Metalloprotein

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Metalloprotein

 

The structure of hemoglobin. The heme cofactor, containing the metal iron, shown in green.

 

Metalloprotein is a generic term for a protein that contains a metal ion cofactor. A large proportion of all proteins are part of this category. For instance, at least 1000 human proteins (out of ~20,000) contain zinc-binding protein domains although there may be up to 3000 human zinc metalloproteins.

Abundance

It is estimated that approximately half of all proteins contain a metal. In another estimate, about one quarter to one third of all proteins are proposed to require metals to carry out their functions. Thus, metalloproteins have many different functions in cells, such as storage and transport of proteins, enzymes and signal transduction proteins, or infectious diseases. The abundance of metal binding proteins may be inherent to the amino acids that proteins use, as even artificial proteins without evolutionary history will readily bind metals.

Most metals in the human body are bound to proteins. For instance, the relatively high concentration of iron in the human body is mostly due to the iron in hemoglobin.

Metal concentrations in humans organs (ppm = ug/g ash)

	Liver	Kidney	Lung	Heart	Brain	Muscle
Mn (manganese)	138	79	29	27	22	<4-40 font="">
Fe (iron)	16,769	7,168	24,967	5530	4100	3,500
Co (cobalt)	<2-13 font="">	<2 font="">	<2-8 font="">	---	<2 font="">	150 (?)
Ni (nickel)	<5 font="">	<5-12 font="">	<5 font="">	<5 font="">	<5 font="">	<15 font="">
Cu (copper)	882	379	220	350	401	85-305
Zn (zinc)	5,543	5,018	1,470	2,772	915	4,688

Coordination chemistry principles

In metalloproteins, metal ions are usually coordinated by nitrogen, oxygen or sulfur centers belonging to amino acid residues of the protein. These donor groups are often provided by side-chains on the amino acid residues. Especially important are the imidazole substituent in histidine residues, thiolate substituents in cysteine residues, and carboxylate groups provided by aspartate. Given the diversity of the metalloproteome, virtually all amino acid residues have been shown to bind metal centers. The peptide backbone also provides donor groups; these include deprotonated amides and the amide carbonyl oxygen centers. Lead(II) binding in natural and artificial proteins has been reviewed.

In addition to donor groups that are provided by amino acid residues, many organic cofactors function as ligands. Perhaps most famous are the tetradentate N₄ macrocyclic ligands incorporated into the heme protein. Inorganic ligands such as sulfide and oxide are also common.

Storage and transport metalloproteins

These are the second stage product of protein hydrolysis obtained by treatment with slightly stronger acids and alkalies.

Oxygen carriers

Hemoglobin, which is the principal oxygen-carrier in humans, has four subunits in which the iron(II) ion is coordinated by the planar macrocyclic ligand protoporphyrin IX (PIX) and the imidazole nitrogen atom of a histidine residue. The sixth coordination site contains a water molecule or a dioxygen molecule. By contrast the protein myoglobin, found in muscle cells, has only one such unit. The active site is located in a hydrophobic pocket. This is important as without it the iron(II) would be irreversibly oxidized to iron(III). The equilibrium constant for the formation of HbO₂ is such that oxygen is taken up or released depending on the partial pressure of oxygen in the lungs or in muscle. In hemoglobin the four subunits show a cooperativity effect that allows for easy oxygen transfer from hemoglobin to myoglobin.

In both hemoglobin and myoglobin it is sometimes incorrectly stated that the oxygenated species contains iron(III). It is now known that the diamagnetic nature of these species is because the iron(II) atom is in the low-spin state. In oxyhemoglobin the iron atom is located in the plane of the porphyrin ring, but in the paramagnetic deoxyhemoglobin the iron atom lies above the plane of the ring. This change in spin state is a cooperative effect due to the higher crystal field splitting and smaller ionic radius of Fe²⁺ in the oxyhemoglobin moiety.

Hemerythrin is another iron-containing oxygen carrier. The oxygen binding site is a binuclear iron center. The iron atoms are coordinated to the protein through the carboxylate side chains of a glutamate and aspartate and five histidine residues. The uptake of O₂ by hemerythrin is accompanied by two-electron oxidation of the reduced binuclear center to produce bound peroxide (OOH⁻). The mechanism of oxygen uptake and release have been worked out in detail.

