QPNC-PAGE: A Milestone for Protein Electrophoresis

Research Paper (postgraduate), 2012
59 Pages

Free online reading



Chapter I: Preparative native continuous polyacrylamide gel electrophoresis (PNC-PAGE): an efficient method for isolating cadmium cofactors in biological systems

Chapter II: Metalloprotein

Chapter III: Protein folding


Chapter V: Inductively coupled plasma mass spectrometry

Chapter VI: Nuclear magnetic resonance spectroscopy

Chapter VII: Isolation of acidic, basic and neutral metalloproteins by QPNC- PAGE

Chapter VIII: Herbal drugs in mirror of Alzheimer’s disease

Chapter IX: Phytochemical approach and bioanalytical strategy to develop chaperone-based medications

Chapter X: Phytopharmaceuticals in the therapy of younger Alzheimer patients

Chapter XI: Quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE)

Chapter XII: QPNC-PAGE (German)


Article sources


Metal cofactors (e.g. Fe, Cu, Zn) play an important role in the etiology of protein-misfolding diseases (e.g. Alzheimer’s disease (AD), Amyotrophic Lateral Sclerosis (ALS) or prion diseases) concerning biometal metabolism, because several metal ions are binding partners of organic ligands (e.g. proteins) in the cytoplasm. For example, pathogenic metal proteins may be causative for several neurodegenerative diseases. Physiological metal proteins (e.g. copper chaperone for superoxide dismutase (CCS) and superoxide dismutase (SOD)) contribute to the homeostatic control of metal ions and thus protect the cells against metal-based oxidative stress. If these intracellular processes do not function properly oxidative stress and misfolding of normal amyloid beta peptide (AP) and amyloid beta precursor protein (ApPP) molecules, may lead to accumulation and aggregation of proteins in the brain of AD patients.

In literature it is reported that protein-misfolding processes are involved in about 50 % of all diseases. Furthermore, researchers estimate that about 25 to 50 % of all known proteins require metal ion cofactors to carry out their functions. Therefore, it is crucial to analyse the structure-function relationships of metal proteins and to investigate the biometal metabolism in clinical samples of probands and patients.

For a deeper understanding and for developing effective medications in the treatment of these protein-misfolding diseases, a novel interdisciplinary approach is presented in this book. By high-resolution methods such as solution nuclear magnetic resonance (NMR) spectroscopy, inductively coupled plasma mass spectrometry (ICP-MS) and quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE), the structure of active (native) and inactive (denatured) metal proteins and protein isomers can be quantified.

This procedure allows to obtain fundamental knowledge on different conformational states of essential metal proteins (e.g. CCS, SOD) in biofluids (e.g. blood or cerebrospinal fluid). Copper chaperones may serve as potential biomarkers in the diagnosis and therapy of protein- misfolding (conformational) diseases. It is indicated that copper chaperones from medicinal plants may be promising agents for treating the causes of AD.

Quantitative preparative native continuous polyacrylamide gel electrophoresis is proposed to be key method for isolating native metalloproteins and for resolving properly and improperly folded metal cofactor-containing proteins in complex protein mixtures. By one-dimensional gel electrophoresis high concentrations of highly purified proteins can be isolated in fractions. As solution protein NMR spectroscopy demands high protein concentrations and high purity, this non-invasive method can be combined with QPNC-PAGE. Physiological concentrations of different metal cofactors can be determined in PAGE fractions via ICP-MS beforehand.

In these analytical processes proteins are separated by isoelectric point based on the use of a commercial preparative-electrophoresis device as described in various articles and protocols. This book may provide an interesting overview of QPNC-PAGE including theoretical as well as important practical aspects of modem protein electrophoresis. At the end of each chapter the reader may find several cross references that may provide valuable information for further reading.

