QPNC-PAGE, or quantitative preparative native continuous polyacrylamide gel electrophoresis, is a high-resolution technique applied in biochemistry

Biochemistry

Biochemistry, sometimes called biological chemistry, is the study of chemical processes in living organisms, including, but not limited to, living matter. Biochemistry governs all living organisms and living processes...

 and bioinorganic chemistry

Bioinorganic chemistry

Bioinorganic chemistry is a field that examines the role of metals in biology. Bioinorganic chemistry includes the study of both natural phenomena such as the behavior of metalloproteins as well artificially introduced metals, including those that are non-essential, in medicine and toxicology...

 to separate protein

Protein

Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

s by isoelectric point

Isoelectric point

The isoelectric point , sometimes abbreviated to IEP, is the pH at which a particular molecule or surface carries no net electrical charge....

. This variant of gel electrophoresis

Gel electrophoresis

Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge...

 is used by biologists to isolate active or native

Native state

In biochemistry, the native state of a protein is its operative or functional form. While all protein molecules begin as simple unbranched chains of amino acids, once completed they assume highly specific three-dimensional shapes; that ultimate shape, known as tertiary structure, is the folded...

 metalloprotein

Metalloprotein

Metalloprotein is a generic term for a protein that contains a metal ion cofactor. Metalloproteins have many different functions in cells, such as enzymes, transport and storage proteins, and signal transduction proteins. Indeed, about one quarter to one third of all proteins require metals to...

s in biological

Biology

Biology is a natural science concerned with the study of life and living organisms, including their structure, function, growth, origin, evolution, distribution, and taxonomy. Biology is a vast subject containing many subdivisions, topics, and disciplines...

 sample

Sample (material)

In general, a sample is a limited quantity of something which is intended to be similar to and represent a larger amount of that thing. The things could be countable objects such as individual items available as units for sale, or a material not countable as individual items. Samples of countable...

s and to resolve properly and improperly folded

Protein folding

Protein folding is the process by which a protein structure assumes its functional shape or conformation. It is the physical process by which a polypeptide folds into its characteristic and functional three-dimensional structure from random coil....

 metal cofactor

Cofactor (biochemistry)

A cofactor is a non-protein chemical compound that is bound to a protein and is required for the protein's biological activity. These proteins are commonly enzymes, and cofactors can be considered "helper molecules" that assist in biochemical transformations....

-containing proteins or protein isoform

Protein isoform

A protein isoform is any of several different forms of the same protein. Different forms of a protein may be produced from related genes, or may arise from the same gene by alternative splicing. A large number of isoforms are caused by single-nucleotide polymorphisms or SNPs, small genetic...

s in complex protein mixture

Mixture

In chemistry, a mixture is a material system made up by two or more different substances which are mixed together but are not combined chemically...

s.

Electrophoresis buffer and device

The QPNC-PAGE procedure is accomplished in a special electrophoresis

Electrophoresis

Electrophoresis, also called cataphoresis, is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. This electrokinetic phenomenon was observed for the first time in 1807 by Reuss , who noticed that the application of a constant electric...

 chamber for separating

Separation process

In chemistry and chemical engineering, a separation process, or simply a separation, is any mass transfer process used to convert a mixture of substances into two or more distinct product mixtures, at least one of which is enriched in one or more of the mixture's constituents. In some cases, a...

 charged biomolecule

Biomolecule

A biomolecule is any molecule that is produced by a living organism, including large polymeric molecules such as proteins, polysaccharides, lipids, and nucleic acids as well as small molecules such as primary metabolites, secondary metabolites, and natural products...

s. Due to the specific properties of the prepared gel

Gel

A gel is a solid, jelly-like material that can have properties ranging from soft and weak to hard and tough. Gels are defined as a substantially dilute cross-linked system, which exhibits no flow when in the steady-state...

 and electrophoresis buffer solution

Buffer solution

A buffer solution is an aqueous solution consisting of a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. It has the property that the pH of the solution changes very little when a small amount of strong acid or base is added to it. Buffer solutions are used as a...

 (which is basic and contains Tris

Tris

Tris is an abbreviation of the organic compound known as trisaminomethane, with the formula 3CNH2. Tris is extensively used in biochemistry and molecular biology. In biochemistry, tris is widely used as a component of buffer solutions, such as in TAE and TBE buffer, especially for solutions of...