Hemocyanins carry oxygen in the blood of most mollusks, and some arthropods such as the horseshoe crab. They are second only to hemoglobin in biological popularity of use in oxygen transport. On oxygenation the two copper(I) atoms at the active site are oxidized to copper(II) and the dioxygen molecules are reduced to peroxide, O²⁻
₂.

Chlorocruorin (as the larger carrier erythrocruorin) is an oxygen-binding hemeprotein present in the blood plasma of many annelids, particularly certain marine polychaetes.

Cytochromes

Oxidation and reduction reactions are not common in organic chemistry as few organic molecules can act as oxidizing or reducing agents. Iron(II), on the other hand, can easily be oxidized to iron(III). This functionality is used in cytochromes, which function as electron-transfer vectors. The presence of the metal ion allows metalloenzymes to perform functions such as redox reactions that cannot easily be performed by the limited set of functional groups found in amino acids. The iron atom in most cytochromes is contained in a heme group. The differences between those cytochromes lies in the different side-chains. For instance cytochrome a has a heme a prosthetic group and cytochrome b has a heme b prosthetic group. These differences result in different Fe²⁺/Fe³⁺ redox potentials such that various cytochromes are involved in the mitochondrial electron transport chain.

Cytochrome P450 enzymes perform the function of inserting an oxygen atom into a C−H bond, an oxidation reaction.

Rubredoxin active site.

Rubredoxin

Rubredoxin is an electron-carrier found in sulfur-metabolizing bacteria and archaea. The active site contains an iron ion coordinated by the sulfur atoms of four cysteine residues forming an almost regular tetrahedron. Rubredoxins perform one-electron transfer processes. The oxidation state of the iron atom changes between the +2 and +3 states. In both oxidation states the metal is high spin, which helps to minimize structural changes.

Plastocyanin

The copper site in plastocyanin

 

Plastocyanin is one of the family of blue copper proteins that are involved in electron transfer reactions. The copper-binding site is described as distorted trigonal pyramidal. The trigonal plane of the pyramidal base is composed of two nitrogen atoms (N₁ and N₂) from separate histidines and a sulfur (S₁) from a cysteine. Sulfur (S₂) from an axial methionine forms the apex. The distortion occurs in the bond lengths between the copper and sulfur ligands. The Cu−S₁ contact is shorter (207 pm) than Cu−S₂ (282 pm). The elongated Cu−S₂ bonding destabilizes the Cu(II) form and increases the redox potential of the protein. The blue color (597 nm peak absorption) is due to the Cu−S₁ bond where S(pπ) to Cu(d_x²−y²) charge transfer occurs.

In the reduced form of plastocyanin, His-87 will become protonated with a pK_a of 4.4. Protonation prevents it acting as a ligand and the copper site geometry becomes trigonal planar.

Metal-ion storage and transfer

Iron

Iron is stored as iron(III) in ferritin. The exact nature of the binding site has not yet been determined. The iron appears to be present as a hydrolysis product such as FeO(OH). Iron is transported by transferrin whose binding site consists of two tyrosines, one aspartic acid and one histidine. The human body has no mechanism for iron excretion. This can lead to iron overload problems in patients treated with blood transfusions, as, for instance, with β-thalassemia. Iron is actually excreted in urine and is also concentrated in bile which is excreted in feces.

Copper

Ceruloplasmin is the major copper-carrying protein in the blood. Ceruloplasmin exhibits oxidase activity, which is associated with possible oxidation of Fe(II) into Fe(III), therefore assisting in its transport in the blood plasma in association with transferrin, which can carry iron only in the Fe(III) state.

Calcium

Osteopontin is involved in mineralization in the extracellular matrices of bones and teeth.

Metalloenzymes

Metalloenzymes all have one feature in common, namely that the metal ion is bound to the protein with one labile coordination site. As with all enzymes, the shape of the active site is crucial. The metal ion is usually located in a pocket whose shape fits the substrate. The metal ion catalyzes reactions that are difficult to achieve in organic chemistry.