Vol. 37, No. 4, pp. 657-665, 2004


Preparative Native Continuous
Polyacrylamide Gel Electrophoresis
(PNC-PAGE): An Efficient Method for
Isolating Cadmium Cofactors in
Biological Systems

Bernd Kastenholz[1]

Research Centre Juelich, Institute for Phytosphere Research (ICG-IH),
Juelich, Germany


A new electrophoretic method [preparative native continuous polyacryl­amide gel electrophoresis (PNC-PAGE)] was developed to isolate cadmium cofactor-containing proteins in biological systems. For this purpose Arabidopsis cytosol was subjected to gel permeation chromatography (GPC) and high molecular mass cadmium proteins (MW 200 kDa) in a GPC fraction of this plant were isolated by PNC- PAGE. Furthermore plant cytosol was directly separated by this method. The cadmium concentrations in all GPC and PAGE fractions were determined by GF-AAS using matrix modifier. As electrophoresis buffer of the PAGE method a Tris-HCl buffer (20mM Tris, ImM NaN3, pH 10.00) was used. The gel (degree of polymerization of the polyacrylamide: 4%; gel length: 4 cm) was polymerized for 69 hr. By these procedures it was revealed that the high molecular mass cadmium proteins of Arabidopsis can be detected in quantitative amounts by using PNC-PAGE. It could be shown that the chemical structure of the cadmium proteins did not change under these PAGE conditions. PNC- PAGE is supposed to be an efficient method for isolating other metal cofactors such as Zn, Cu, Ni, Pd, Co, Fe, Mn, Pt, Cr, and Mo and might be suitable for subsequent structural determinations of metalloproteins present in PAGE fractions.

Key Words: Cadmium cofactors; Arabidopsis; PNC-PAGE.


Protein and enzyme research play an important role in today’s scientific discussions at questions of the relationships between structure and function of proteins and enzymes in biological systems.1-11 About one-third of all known proteins contain metal cofactors and the vast majority of these function as essential metalloenzymes.121 Cofactors are required to perform the biological activity of many proteins131 and the presence of a cofactor often stabilizes native protein molecules.141 The specific characteristics of metal ions, like e.g., Fe, Ni, Cu, Mn, or Mo in particular, as cofactors of proteins and enzymes are of biochemical relevance, because they play a key role in enzymatic catalytic process.151 The qualitative determinations of these cofactors and the analysis of the metal cofactor/protein stoichiometries represent an important step when metal enzymes and metalloproteins are classified.161

Because of difficult and time consuming isolations and cleaning proce­dures enzyme samples are often available in small amounts only.151 For the elucidation of the structures of certain metal proteins present in organisms larger quantities of these compounds have to be isolated in biological matrices by applying analytical methods such as extraction, gel permeation chromatography (GPC) and preparative native continuous polyacrylamide gel electrophoresis (PNC-PAGE).171 By these analytical processes proteins and enzymes must not be denatured because otherwise the function of a certain biomolecule can not be assessed. For those reasons preparative and native analytical methods for separating proteins and enzymes in biological samples are developed.

For example, an off-line coupling of GPC and PNC-PAGE was used to isolate high molecular mass cadmium proteins in different vegetable foodstuffs.171 Plant cytosols of these matrices were subjected to GPC and the cadmium elution maxima were detected in the range of about 200 kDa. The cadmium proteins with molecular mass 200 kDa were further separated by a PNC-PAGE method. The results obtained from the electrophoretic separation of the high molecular mass cadmium proteins had to be regarded as semiquantitative because the cadmium concentrations detected in the respective PAGE fractions were very small and not reproducible.1-7'1

Therefore, a new preparative native continuous PAGE method was developed for the quantitative analysis of high molecular mass cadmium proteins in plants. Exemplary, this analytical system was used for separating protein molecules in cytosol samples of the model plant Arabidopsis thaliana.

