniques are highly relevant as initial step of protein and trace metal speciation analysis of biological specimen, subsequently, followed by modern mass spectrometric and magnetic resonance methods for quantifying and identifying the soluble proteins of interest. [6]

Method

Separation and buffering mechanisms

Equipment for bioanalytical continuous-elution GE: electrophoresis chamber, peristaltic pump, fraction collector, buffer recirculation pump and UV detector (in the refrigerator), power supply and recorder (on the table). Electrphoresis Tools.TIF
Equipment for bioanalytical continuous-elution GE: electrophoresis chamber, peristaltic pump, fraction collector, buffer recirculation pump and UV detector (in the refrigerator), power supply and recorder (on the table).

In gel electrophoresis (GE), ampholytes or zwitterions, such as proteins and peptides, are normally separated by charge, size, or shape. [8] The aim of isoelectric focusing (IEF), for example, is to separate proteins according to their isoelectric point (pI), thus, according to their net charge at different pH values using immobilized pH gradient gels. [9] Here, a similar mechanism is accomplished in a commercially available electrophoresis chamber with integrated elution chamber for separating charged biomolecules, for example, superoxide dismutase (SOD) [10] or allergens, [11] at constantly higher pH conditions and different migration rates depending on different isoelectric points in an optimized resolving gel. The separated (metal) proteins elute sequentially, almost independently of their molecular mass, folding state or hydrodynamic volume, starting with the lowest (pI > 2–4) and ending with the highest isoelectric point (pI < 10.0) of the dissolved protein molecules to be analyzed. [12]

Due to the specific properties of the prepared gel and electrophoresis buffer solution which is basic and contains Tris-HCl and NaN3, [7] most proteins of a biological system (e.g., Helicobacter pylori [13] ) are charged negatively in the solution, and will migrate almost frictionlessly from the cathode to the anode due to the electric field. In general, reaction equation (1) shows that the carboxyl side group of a proteinogenic amino acid is negatively charged, equation (2) that the amino side groups are electrically neutral under these conditions:

(1) R-COOH + OH− → R-COO− + H2O

(2) R-NH3+ + OH− → R-NH2 + H2O

At the anode, electrochemically-generated hydrogen ions react with Tris molecules to form monovalent Tris ions (3). The positively charged Tris ions migrate through the gel to the cathode where they neutralise hydroxide ions to form Tris molecules and water (4):

(3) (HOCH2)3CNH2 + H+ → [(HOCH2)3CNH3]+

(4) [(HOCH2)3CNH3]+ + OH− → (HOCH2)3CNH2 + H2O

Thus, the Tris-based buffering mechanism causes a constant pH in the continuous buffer system with a high buffer capacity. [14]

At 25 °C Tris buffer has an effective pH range between 7.5 and 9.0. Under the conditions given here (addressing the concentration of buffer components, buffering mechanism, pH and temperature) the effective pH is shifted in the range of about 10.0 to 10.5. Native buffer systems all have low conductivity and range in pH from 3.8 to 10.2. Continuous native buffer systems are thus used to separate proteins according their pI. [15]

Although the constant 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 (zones) are continuously eluted from the gel column into a physiological buffer solution (pH 8.00) and isolated in different fractions. [7] Provided that irreversible denaturation cannot be demonstrated by an independent procedure, most protein molecules are stable in aqueous solution, at pH values from 3 to 10 if the temperature is below 50 °C. [16] As the Joule heat and temperature generated during electrophoresis may exceed 50 °C, [17] and thus, have a negative impact on the stability and migration behavior of proteins in the gel, the separation system, consisting of the electrophoresis chamber, fraction collector and other devices, is cooled in a refrigerator at 4 °C, thus, greatly reducing the risk of heat convection currents. [18] Overheating of the gel is prevented by an additional internal cooling circuit as an integrated part of the electrophoresis chamber and by generating a constant power by the power supply. [19]