-HCl

HCL

HCL or HCl can stand for:* Hairy cell leukemia, an uncommon and slowly progressing B cell leukemia* Hardware compatibility list...

 and NaN₃), most proteins of a biological system

Biological system

In biology, a biological system is a group of organs that work together to perform a certain task. Common systems, such as those present in mammals and other animals, seen in human anatomy, are those such as the circulatory system, the respiratory system, the nervous system, etc.A group of systems...

 are charged negatively in the solution, and will migrate from the cathode

Cathode

A cathode is an electrode through which electric current flows out of a polarized electrical device. Mnemonic: CCD .Cathode polarity is not always negative...

 to the anode

Anode

An anode is an electrode through which electric current flows into a polarized electrical device. Mnemonic: ACID ....

 due to the electric field

Electric field

In physics, an electric field surrounds electrically charged particles and time-varying magnetic fields. The electric field depicts the force exerted on other electrically charged objects by the electrically charged particle the field is surrounding...

 (see Figure "Electrophoresis chamber").

Although the pH

PH

In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...

 value (10.00) of the electrophoresis buffer does not correspond to a physiological

Physiology

Physiology is the science of the function of living systems. This includes how organisms, organ systems, organs, cells, and bio-molecules carry out the chemical or physical functions that exist in a living system. The highest honor awarded in physiology is the Nobel Prize in Physiology or...

 pH value within a cell

Cell (biology)

The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....

 or tissue

Biological tissue

Tissue is a cellular organizational level intermediate between cells and a complete organism. A tissue is an ensemble of cells, not necessarily identical, but from the same origin, that together carry out a specific function. These are called tissues because of their identical functioning...

 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

Fraction (chemistry)

A fraction in chemistry is a quantity collected from a sample or batch of a substance in a fractionating separation process. In such a process, a mixture is separated into fractions, which have compositions that vary according to a gradient. A fraction can be defined as a group of chemicals that...

 collector, is cooled in a refrigerator.

Gel and gel properties

The time of polymerization of the gel may affect the peak-elution times of separated metalloproteins in the electropherogram

Electropherogram

An electropherogram is a plot of results from an analysis done by electrophoresis automatic sequencing.Electropherograms may be used for deriving results from:*genealogical DNA testing*paternity testing*DNA sequencing*Genetic fingerprinting...

 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

Room temperature

-Comfort levels:The American Society of Heating, Refrigerating and Air-Conditioning Engineers has listings for suggested temperatures and air flow rates in different types of buildings and different environmental circumstances. For example, a single office in a building has an occupancy ratio per...

. As a result, the prepared gel is homogeneous

Homogeneous (chemistry)

A substance that is uniform in composition is a definition of homogeneous. This is in contrast to a substance that is heterogeneous.The definition of homogeneous strongly depends on the context used. In Chemistry, a homogeneous suspension of material means that when dividing the volume in half, the...

, mechanically stable and free of monomer

Monomer

A monomer is an atom or a small molecule that may bind chemically to other monomers to form a polymer; the term "monomeric protein" may also be used to describe one of the proteins making up a multiprotein complex...

s or radicals

Radical (chemistry)

Radicals are atoms, molecules, or ions with unpaired electrons on an open shell configuration. Free radicals may have positive, negative, or zero charge...

. 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 biomolecule

Biomolecule

A biomolecule is any molecule that is produced by a living organism, including large polymeric molecules such as proteins, polysaccharides, lipids, and nucleic acids as well as small molecules such as primary metabolites, secondary metabolites, and natural products...

s can be neglected. The separated metalloprotein

Metalloprotein

Metalloprotein is a generic term for a protein that contains a metal ion cofactor. Metalloproteins have many different functions in cells, such as enzymes, transport and storage proteins, and signal transduction proteins. Indeed, about one quarter to one third of all proteins require metals to...

s (e.g., metal chaperones, prion

Prion

A prion is an infectious agent composed of protein in a misfolded form. This is in contrast to all other known infectious agents which must contain nucleic acids . The word prion, coined in 1982 by Stanley B. Prusiner, is a portmanteau derived from the words protein and infection...

s, metal transport proteins, amyloid

Amyloid

Amyloids are insoluble fibrous protein aggregates sharing specific structural traits. Abnormal accumulation of amyloid in organs may lead to amyloidosis, and may play a role in various neurodegenerative diseases.-Definition:...