Gel Permeation Chromatography

Plant cytosol of Arabidopsis was separated by gel permeation chromatography. All parameters and experimental conditions of the GPC are pointed out in a study of Alt, Weber et al.[8! A Sephacryl S-400 Superfine (HR) column was calibrated with 4 high molecular weight globular proteins (aldolase 158 kDa, catalase 232 kDa, ferritin 449 kDa, and thyroglobuline 669 kDa, high molecular weight gel filtration calibration kit, Amersham Pharmacia). Gel permeation chromatography system (Amersham Pharmacia, Freiburg, Germany): Peristaltic pump (LKB pump P-1), one channel UV monitor (Uvicord S H, Pharmacia), automatic fraction collector (Recorder REC 102, LKB). Parameters: Column length: 700 mm; column diameter: 30mm; buffer: Tris-HCl 20mmol/L, NaN3 lmmol/L, pH 8.00; flow rate: 13.8mL/hr; gel volume: 500 mL; fraction volume: 8.6 mL; detection wavelength: 254 nm.t8] Temperature of the separation system: 4°C; sample volume of the cytosol sample: 5 mL. The size-range of the GPC method used was about 20-8000 kDa for globular proteins. The cadmium concentrations of the resulted GPC fractions were analyzed by a GF-AAS method.

Electrophoretic Separation

Samples of Arabidopsis cytosol were either directly separated by a preparative native continuous PAGE method or first chromatographed on Sephacryl S-400 HR. Native method means that the metal cofactor-containing proteins to be separated must not be denatured nor that a metal cofactor is dissociated during the electrophoretic run. Continuous method refers to the electrophoresis buffer used in PNC-PAGE. Only one type of buffer with the same pH and composition, a Tris-HCl-buffer (pH 10.00; 20mM Tris; 1 mM NaN3) is applied to the anode chamber, cathode chamber and gel column of the Model 491 Prep Cell from Bio Rad.[9] Peripheral tools are PowerPAC 1000 (5W constant, 8 hr), Model EP-1 Econo Pump (lmL/min, 5mL/ fraction, 80 fractions, 80 mL prerun volume, 480 mL total volume), Model 2110 (fraction collector), Model EM-1 Econo UV Monitor (AUFS 1.0; A = 254nm), Model 1327 Econo Recorder (100 mV; 6 cm/hr), Buffer Recirculation Pump (95 mL/min), gel column (28 mm inner diameter), all Bio Rad. Degree of polymerization of the polyacrylamide: 4%. Gel length: 4 cm.

To prepare 40 mL of a gel (4%) the following chemicals were used: 32 mL purest water, 4mL Tris-HCl stock solution (200mmol/L Tris, lOmmol/L NaN3, pH 10.00), 4mL acrylamide/bis stock solution (40%), 20 p,L TEMED, 200 pL APS. Before usage all chemicals were adapted to room temperature, APS solution (10%) and gels were renewed at each trial. The gel solution was loaded with 3 mL 2-propanol and after a polymerization of 60 min the gel surface was rinsed with 8x4 mL electrophoresis buffer (pH 10.00; 20 mM Tris; 1 mM NaN3). Then the surface was covered with 4mL electrophoresis buffer (20mmol/L Tris, 1 mmol/L NaN3, pH 10.00).

For a total of 69 hr the gel was given time to be polymerized at room temperature before the electrophoretic prerun was started. As eluent a Tris- HCl buffer (20 mM Tris, 1 mM NaN3, pH 8.00) was used. The separation system was cooled in a refrigerator at 4°C. Before the electrophoretic separation started the PAGE system was equilibrated and stabilized for 80 min. After 75 min of the electrophoretic prerun a cooled mixture of the cytosol sample (2.7 mL) or a respective GPC fraction (2.7 mL) and glycerol (0.3 mL) were carefully layered under the upper electrophoresis buffer on the gel surface.

Cadmium Determination

Cadmium concentrations in the resulted GPC and PAGE fractions were determined by use of GF-AAS (Perkin Elmer SIMAA 6000; Software: AAWinLab 2.50, Auto-Sampler: PE AS 72). All fractions were analyzed for cadmium without digestion using Pd-Mg matrix modifier. Small graphite tubes with L’vov platform were applied. So-called “end cap tubes” were inserted in a transverse heated graphite atomizer of the SIMAA 6000. By this procedure a higher sensitivity of the detection method was achieved as compared to measurements with standard graphite tubes.