Carbonic anhydrase

Active site of carbonic anhydrase. The three coordinating histidine residues are shown in green, hydroxide in red and white, and the zinc in gray.

 

In aqueous solution, carbon dioxide forms carbonic acid

CO₂ + H₂O ⇌ H₂CO₃

This reaction is very slow in the absence of a catalyst, but quite fast in the presence of the hydroxide ion

CO₂ + OH⁻ ⇌ HCO⁻
₃

A reaction similar to this is almost instantaneous with carbonic anhydrase. The structure of the active site in carbonic anhydrases is well known from a number of crystal structures. It consists of a zinc ion coordinated by three imidazole nitrogen atoms from three histidine units. The fourth coordination site is occupied by a water molecule. The coordination sphere of the zinc ion is approximately tetrahedral. The positively-charged zinc ion polarizes the coordinated water molecule, and nucleophilic attack by the negatively-charged hydroxide portion on carbon dioxide (carbonic anhydride) proceeds rapidly. The catalytic cycle produces the bicarbonate ion and the hydrogen ion as the equilibrium

H₂CO₃ ⇌ HCO⁻
₃ + H⁺

favours dissociation of carbonic acid at biological pH values.

Vitamin B₁₂-dependent enzymes

The cobalt-containing Vitamin B₁₂ (also known as cobalamin) catalyzes the transfer of methyl (−CH₃) groups between two molecules, which involves the breaking of C−C bonds, a process that is energetically expensive in organic reactions. The metal ion lowers the activation energy for the process by forming a transient Co−CH₃ bond. The structure of the coenzyme was famously determined by Dorothy Hodgkin and co-workers, for which she received a Nobel Prize in Chemistry. It consists of a cobalt(II) ion coordinated to four nitrogen atoms of a corrin ring and a fifth nitrogen atom from an imidazole group. In the resting state there is a Co−C sigma bond with the 5′ carbon atom of adenosine. This is a naturally occurring organometallic compound, which explains its function in trans-methylation reactions, such as the reaction carried out by methionine synthase.

Nitrogenase (nitrogen fixation)

The fixation of atmospheric nitrogen is a very energy-intensive process, as it involves breaking the very stable triple bond between the nitrogen atoms. The enzyme nitrogenase is one of the few enzymes that can catalyze the process. The enzyme occurs in Rhizobium bacteria. There are three components to its action: a molybdenum atom at the active site, iron–sulfur clusters that are involved in transporting the electrons needed to reduce the nitrogen, and an abundant energy source in the form of magnesium ATP. This last is provided by a symbiotic relationship between the bacteria and a host plant, often a legume. The relationship is symbiotic because the plant supplies the energy by photosynthesis and benefits by obtaining the fixed nitrogen. The reaction may be written symbolically as

N₂ + 16 MgATP + 8 e⁻ → 2 NH₃ + 16 MgADP +16 P_i + H₂

where P_i stands for inorganic phosphate. The precise structure of the active site has been difficult to determine. It appears to contain a MoFe₇S₈ cluster that is able to bind the dinitrogen molecule and, presumably, enable the reduction process to begin. The electrons are transported by the associated "P" cluster, which contains two cubical Fe₄S₄ clusters joined by sulfur bridges.

Superoxide dismutase

Structure of a human superoxide dismutase 2 tetramer

 

The superoxide ion, O⁻
₂ is generated in biological systems by reduction of molecular oxygen. It has an unpaired electron, so it behaves as a free radical. It is a powerful oxidizing agent. These properties render the superoxide ion very toxic and are deployed to advantage by phagocytes to kill invading microorganisms. Otherwise, the superoxide ion must be destroyed before it does unwanted damage in a cell. The superoxide dismutase enzymes perform this function very efficiently.

The formal oxidation state of the oxygen atoms is −​¹⁄₂. In solutions at neutral pH, the superoxide ion disproportionates to molecular oxygen and hydrogen peroxide.

2 O⁻
₂ + 2 H⁺ → O₂ + H₂O₂

In biology this type of reaction is called a dismutation reaction. It involves both oxidation and reduction of superoxide ions. The superoxide dismutase (SOD) group of enzymes increase the rate of reaction to near the diffusion-limited rate. The key to the action of these enzymes is a metal ion with variable oxidation state that can act either as an oxidizing agent or as a reducing agent.