Gel properties and polymerization time

Best polymerization conditions for polyacrylamide gels (PAG) are obtained at 25–30 °C [20] and polymerization seems terminated after 20–30 min of reaction although residual monomers (10–30%) are detected after this time. [21] The co-polymerization of acrylamide (AA) monomer/N,N'-Methylenebisacrylamide (Bis-AA) cross-linker initiated by ammonium persulfate (APS)/tetramethylethylenediamine (TEMED) reactions, is most efficient at alkaline pH of the acrylamide solution. Thereby, acrylamide chains are created and cross-linked at a time. Due to the properties of the electrophoresis buffer, the gel polymerization is conducted at pH 10.00 making sure an efficient use of TEMED and APS as catalysts of the polymerization reaction, and concurrently, suppressing a competitive hydrolysis of the produced acrylamide polymer network (gel matrix). Polymer networks are three-dimensionally linked polymer chains. Otherwise, proteins could be modified by reaction with unpolymerized monomers of acrylamide, forming covalent acrylamide adduction products that may result in multiple bands. [22]

Additionally, the time of polymerization of a gel can directly affect the peak-elution times of separated metalloproteins in the electropherogram due to the compression and dilatation of the gels and their pores if the incubation times for the reaction mixture used to prepare a gel are not optimized. In order to ensure 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 (RT) in a graduated glass column (gel tube) enclosing the cooling core on the casting stand. [19] The exothermic heat generated by the polymerization processes is dissipated constantly while the temperature may rise rapidly to over 75 °C in the first minutes, after which it falls slowly. [23] According to empirical studies, after 69 hr, the gel has reached room temperature and is in its lowest energy state, as the basic chemical reactions and gelation are complete. [19] Gelation means that the solvent (water) gets immobilized within the polymer network by means of hydrogen bonds and also van der Waals forces. As a result, the prepared gel is homogeneous (in terms of homogeneous distribution of cross-links throughout the gel sample [24] ), inherently stable and free of monomers or radicals. Fresh polyacrylamide gels are further hydrophilic, electrically neutral and do not bind proteins. [25] Sieving effects due to gravity-induced compression of the gel column can be excluded for the same reasons. Thus, in a medium without molecular sieving properties a high-resolution can be expected. [26]

Before an electrophoretic run is started the prepared 4% T (total polymer content (T)), 2.67% C (cross-linker concentration (C)) resolving gel, which has the shape of a hollow cylinder (gel column) and encapsulates the cooling core, is pre-run to equilibrate it. [7] It is essentially non-sieving and optimal for electrophoresis of proteins greater than or equal to 200 ku. Proteins migrate in it more or less on the basis of their free mobility. [27] For these reasons interactions of the gel with these biomolecules are negligibly low, and thus, the proteins separate cleanly and predictably in an anticonvective medium at a polymerization time of 69 hr. [7] The separated metalloproteins including biomolecules ranging from approximately < 1 ku to greater than 30 ku (e.g., metal chaperones, prions, metal transport proteins, amyloids, metalloenzymes, metallopeptides, metallothionein, phytochelatins) are not dissociated into apoproteins and metal ion cofactors. [28]

Reproducibility and recovery

Electropherogram showing four runs of a high molecular weight plant protein ([?] 200 kDa) as a function of time of polymerization of PAG. Detection method of Cd cofactor concentrations (in mg/L): GF-AAS. Preparative Electroph.TIF
Electropherogram showing four runs of a high molecular weight plant protein (≈ 200 kDa) as a function of time of polymerization of PAG. Detection method of Cd cofactor concentrations (in μg/L): GF-AAS.

The bioactive structures (native or 3-D conformation or shape) of the isolated protein molecules do not undergo any significant conformational changes. Thus, active metal cofactor-containing proteins can be isolated reproducibly in the same fractions after a PAGE run. [12] A shifting peak in the respective electropherogram indicates that the standardized time of gel polymerization (69 hr, RT) is not implemented in a PAGE experiment. A shortening of the standardized polymerization time (< 69 hr) stands for incomplete polymerization, and thus, for inherent instability due to gel softening during the cross-linking of polymers as the material reaches swelling equilibrium, [29] whereas exceeding this time limit (> 69 hr) is an indicator of gel aging. [30] The phenomenon of gel aging is closely connected to long-term viscosity decrease of aqueous polyacrylamide solutions [31] and increased swelling of hydrogels. [32]

Under these 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%. [19] By preparative SDS-PAGE standard proteins (cytochrome c, aldolase, ovalbumin and bovine serum albumin) with molecular masses of 14–66 ku can be recovered with an average yield of about 73.6%. [33] Preparative isotachophoresis (ITP) is applied for isolating palladium-containing proteins with molecular masses of 362 ku (recovery: 67%) and 158 ku (recovery: 97%). [34]