Parameters: Resonance line: 228.8 nm; sample volume: 40 p,L; modifier volume: 5 pL; Cd was analyzed in a linear mode with calculated intercept. Graphite furnace temperature program: Temperature (°C) (110; 130 drying);

(500 matrix separation); (1500 atomizing); (2450 cleaning); ramp time (s) (1; 15; 10; 0; 1); hold time (40; 40; 20; 5; 3); all necessary parameters were read in the computer program. Cadmium concentrations in each fraction were determined 3-fold.


An Arabidopsis cytosol sample was electrophoretically separated by PNC-PAGE. The UV absorption profile received showed two significant UV peaks in the range of PAGE fraction 21-25. Also in the elution range from fraction 6-20 a clear UV absorption was detected. The determination of cadmium in PAGE fractions 1-77 using GF-AAS revealed that there are cadmium concentrations up to about 2ppb Cd analyzed in fraction 9-20. Then a cadmium peak with a concentration maximum of about lOppb Cd in fraction 24 followed. The results for Cd are illustrated in Fig. 1. The total amount of Cd recovered in the PAGE fractions added up about 250 ng Cd.

The cytosol fraction of this plant contained 97 ppb Cd according to an absolute amount of 262 ng Cd present in the cytosol sample (2.7 mL) to be separated by PNC-PAGE. The ratio of the cadmium amount (250 ng) recovered in the PAGE fractions and the cadmium available in the respective plant cytosol sample (262 ng Cd) results in a cadmium recovery rate of about 95% after PNC-PAGE. Consequently, nearly the whole cadmium present in Arabidopsis cytosol is eluted after separation by a PAGE method. Therefore, the results for cadmium obtained by this method have to be regarded as quantitative.

illustration not visible in this excerpt

Figure 1. Cadmium elution profile after electrophoretic separation of Arabidopsis cytosol using PNC-PAGE. Determination of Cd in PAGE fractions in p,g/L was carried out by use of GF-AAS.

A PAGE fraction containing the highest cadmium concentration (fraction 24, Fig. 1) was further separated by a GPC method to determine the molecular mass of the cadmium species present in this PAGE fraction. As a result cadmium eluted in a single peak in the high molecular mass range with an elution maximum of about 200 kDa after GPC. This result means that the cadmium proteins of Arabidopsis are chemically stable when separated by PNC-PAGE. Furthermore quantitative amounts of the high molecular mass cadmium proteins present in Arabidopsis cytosol are eluted in a few fractions by PNC-PAGE.

A cytosol sample of Arabidopsis was subjected to GPC and cadmium was determined in the different GPC fractions. High and low molecular mass cadmium species eluted over a broad molecular mass range in GPC. The most important cadmium species with a very low UV absorption were detected to be high molecular mass cadmium proteins having a molecular mass of about 200 kDa. A sample (2.7 mL) of the cadmium protein fraction with the highest cadmium concentration was directly separated by PNC-PAGE.

The electropherogram of this PAGE trial showed an extreme low UV absorption around fraction 23 and 24. PAGE fractions 1 to 77 were analyzed for Cd using GF-AAS. As result in Fig. 2 it is presented that cadmium was eluted in a single peak with a maximum cadmium concentration of about 0. 5 ppb Cd detected in PAGE fraction 24. These results were reproduced. PAGE experiments revealed that the high molecular mass cadmium proteins of Arabidopsis present in a GPC fraction can be eluted in detectable amounts by PNC-PAGE. From the results obtained by the PAGE and GPC examinations it can be concluded that the chemical structure of the cadmium proteins of this plant did not change during the electrophoretic run.

This is an important result because data on the functions of metallo- proteins in biological organisms mainly depend on the structural determi­nation of the native forms of metal cofactor-containing proteins isolated by electrophoretic and chromatographic methods.


In this study a new PNC-PAGE method to separate cadmium cofactor- containing proteins in biological systems is presented. As example, high molecular mass cadmium proteins (MW » 200 kDa) were isolated by this method using Arabidopsis cytosol as sample. The cytosol sample was either directly separated by PNC-PAGE or subjected to GPC before applying this electrophoretic system. It was shown that PNC-PAGE is a very efficient method for separating high molecular mass cadmium proteins in plants. These compounds eluted in a single peak separated from the base line in the resulted electropherogram after the PAGE experiments.

illustration not visible in this excerpt

Figure 2. Cadmium elution profile after electrophoretic separation of a GPC fraction containing high molecular mass cadmium proteins (MW ~ 200 kDa) of Arabidopsis. Determination of Cd in PAGE fractions in /xg/L was carried out by use of GF-AAS.