Oxidation: M⁽ⁿ⁺¹⁾⁺ + O⁻
₂ → Mⁿ⁺ + O₂

Reduction: Mⁿ⁺ + O⁻
₂ + 2 H⁺ → M⁽ⁿ⁺¹⁾⁺ + H₂O₂.

In human SOD the active metal is copper, as Cu(II) or Cu(I), coordinated tetrahedrally by four histidine residues. This enzyme also contains zinc ions for stabilization and is activated by copper chaperone for superoxide dismutase (CCS). Other isozymes may contain iron, manganese or nickel. Ni-SOD is particularly interesting as it involves nickel(III), an unusual oxidation state for this element. The active site nickel geometry cycles from square planar Ni(II), with thiolate (Cys₂ and Cys₆) and backbone nitrogen (His₁ and Cys₂) ligands, to square pyramidal Ni(III) with an added axial His₁ side chain ligand.

Chlorophyll-containing proteins

Hemoglobin (left) and chlorophyll (right), two extremely different molecules when it comes to function, are quite similar when it comes to its atomic shape. There are only three major structural differences; a magnesium atom (Mg) in chlorophyll, as opposed to iron (Fe) in hemoglobin. Additionally, chlorophyll has an extended isoprenoid tail and an additional aliphatic cyclic structure off the macrocycle.

 

Chlorophyll plays a crucial role in photosynthesis. It contains a magnesium enclosed in a chlorin ring. However, the magnesium ion is not directly involved in the photosynthetic function and can be replaced by other divalent ions with little loss of activity. Rather, the photon is absorbed by the chlorin ring, whose electronic structure is well-adapted for this purpose. 

Initially, the absorption of a photon causes an electron to be excited into a singlet state of the Q band. The excited state undergoes an intersystem crossing from the singlet state to a triplet state in which there are two electrons with parallel spin. This species is, in effect, a free radical, and is very reactive and allows an electron to be transferred to acceptors that are adjacent to the chlorophyll in the chloroplast. In the process chlorophyll is oxidized. Later in the photosynthetic cycle, chlorophyll is reduced back again. This reduction ultimately draws electrons from water, yielding molecular oxygen as a final oxidation product.

Hydrogenase

Hydrogenases are subclassified into three different types based on the active site metal content: iron–iron hydrogenase, nickel–iron hydrogenase, and iron hydrogenase. All hydrogenases catalyze reversible H₂ uptake, but while the [FeFe] and [NiFe] hydrogenases are true redox catalysts, driving H₂ oxidation and H⁺ reduction

H₂ ⇌ 2 H⁺ + 2 e⁻

the [Fe] hydrogenases catalyze the reversible heterolytic cleavage of H₂.

H₂ ⇌ H⁺ + H⁻

The active site structures of the three types of hydrogenase enzymes.

Ribozyme and deoxyribozyme

Since discovery of ribozymes by Thomas Cech and Sidney Altman in the early 1980s, ribozymes have been shown to be a distinct class of metalloenzymes. Many ribozymes require metal ions in their active sites for chemical catalysis; hence they are called metalloenzymes. Additionally, metal ions are essential for structural stabilization of ribozymes. Group I intron is the most studied ribozyme which has three metals participating in catalysis. Other known ribozymes include group II intron, RNase P, and several small viral ribozymes (such as hammerhead, hairpin, HDV, and VS) and the large subunit of ribosomes. Recently, four new classes of ribozymes have been discovered (named twister, twister sister, pistol and hatchet) which are all self-cleaving ribozymes.

Deoxyribozymes, also called DNAzymes or catalytic DNA, are artificial catalytic DNA molecules that were first produced in 1994 and gained a rapid increase of interest since then. Almost all DNAzymes require metal ions in order to function; thus they are classified as metalloenzymes. Although ribozymes mostly catalyze cleavage of RNA substrates, a variety of reactions can be catalyzed by DNAzymes including RNA/DNA cleavage, RNA/DNA ligation, amino acid phosphorylation and dephosphorylation, and carbon–carbon bond formation. Yet, DNAzymes that catalyze RNA cleavage reaction are the most extensively explored ones. 10-23 DNAzyme, discovered in 1997, is one of the most studied catalytic DNAs with clinical applications as a therapeutic agent. Several metal-specific DNAzymes have been reported including the GR-5 DNAzyme (lead-specific), the CA1-3 DNAzymes (copper-specific), the 39E DNAzyme (uranyl-specific) and the NaA43 DNAzyme (sodium-specific).