s, metalloenzymes, metallopeptides) are not dissociated into apoprotein

Apoprotein

Apoprotein can refer to:*Apoenzyme, the protein part of an enzyme without its characteristic prosthetic group.*Apolipoprotein, a lipid-binding protein that is a constituent of the plasma lipoprotein....

s and metal

Metal

A metal , is an element, compound, or alloy that is a good conductor of both electricity and heat. Metals are usually malleable and shiny, that is they reflect most of incident light...

 cofactor

Cofactor (biochemistry)

A cofactor is a non-protein chemical compound that is bound to a protein and is required for the protein's biological activity. These proteins are commonly enzymes, and cofactors can be considered "helper molecules" that assist in biochemical transformations....

s.

Reproducibility and recovery

The bioactive structure

Structure

Structure is a fundamental, tangible or intangible notion referring to the recognition, observation, nature, and permanence of patterns and relationships of entities. This notion may itself be an object, such as a built structure, or an attribute, such as the structure of society...

s (native

Native state

In biochemistry, the native state of a protein is its operative or functional form. While all protein molecules begin as simple unbranched chains of amino acids, once completed they assume highly specific three-dimensional shapes; that ultimate shape, known as tertiary structure, is the folded...

 or 3D

Three-dimensional space

Three-dimensional space is a geometric 3-parameters model of the physical universe in which we live. These three dimensions are commonly called length, width, and depth , although any three directions can be chosen, provided that they do not lie in the same plane.In physics and mathematics, a...

 conformation

Protein structure

Proteins are an important class of biological macromolecules present in all organisms. Proteins are polymers of amino acids. Classified by their physical size, proteins are nanoparticles . Each protein polymer – also known as a polypeptide – consists of a sequence formed from 20 possible L-α-amino...

) of the isolated protein

Protein

Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

 molecule

Molecule

A molecule is an electrically neutral group of at least two atoms held together by covalent chemical bonds. Molecules are distinguished from ions by their electrical charge...

s do not undergo any significant conformational changes. Active metal cofactor-containing proteins can be isolated reproducibly

Reproducibility

Reproducibility is the ability of an experiment or study to be accurately reproduced, or replicated, by someone else working independently...

 in the same fraction after a PAGE run (see Figure "Electropherogram"). A shifting peak in the respective electropherogram

Electropherogram

An electropherogram is a plot of results from an analysis done by electrophoresis automatic sequencing.Electropherograms may be used for deriving results from:*genealogical DNA testing*paternity testing*DNA sequencing*Genetic fingerprinting...

 may indicate that a denatured metalloprotein

Metalloprotein

Metalloprotein is a generic term for a protein that contains a metal ion cofactor. Metalloproteins have many different functions in cells, such as enzymes, transport and storage proteins, and signal transduction proteins. Indeed, about one quarter to one third of all proteins require metals to...

 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 %.

Quantification and identification

Low concentrations (ppb

Parts-per notation

In science and engineering, the parts-per notation is a set of pseudo units to describe small values of miscellaneous dimensionless quantities, e.g. mole fraction or mass fraction. Since these fractions are quantity-per-quantity measures, they are pure numbers with no associated units of measurement...

-range) of Fe, Cu, Zn, Ni, Mo, Pd, Co, Mn, Pt, Cr, Cd and other metal

Metal

A metal , is an element, compound, or alloy that is a good conductor of both electricity and heat. Metals are usually malleable and shiny, that is they reflect most of incident light...

 cofactor

Cofactor (biochemistry)

A cofactor is a non-protein chemical compound that is bound to a protein and is required for the protein's biological activity. These proteins are commonly enzymes, and cofactors can be considered "helper molecules" that assist in biochemical transformations....

s can be identified and absolutely quantified by inductively coupled plasma mass spectrometry (ICP-MS), for example. Because of high purity and optimized concentration

Concentration

In chemistry, concentration is defined as the abundance of a constituent divided by the total volume of a mixture. Four types can be distinguished: mass concentration, molar concentration, number concentration, and volume concentration...

 of the separated metalloprotein

Metalloprotein

Metalloprotein is a generic term for a protein that contains a metal ion cofactor. Metalloproteins have many different functions in cells, such as enzymes, transport and storage proteins, and signal transduction proteins. Indeed, about one quarter to one third of all proteins require metals to...

s, for example, therapeutic recombinant plant-made pharmaceuticals

Pharming (genetics)

Pharming is a portmanteau of farming and "pharmaceutical" and refers to the use of genetic engineering to insert genes that code for useful pharmaceuticals into host animals or plants that would otherwise not express those genes. As a consequence, the host animals or plants then make the...

 such as copper chaperone for superoxide dismutase (CCS) from medicinal plants

Herbalism

Herbalism is a traditional medicinal or folk medicine practice based on the use of plants and plant extracts. Herbalism is also known as botanical medicine, medical herbalism, herbal medicine, herbology, herblore, and phytotherapy...