Quantification and identification

Physiological concentrations (ppb-range) of iron, copper, zinc, nickel, molybdenum, palladium, cobalt, manganese, platinum, chromium, cadmium and other metal cofactors can be identified and absolutely quantified in an aliquot of an analytical fraction by inductively coupled plasma mass spectrometry (ICP-MS) [35] or total reflection X-ray fluorescence (TXRF), [36] for example. In case of ICP-MS the structural information of the functionally associated metallobiomolecules is irreversibly lost due to ionization of the sample with plasma. [37] [38] Another established high sensitive detection method for the determination of trace elements in biological samples is graphite furnace atomic absorption spectroscopy (GF-AAS). [39] Because of high purity and optimized concentration of the separated properly folded 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 bioactive analytes can be elucidated quantitatively by using solution NMR spectroscopy under non-denaturing conditions. [40]

Applications

Improperly folded metal proteins, for example, CCS or Cu-Zn-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). [41] Functional CCS or SOD molecules contribute to intracellular homeostatic control of essential metal ion species (e.g., Cu1+/2+, Zn2+, Fe2+/3+, Mn2+, Ni3+) in organisms, and thus, these biomolecules can balance pro-oxidative and antioxidative processes in the cytoplasm. [42] Otherwise, free or loosely bound transition metal ions take part in Fenton-like reactions in which deleterious hydroxyl radical is formed, which unrestrained would be destructive to proteins. [43] The loss of active CCS increases the amyloid-β production in neurons which, in turn, is a major pathological hallmark of AD. [44] Therefore, copper chaperone for superoxide dismutase is proposed to be one of the most promising biomarkers of copper toxicity in these diseases. [45] CCS should be analysed primarily in blood because a meta-analysis of serum data showed that AD patients have higher levels of serum Cu than healthy controls. [46]

Limitations

The main limitations of Quantitative Preparative Native Continuous Polyacrylamide Gel Electrophoresis are its technical complexity, demanding protocol, technical expertise, specialized equipment, multi-platform experimental design, long runtime, sample capacity, sample loss due to multianalytical processing steps, manual operator intervention, co-migrating proteins, and high cost, which may limit its availability for routine laboratory and high-throughput applications. [7]

History

In the late 1990s, QPNC-PAGE was developed at the Jülich Research Centre to optimize the chemical composition and physico-chemical properties of the acrylamide gel matrix, as incomplete polymerization processes during electrophoresis lead to dynamic compression and decompression effects in the gel, which impair the results of protein purification. [47] A new gel design made it possible to elucidate the relationships between gel properties and polymerization time on the one hand, and reproducibility and yield on the other. For the first time in the history of electrophoresis, an independent method confirmed that physiological concentrations of proteins containing bioactive metal cofactors do not denature during preparative native gel electrophoresis runs. [12] Metal proteins of any size or folding state could be isolated in quantitative amounts for further analysis or structure determination in a combined procedure. [28] As a groundbreaking technique compatible with sensitive downstream analyses, this was finally published in the Nature Portfolio in 2010 [7] and patented by the DPMA in 2014. [48]

Persons

This unique, highly specialized electrophoretic technique for investigating the relationship between metal metabolism and protein folding in clinical research, as well as the structure-function relationships of fragile metal proteins, was invented by Bernd Kastenholz and was significantly influenced by the pioneering work of David E. Garfin and several other specialist authors from various fields, such as Jürgen Horst and Kerstin A. Nagel. Building on existing findings in natural product pharmacology and metal-related neurobiology, Kastenholz, Garfin, Horst, and Nagel were among the first to specifically propose the use of plant copper chaperones for Alzheimer's therapy. [42]

See also

References

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  47. ↑ Günther K, Ji G, Kastenholz B (2000). "Characterization of high molecular weight cadmium species in contaminated vegetable food" . Fresenius' Journal of Analytical Chemistry. 368 (2): 281–7. doi:10.1007/s002160000431. PMID   11220593.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  48. ↑ "Method of isolating metal cofactors from biological organic systems using preparative native polyacrylamide continuous gel electrophoresis (PNC-PAGE)". Google Patents. Retrieved 9 October 2025.

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