Signal-transduction metalloproteins

Calmodulin

EF-hand motif

 

Calmodulin is an example of a signal-transduction protein. It is a small protein that contains four EF-hand motifs, each of which is able to bind a Ca²⁺ ion. 

In an EF-hand loop the calcium ion is coordinated in a pentagonal bipyramidal configuration. Six glutamic acid and aspartic acid residues involved in the binding are in positions 1, 3, 5, 7 and 9 of the polypeptide chain. At position 12, there is a glutamate or aspartate ligand that behaves as a (bidentate ligand), providing two oxygen atoms. The ninth residue in the loop is necessarily glycine due to the conformational requirements of the backbone. The coordination sphere of the calcium ion contains only carboxylate oxygen atoms and no nitrogen atoms. This is consistent with the hard nature of the calcium ion.

The protein has two approximately symmetrical domains, separated by a flexible "hinge" region. Binding of calcium causes a conformational change to occur in the protein. Calmodulin participates in an intracellular signaling system by acting as a diffusible second messenger to the initial stimuli.

Troponin

In both cardiac and skeletal muscles, muscular force production is controlled primarily by changes in the intracellular calcium concentration. In general, when calcium rises, the muscles contract and, when calcium falls, the muscles relax. Troponin, along with actin and tropomyosin, is the protein complex to which calcium binds to trigger the production of muscular force.

Transcription factors

Zinc finger. The zinc ion (green) is coordinated by two histidine residues and two cysteine residues.

 

Many transcription factors contain a structure known as a zinc finger, this is a structural module where a region of protein folds around a zinc ion. The zinc does not directly contact the DNA that these proteins bind to. Instead, the cofactor is essential for the stability of the tightly-folded protein chain. In these proteins, the zinc ion is usually coordinated by pairs of cysteine and histidine side-chains.

Other metalloenzymes

There are two types of carbon monoxide dehydrogenase: one contains iron and molybdenum, the other contains iron and nickel. Parallels and differences in catalytic strategies have been reviewed.

Pb²⁺ (lead) can replace Ca²⁺ (calcium) as, for example, with calmodulin or Zn²⁺ (zinc) as with metallocarboxypeptidases.

 

Some other metalloenzymes are given in the following table, according to the metal involved.

 

Ion	Examples of enzymes containing this ion
Magnesium	Glucose 6-phosphatase Hexokinase DNA polymerase
Vanadium	vanabins
Manganese	Arginase Oxygen-evolving complex
Iron	Catalase Hydrogenase IRE-BP Aconitase
Cobalt	Nitrile hydratase Methionyl aminopeptidase Methylmalonyl-CoA mutase Isobutyryl-CoA mutase
Nickel	Urease Hydrogenase Methyl-coenzyme M reductase (MCR)
Copper	Cytochrome oxidase Laccase Nitrous-oxide reductase Nitrite reductase
Zinc	Alcohol dehydrogenase Carboxypeptidase Aminopeptidase Beta amyloid
Cadmium	Metallothionein Thiolate proteins
Molybdenum	Nitrate reductase Sulfite oxidase Xanthine oxidase DMSO reductase
Tungsten	Acetylene hydratase
various	Metallothionein Phosphatase

Gel electrophoresis of proteins

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Gel_electrophoresis_of_proteins

 

Proteins separated by SDS-PAGE, Coomassie Brilliant Blue staining

 

Protein electrophoresis is a method for analysing the proteins in a fluid or an extract. The electrophoresis may be performed with a small volume of sample in a number of alternative ways with or without a supporting medium: SDS polyacrylamide gel electrophoresis (in short: gel electrophoresis, PAGE, or SDS-electrophoresis), free-flow electrophoresis, electrofocusing, isotachophoresis, affinity electrophoresis, immunoelectrophoresis, counterelectrophoresis, and capillary electrophoresis. Each method has many variations with individual advantages and limitations. Gel electrophoresis is often performed in combination with electroblotting immunoblotting to give additional information about a specific protein. Because of practical limitations, protein electrophoresis is generally not suited as a preparative method.