It would be interesting to identify the isolated cadmium compounds and to elucidate the exact chemical structure of the high molecular mass cadmium proteins of the investigated plant. For this purpose MALDI-TOF-MS can be applied for an identification of the cadmium proteins present in the respective PAGE fractions. Also element species other than cadmium binding forms can be isolated by PNC-PAGE. For example, proteins containing metal cofactors such as Zn, Cu, Ni, Pd, Co, Fe, Mn, Pt, Cr, and Mo could be analyzed in PAGE fractions of plants using inductively coupled plasma mass spectrometry (ICP- MS) or total reflection x-ray fluorescence spectrometry (TXRF) as detection methods. Total reflection x-ray fluorescence spectrometry1105 is well suited to the quantitative determination of metals in proteins and enzymes.

There are different methods to determine the structures of cofactor- containing proteins in biological systems. For example, 3-D structures of protein cofactor complexes are elucidated by x-ray crystallography.5115 The availability of well ordered three-dimensional crystals is a prerequisite to obtain detailed knowledge on the structure of sized biological macromolecules detected by high resolution x-ray crystallography.112-1

In the future other biomatrices should be analyzed by PNC-PAGE. Metal proteins and metalloenzymes present in cytosol samples of human matrices and animal matrices as well as cytosols of microorganisms can be separated by GPC. Metal cofactor-containing proteins of these matrices which are eluted in the high molecular (> 10 kDa) or low molecular (< 10 kDa) mass range by this chromatographic method could be further isolated by PNC-PAGE. Using NMR spectroscopy as detection method it might be possible to elucidate the structures of high molecular and low molecular mass metal cofactor- containing proteins in PAGE fractions of biological samples.

NMR spectroscopy is used for three-dimensional structure determination of proteins and nucleic acids at atomic resolution but also structural, thermodynamic, and kinetic aspects of interactions between proteins and other components can be measured directly in solution applying this method. Solution NMR spectra have been recorded for structures with molecular weights up to 870,000 Da.[13J

In aqueous extracts of medicinal plants the binding forms of essential elements were analyzed because there is only little knowledge about the potential influence of metals on the pharmacological effects of natural drugs obtained from these organism. It was revealed that high amounts of metals Mn and Zn are present as low molecular weight species (<5000 Da) in the investigated plant extracts.114-1 For isolating pharmacologically active compounds in phytosystems PNC-PAGE is supposed to play an important role.


I am grateful to Prof. Dr. U. Schurr, Prof. Dr. K. Gunther, B. Muktiono, Dr. U. Katscher, Dr. M. von Sparr, Research Centre Jiilich, my wife Claudia and our little son Noah.


1. Finney, L:A.; O’Halloran, T.V. Transition metal speciation in the cell: insights from the chemistry of metal ion receptors. Science 2003, 300, 931-936.

2. Bartnikas, T.B.; Gitlin, J.D. How to make a metalloprotein. Nature Struct. Biol. 2001, 8, 733-734.

3. Pozdnyakova, I.; Wittung-Stafshede, P. Biological relevance of metal binding before protein folding. J. Am. Chem. Soc. 2001, 123, 10135-10136.

4. Wittung-Stafshede, P. Role of cofactors in protein folding. Acc. Chem. Res. 2002, 35, 201-208.

5. Mertens, M.; Rittmeyer, C.; Kolbesen, B.O. Evaluation of the protein concentration in enzymes via determination of sulfur by total reflection X-ray fluorescence spectrometry-limitations of the method. Spectro- chim. Acta B 2001, 56, 2157-2164.