, in specific PAGE fractions, the related structure

Structure

Structure is a fundamental, tangible or intangible notion referring to the recognition, observation, nature, and permanence of patterns and relationships of entities. This notion may itself be an object, such as a built structure, or an attribute, such as the structure of society...

s of these analyte

Analyte

An analyte, or component , is a substance or chemical constituent that is of interest in an analytical procedure. Grammatically, it is important to note that experiments always seek to measure properties of analytes—and that analytes themselves can never be measured. For instance, one cannot...

s can be elucidated by using solution NMR spectroscopy

NMR spectroscopy

Nuclear magnetic resonance spectroscopy, most commonly known as NMR spectroscopy, is a research technique that exploits the magnetic properties of certain atomic nuclei to determine physical and chemical properties of atoms or the molecules in which they are contained...

 under non-denaturing conditions.

Applications

Improperly folded

Protein folding

Protein folding is the process by which a protein structure assumes its functional shape or conformation. It is the physical process by which a polypeptide folds into its characteristic and functional three-dimensional structure from random coil....

 metal proteins, for example, CCS

CCS (gene)

Copper chaperone for superoxide dismutase is a metalloprotein that in humans is encoded by the CCS gene.-Interactions:CCS has been shown to interact with SOD1 and APBA1.-Further reading:...

 or superoxide dismutase (SOD1

SOD1

Superoxide dismutase [Cu-Zn] also known as superoxide dismutase 1 or SOD1 is an enzyme that in humans is encoded by the SOD1 gene. SOD1 is one of three human superoxide dismutases.- Function :...

) present in brain

Brain

The brain is the center of the nervous system in all vertebrate and most invertebrate animals—only a few primitive invertebrates such as sponges, jellyfish, sea squirts and starfishes do not have one. It is located in the head, usually close to primary sensory apparatus such as vision, hearing,...

, blood

Blood

Blood is a specialized bodily fluid in animals that delivers necessary substances such as nutrients and oxygen to the cells and transports metabolic waste products away from those same cells....

 or other clinical samples, are indicative of neurodegenerative diseases like Alzheimer’s disease or Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis , also referred to as Lou Gehrig's disease, is a form of motor neuron disease caused by the degeneration of upper and lower neurons, located in the ventral horn of the spinal cord and the cortical neurons that provide their efferent input...

. Active CCS or SOD

Superoxide dismutase

Superoxide dismutases are a class of enzymes that catalyze the dismutation of superoxide into oxygen and hydrogen peroxide. As such, they are an important antioxidant defense in nearly all cells exposed to oxygen...

 molecules contribute to intracellular homeostatic

Homeostasis

Homeostasis is the property of a system that regulates its internal environment and tends to maintain a stable, constant condition of properties like temperature or pH...

 control of essential metal ions

Metal Ions in Life Sciences

Metal Ions in Life Sciences is a series of books edited by Astrid, Helmut and Roland Sigel, now published by the Royal Society of Chemistry which reflects the interdisciplinary nature of biological inorganic chemistry. Six volumes have been produced, a seventh volume is in the press and two more...

 (e.g., Cu^1+/2+

Copper in health

Copper is an essential trace element that is vital to the health of all living things . In humans, copper is essential to the proper functioning of organs and metabolic processes. The human body has complex homeostatic mechanisms which attempt to ensure a constant supply of available copper, while...

, Zn²⁺, Fe^2+/3+) in organisms and thus, these biomolecules can balance pro-oxidative and antioxidative processes in the cytoplasm

Cytoplasm

The cytoplasm is a small gel-like substance residing between the cell membrane holding all the cell's internal sub-structures , except for the nucleus. All the contents of the cells of prokaryote organisms are contained within the cytoplasm...

.

External links

• Kastenholz B, Garfin DE. "Isolation of Acidic, Basic and Neutral Metalloproteins by QPNC-PAGE". (preprint) in Nature Precedings