Denaturing gel methods

SDS-PAGE

SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, describes a collection of related techniques to separate proteins according to their electrophoretic mobility (a function of the molecular weight of a polypeptide chain) while in the denatured (unfolded) state. In most proteins, the binding of SDS to the polypeptide chain imparts an even distribution of charge per unit mass, thereby resulting in a fractionation by approximate size during electrophoresis. 

SDS is a strong detergent agent used to denature native proteins to unfolded, individual polypeptides. When a protein mixture is heated to 100 °C in presence of SDS, the detergent wraps around the polypeptide backbone. In this process, the intrinsic charges of polypeptides becomes negligible when compared to the negative charges contributed by SDS. Thus polypeptides after treatment become rod-like structures possessing a uniform charge density, that is same net negative charge per unit length. The electrophoretic mobilities of these proteins will be a linear function of the logarithms of their molecular weights.

Native gel methods

Native gels, also known as non-denaturing gels, analyze proteins that are still in their folded state. Thus, the electrophoretic mobility depends not only on the charge-to-mass ratio, but also on the physical shape and size of the protein.

Blue native PAGE

BN-PAGE is a native PAGE technique, where the Coomassie Brilliant Blue dye provides the necessary charges to the protein complexes for the electrophoretic separation. The disadvantage of Coomassie is that in binding to proteins it can act like a detergent causing complexes to dissociate. Another drawback is the potential quenching of chemoluminescence (e.g. in subsequent western blot detection or activity assays) or fluorescence of proteins with prosthetic groups (e.g. heme or chlorophyll) or labelled with fluorescent dyes.

Clear native PAGE

CN-PAGE (commonly referred to as Native PAGE) separates acidic water-soluble and membrane proteins in a polyacrylamide gradient gel. It uses no charged dye so the electrophoretic mobility of proteins in CN-PAGE (in contrast to the charge shift technique BN-PAGE) is related to the intrinsic charge of the proteins. The migration distance depends on the protein charge, its size and the pore size of the gel. In many cases this method has lower resolution than BN-PAGE, but CN-PAGE offers advantages whenever Coomassie dye would interfere with further analytical techniques, for example it has been described as a very efficient microscale separation technique for FRET analyses. Also CN-PAGE is milder than BN-PAGE so it can retain labile supramolecular assemblies of membrane protein complexes that are dissociated under the conditions of BN-PAGE.

Quantitative native PAGE

The folded protein complexes of interest separate cleanly and predictably due to the specific properties of the polyacrylamide gel. The separated proteins are continuously eluted into a physiological eluent and transported to a fraction collector. In four to five PAGE fractions each the metal cofactors can be identified and absolutely quantified by high-resolution ICP-MS. The respective structures of the isolated metalloproteins can be determined by solution NMR spectroscopy.

Buffer systems

Postulated migration of proteins in a Laemmli gel system A: Stacking gel, B: Resolving gel, o: sample application c: discontinuities in the buffer and electrophoretic matrix

 

Most protein separations are performed using a "discontinuous" (or DISC) buffer system that significantly enhances the sharpness of the bands within the gel. During electrophoresis in a discontinuous gel system, an ion gradient is formed in the early stage of electrophoresis that causes all of the proteins to focus into a single sharp band. The formation of the ion gradient is achieved by choosing a pH value at which the ions of the buffer are only moderately charged compared to the SDS-coated proteins. These conditions provide an environment in which Kohlrausch's reactions determine the molar conductivity. As a result, SDS-coated proteins are concentrated to several fold in a thin zone of the order of 19 μm within a few minutes. At this stage all proteins migrate at the same migration speed by isotachophoresis. This occurs in a region of the gel that has larger pores so that the gel matrix does not retard the migration during the focusing or "stacking" event. Separation of the proteins by size is achieved in the lower, "resolving" region of the gel. The resolving gel typically has a much smaller pore size, which leads to a sieving effect that now determines the electrophoretic mobility of the proteins. At the same time, the separating part of the gel also has a pH value in which the buffer ions on average carry a greater charge, causing them to "outrun" the SDS-covered proteins and eliminate the ion gradient and thereby the stacking effect.