6. Wittershagen, A.; Rostam-Khani, P.; Rittmeyer, C.; Bublak, D.; Stahl, G.; Riiteijans, H.; Kolbesen, B.O. Detektion von Metall-Cofaktoren in biologisch-organischen Systemen. Ein Vergleich zwischen ICP-OES und Totalreflexions-Rontgenfluoreszenzanalyse (TXRF). In CANAS ’95 Colloquium Analytische Atomspektroskopie\ Welz, B., Ed.; 1996; 697-700.

7. Gunther, K.; Ji, G.; Kastenholz, B. Characterization of high molecular weight cadmium species in contaminated vegetable food. Fresenius J. Anal. Chem. 2000, 368, 281-287.

8. Alt, F.; Weber, G.; Messerschmidt, J.; von Bohlen, A.; Kastenholz, B.; Guenther, K. Bonding states of palladium in phytosystems: first results for endive. Anal. Lett. 2002, 35, 1349-1359.

9. Bio Rad, Model 491 Prep Cell, Instruction Manual, 1-47.

10. Wittershagen, A.; Rostam-Khani, P.; Klimmek, O.; Grofl, R.; Zickermann, V.; Zickermann, I.; Gemeinhardt, S.; Kroger, A.; Ludwig, B.; Kolbesen, B.O. Determination of metal-cofactors in enzyme complexes by total-reflection X-ray fluorescence spectrometry. Spectro- chim. Acta B 1997, 52, 1033-1038.

11. Huber, R. Eine strukturelle Gmndlage fur die Ubertragung von Lichtenergie und Elektronen in der Biologie (Nobel-Vortrag). Angew. Chem. 1989,101, 849-871.

12. Deisenhofer, J.; Michel, H. Das photosynthetische Reaktionszentrum des Purpurbakteriums Rhodopseudomonas viridis (Nobel-Vortrag). Angew. Chem. 1989,101, 872-892.

13. Wiithrich, K. NMR studies of structure and function of biological macromolecules (Nobel Lecture). J. Biomol. NMR 2003, 27, 13-39.

14. Weber, G.; Konieczynski, P. Speciation of Mg, Mn and Zn in extracts of medicinal plants. Anal. Bioanal. Chem. 2003, 375, 1067-1073.

Received October 13, 2003 Accepted November 11, 2003


Metalloprotein is a generic term for a protein that contains a metal ion cofactor. [illustration not visible in this excerpt] A large fraction of all proteins are members of this category, so the area is very large.

Metalloenzymes are widespread and diverse in function

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 require metals to carry out their functionsThus, metalloproteins have many different functions in cells, such as enzymes, transport and storage proteins, and signal transduction proteins.

Coordination chemistry principles

In metalloproteins, metal ions are usually coordinated by nitrogen, oxygen or sulfur centres 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 cysteinyl 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 centres.

In addition to donor groups that are provided by amino acid residues, a large number of 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

Oxygen carriers

Hemoglobin, which is the principal oxygen carrier in humans has four sub-units 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, myoglobin has only one such unit. The active site is located in an hydrophobic pocket. This is important as, without it, the iron(II) would be irreversibly oxidised to iron(III). The equilibrium constant for the formation of Hb02 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 sub-units show a cooperativity effect which 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(IH). It is now known that the diamagnetic nature of these species is due to the fact that the iron(II) 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 ringJ4^ The change in spin state is a cooperative effect of higher crystal field splitting and smaller ionic radius of Fe2+ in the oxy- 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 02 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 molluscs, 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 oxidised to copper(II) and the dioxygen molecules is reduced to peroxide,[illustration not visible in this excerpt]


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 [11][ 12]


Rubredoxin is an electron-carrier found in sulfur-metabolizing bacteria and archaea. The active site contains an iron ion which is coordinated by the sulphur 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.

illustration not visible in this excerpt


Plastocyan is one of the family of blue copper proteins which are involved in electron transfer reactions. The copper binding site is described as a distorted trigonal pyramidal ,l J The trigonal plane of the pyramidal base is composed of two nitrogen atoms (N and N ) from separate histidines and a sulfur (S^ from a cysteine. Sulfur (S ) from an axial methionine forms the apex. The ‘distortion’ occurs in the bond lengths between the copper and sulfur ligands. The Cu-S ^ contact is shorter (207 picometers) than Cu-S (282 pm). The elongated [illustration not visible in this excerpt]Cu-S bonding destabilises the Cu form and increases the redox potential of the protein. The blue colour (597 nm peak absorption) is due to the [illustration not visible in this excerpt] In the reduced form of plastocyanin, His-87 will become protonated with a [illustration not visible in this excerpt] 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 an hydrolysis product such as FeO(OH). Iron is transported by transferrin whose binding site consists of two tyrosines, one aspartic acid and one [illustration not visible in this excerpt] 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 (3-thallasemia.