A very widespread discontinuous buffer system is the tris-glycine or "Laemmli" system that stacks at a pH of 6.8 and resolves at a pH of ~8.3-9.0. A drawback of this system is that these pH values may promote disulfide bond formation between cysteine residues in the proteins because the pKa of cysteine ranges from 8-9 and because reducing agent present in the loading buffer doesn't co-migrate with the proteins. Recent advances in buffering technology alleviate this problem by resolving the proteins at a pH well below the pKa of cysteine (e.g., bis-tris, pH 6.5) and include reducing agents (e.g. sodium bisulfite) that move into the gel ahead of the proteins to maintain a reducing environment. An additional benefit of using buffers with lower pH values is that the acrylamide gel is more stable at lower pH values, so the gels can be stored for long periods of time before use.

SDS gradient gel electrophoresis of proteins

As voltage is applied, the anions (and negatively charged sample molecules) migrate toward the positive electrode (anode) in the lower chamber, the leading ion is Cl⁻ ( high mobility and high concentration); glycinate is the trailing ion (low mobility and low concentration). SDS-protein particles do not migrate freely at the border between the Cl⁻ of the gel buffer and the Gly⁻ of the cathode buffer. Friedrich Kohlrausch found that Ohm's law also applies to dissolved electrolytes. Because of the voltage drop between the Cl⁻ and Glycine-buffers, proteins are compressed (stacked) into micrometer thin layers. The boundary moves through a pore gradient and the protein stack gradually disperses due to a frictional resistance increase of the gel matrix. Stacking and unstacking occurs continuously in the gradient gel, for every protein at a different position. For a complete protein unstacking the polyacrylamide-gel concentration must exceed 16% T. The two-gel system of "Laemmli" is a simple gradient gel. The pH discontinuity of the buffers is of no significance for the separation quality, and a "stacking-gel" with a different pH is not needed.

Visualization

The most popular protein stain is Coomassie Brilliant Blue. It is an anionic dye, which non-specifically binds to proteins. Proteins in the gel are fixed by acetic acid and simultaneously stained. The excess dye incorporated into the gel can be removed by destaining with the same solution without the dye. The proteins are detected as blue bands on a clear background.

When more sensitive method than staining by Coomassie is needed silver staining is usually used. Silver staining is a sensitive procedure to detect trace amounts of proteins in gels, but can also visualize nucleic acid or polysaccharides.

Visualization methods without using a dye such as Coomassie and silver are available on the market. For example Bio-Rad Laboratories markets ”stain-free” gels for SDS-PAGE gel electrophoresis.

Similarly as in nucleic acid gel electrophoresis, tracking dye is often used. Anionic dyes of a known electrophoretic mobility are usually included in the sample buffer. A very common tracking dye is Bromophenol blue. This dye is coloured at alkali and neutral pH and is a small negatively charged molecule that moves towards the anode. Being a highly mobile molecule it moves ahead of most proteins.

Medical applications

Schematic representation of a protein electrophoresis gel.

 

Serum protein electrophoresis showing a paraprotein (peak in the gamma zone) in a patient with multiple myeloma.

 

In medicine, protein electrophoresis is a method of analysing the proteins mainly in blood serum. Before the widespread use of gel electrophoresis, protein electrophoresis was performed as free-flow electrophoresis (on paper) or as immunoelectrophoresis. 

Traditionally, two classes of blood proteins are considered: serum albumin and globulin. They are generally equal in proportion, but albumin as a molecule is much smaller and lightly, negatively-charged, leading to an accumulation of albumin on the electrophoretic gel. A small band before albumin represents transthyretin (also named prealbumin). Some forms of medication or body chemicals can cause their own band, but it usually is small. Abnormal bands (spikes) are seen in monoclonal gammopathy of undetermined significance and multiple myeloma, and are useful in the diagnosis of these conditions. 