Ceruloplasmin is the major copper-carrying protein in the blood. Ceruloplasmin exhibits oxidase activity, which is associated with possible oxidation of [illustration not visible in this excerpt] (ferrous iron) into Fe3+ (ferric iron), therefore assisting in its transport in the plasma in association with transferrin, which can only carry iron in the ferric state.


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 which 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.

illustration not visible in this excerpt

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

illustration not visible in this excerpt

A reaction similar to this is almost instantaneaous 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 hydoxide portion on carbon dioxide (carbonic anhydride) proceeds rapidly. The catalytic cycle produces the bicarbonate ion and the hydrogen [illustration not visible in this excerpt] as the equilibrium

illustration not visible in this excerpt

Vitamin BJ2 catalyzes the transfer of methyl [illustration not visible in this excerpt] 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 [illustration not visible in this excerpt] bond.1 J The structure of the coenzyme was famously determined by Dorothy Hodgkin and co-workers, for which she received a Nobel prized18-' It constists of a cobalt(H) ion coordinated by four nitrogen atoms of a corrin rings and a fifth Nitrogen atom from an imidazole group. In the resting state there is a [illustration not visible in this excerpt] bond with the [illustration not visible in this excerpt] carbon atom of adenosineJ19^ 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 certain bacteria. There are three components to its action: a molybdenum atom at the active site, Iron-sulfur clusters which are involved in transporting the electrons needed to reduce the nitrogen and an abundant energy source. The energy 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

illustration not visible in this excerpt

where P. stands for inorganic phosphate. The precise structure of the active appears to contain a MoFe Sg cluster which is able to bind the dinitrogen reduction process to begin. The electrons are transported by the associated T211 Fe4S4 clusters joined by sulphur bridges.

Superoxide dismutase

The superoxide ion, O ‘ 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 oxidising agent. These properties render the superoxide ion very toxic and are deployed to advantage by phagocytes to kill invading micro organisms. Otherwise, the superoxide ion must be destroyed before it does unwanted damage in a cell. The superoxide dismutase enzymes perform this function very efficiently J22-'

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

illustration not visible in this excerpt

In biology this type of reaction is called a dismutation reaction. It Structure of a human superoxide dismutase 2 involves both oxidation and reduction of superoxide ions. The tetramer superoxide dismutase group of enzymes, abbreviated as SOD, increase the rate of reaction to near the diffusion limited [illustration not visible in this excerpt] The key to the action of these enzymes is a metal ion with variable oxidation state which can act as either an oxidizing agent or as a reducing agent.

illustration not visible in this excerpt

In human SOD the active metal is copper, as Cu2+ or Cu+, 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 Ni geometry cycles from square planar Ni(II), with thiolate (Cys2 and Cys6) and backbone nitrogen (Hisl and Cys2) ligands, to square pyramidal Ni(III) with an added axial Hisl side chain ligand


[1] Correspondence: Bernd Kastenholz, Research Centre Juelich, Institute for Phyto­sphere Research (ICG-III), 52425, Juelich, Germany; E-mail; b.kastenholz@ fz-juelich.de.

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QPNC-PAGE: A Milestone for Protein Electrophoresis
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NMR, metal cofactors, blood-brain barrier, etiological treatment, ICP-MS, Ginkgo biloba, gel electrophoresis
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Bernd Kastenholz (Author), 2012, QPNC-PAGE: A Milestone for Protein Electrophoresis, Munich, GRIN Verlag, https://www.grin.com/document/193809


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