The globulins are classified by their banding pattern (with their main representatives):

• The alpha (α) band consists of two parts, 1 and 2:
  • α₁ - α₁-antitrypsin, α₁-acid glycoprotein.
  • α₂ - haptoglobin, α₂-macroglobulin, α₂-antiplasmin, ceruloplasmin.
• The beta (β) band - transferrin, LDL, complement
• The gamma (γ) band - immunoglobulin (IgA, IgD, IgE, IgG and IgM). Paraproteins (in multiple myeloma) usually appear in this band.

Normal present medical procedure involves determination of numerous proteins in plasma including hormones and enzymes, some of them also determined by electrophoresis. However, gel electrophoresis is mainly a research tool, also when the subject is blood proteins.

What are common treatments for phenylketonuria (PKU)?

 https://www.nichd.nih.gov/health/topics/pku/conditioninfo/treatments

There is no cure for PKU, but treatment can prevent intellectual disabilities and other health problems.¹ A person with PKU should receive treatment at a medical center that specializes in the disorder. (Visit the Resources and Publications section for ways to locate a center.)

The PKU Diet

People with PKU need to follow a diet that limits foods with phenylalanine. The diet should be followed carefully and be started as soon after birth as possible. In the past, experts believed that it was safe for people to stop following the diet as they got older. However, they now recommend that people with PKU stay on the diet throughout their lives for better physical and mental health.

It is especially important for a pregnant woman with PKU to strictly follow the low-phenylalanine diet throughout her pregnancy to ensure the healthy development of her infant.

People with PKU need to avoid various high-protein foods, including:

• Milk and cheese
• Eggs
• Nuts
• Soybeans
• Beans
• Chicken, beef, or pork
• Fish
• Peas
• Beer

People with PKU also need to avoid the sweetener aspartame, which is in some foods, drinks, medications, and vitamins. Aspartame releases phenylalanine when it is digested, so it raises the level of phenylalanine in a person's blood.

Often, people with PKU also have to limit their intake of lower-protein foods, such as certain fruits and vegetables. However, a PKU diet can include low-protein noodles and other special products.

The amount of phenylalanine that is safe to consume differs for each person. Therefore, a person with PKU needs to work with a health care professional to develop an individualized diet. The goal is to eat only the amount of phenylalanine necessary for healthy growth and body processes but not any extra. Frequent blood tests and doctor visits are necessary to help determine how well the diet is working. Some relaxation of the diet may be possible as a child gets older, but the recommendation today is lifelong adherence to the diet. Following the diet is especially important during pregnancy.

However, the PKU diet can be very challenging. Getting support from friends and family or a support group can help. Sticking with the diet ensures better functioning and improved overall health.

A PKU Formula

People who follow the PKU diet will not get enough essential nutrients from food. Therefore, they must drink a special formula.

A newborn who is diagnosed with PKU should receive special infant formula. The formula may be mixed with a small amount of breast milk or regular infant formula to make sure the child gets enough phenylalanine for normal development but not enough to cause harm.

Older children and adults receive a different formula to meet their nutritional needs. This formula should be consumed every day throughout a person's life.

In addition to the formula, health care professionals may recommend other supplements. For example, fish oil may be recommended to help with fine motor coordination and other aspects of development.¹

Medication for PKU

The U.S. Food and Drug Administration (FDA) has approved the drug sapropterin dihydrochloride (Kuvan®) for the treatment of PKU. Kuvan® is a form of BH4, which is a substance in the body that helps break down phenylalanine. However, having too little BH4 is only one reason a person may not break down phenylalanine. Therefore, Kuvan® only helps some people reduce the phenylalanine in their blood. Even if the medication helps, it will not decrease the phenylalanine to the desired amount and must be used together with the PKU diet.

When the FDA approved Kuvan®, the agency suggested that research on the medication continue to determine its long-term safety and effectiveness.

Other Treatments for PKU

NICHD-supported researchers and other scientists are exploring additional treatments for PKU. These treatments include large neutral amino acid supplementation, which may help prevent phenylalanine from entering the brain, and enzyme replacement therapy, which uses a substance similar to the enzyme that usually breaks down phenylalanine. Researchers are also investigating the possibility of using gene therapy, which involves injecting new genes to break down phenylalanine. That would result in the breakdown of phenylalanine and decreased blood phenylalanine levels.