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Endnote Referencing Essay

Laura Jenkins – 17428794

BCH3BMA

“Discuss how electrophoretic techniques can be used to analyze defects in mitochondrial respiratory

chain complexes.”

Mitochondria are classified as the power house of eukaryotic cells, providing majority of cellular energy

in the form of ATP (McKenzie, Lazarou et al. 2007). Mitochondria produce ATP by oxidative

phosphorylation via an electrochemical gradient produced by the mitochondrial respiratory chain. The

respiratory chain is comprised of five complexes (I, II, III, IV and V) embedded into the inner

mitochondrial membrane, each complex is comprised of multiple sub-units hence the complexes are

highly ordered and believed to be supercomplexes (Zeviana and Donato 2004). Defects in these

complexes or sub units as a whole or singularly have been associated with various neuromuscular

diseases, these conditions have an incidence of 1 in 5000 births, onset begins at any age however the most

severe cases are found in children (Fernandez-Vizarra, Tiranti et al. 2009). Detecting the defects in the

mitochondrial respiratory chain complex associated with the neuromuscular disorders are analyzed in

many cases using Blue Native polyacrylamide gel electrophoresis (BN-PAGE), to obtain additional

information a two dimensional BN-PAGE/SDS PAGE is performed (Calvaruso, Smeitink et al. 2008).

BN-PAGE was developed in 1991 for the purpose of analyzing the respiratory chain complexes involved

in the oxidative phosphorylation process, the technique involves the separation of the complexes whilst

keeping them in their native form, as the complexes have not been denatured they remain enzymatically

active (Coster, Smet et al. 2001). To keep the complexes in their native form the correct detergent must be

used for solubilization, due to the complexes being integral membrane proteins they require a strong

enough detergent to detach it from the membrane. In BN-PAGE, solubilization is achieved by using non-

ionic detergents as they only interrupt lipid-lipid or lipid-protein interactions and not protein-protein

interactions therefore the proteins are not denatured, common non-ionic detergents are triton x-100 and

digitonin. When performing BN-PAGE for the respiratory chain complexes the detergent commonly used

is digitonin as it provides a large concentration range and destroys no protein-protein interactions

(Reisinger and Eichaker 2008).

Gene defects in respiratory chain complexes lead to a decrease in enzymes within the protein causing a

change in the overall composition, BN-PAGE is used to detect the abundance and composition of protein

complexes therefore defects are easily evaluated. When undertaking BN-PAGE a small amount of heart

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and skeletal muscle are taken for defect analysis, where the tissue will be solubilized using detergent

previously mentioned and centrifuged, then stained and ran through a gel where it will later be analyzed.

The advantages of using BN-PAGE include; only a small tissue sample size is required for the process,

complexes are able to be analyzed in their active form and staining makes analysis of the complex easy

(Coster, Smet et al. 2001).

Although BN-PAGE is a useful technique in analysis for defects it has its limitations therefore it is often

accompanied by western blotting or a second dimension of SDS-PAGE. When undertaking the western

blotting technique, electrophoresis must be done in two dimensions as BN-PAGE provides limitations to

the technique of which include; the epitopes are unrecognizable as the protein complex is not denatured

and are therefore hidden by the complex, the other limitation being, the dye associated with BN-PAGE

may interrupt the binding of antibody to the target protein (Nijtmans, Henderson et al. 2002). SDS-PAGE

is the second dimension of the electrophoretic technique and unlike BN-PAGE, SDS-PAGE requires

denaturation of protein complexes, which is achieved by ionic detergents such as SDS and DDM,

respectively the role of each detergent is to; interrupt the protein-protein interactions within the

membrane and break cysteine linkages. SDS-PAGE separates proteins based on their mass: charge ratio,

making it a useful technique in analyzing the molecular weight of complexes as well as the weights of the

various sub-units that the complexes are comprised of. SDS-PAGE provides to be a useful resource in

detecting defects, as it is possible that the defects in the chain complexes are found in one of the sub-units

that comprise any of the five complexes. As proteins are denatured with SDS-PAGE epitope availability

increases, also the absence of dye reduces any interruption for antibody-protein binding, therefore western

blotting can be used to its full potential (Calvaruso, Smeitink et al. 2008).

Through these electrophoretic techniques as well as accompanying techniques such as western blotting or

mass spectrometry testing for defects in such things as the mitochondrial respiratory chain complexes has

been made something that can be done in most science labs, and are useful in screening for clinical

purposes.BN-PAGE is the most useful technique for such analyses, however in most cases it is

accompanied by the second dimension, SDS-PAGE which is followed by immunoblotting this provides

as much information of the complexes as possible. When undertaking such procedures many aspects must

be taken into consideration such as the type of detergent used for solubilization, the type of dye that is

used as many dyes only compensate for a particular protein size, these points must be taken into account

for the specific sample type and size (Coster, Smet et al. 2001).

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References

Calvaruso, M. A., J. Smeitink and L. Nijtmans (2008). "Electrophoretic techniques to investigate defects in oxidative phosphorylation." Methods 46: 7.

Coster, R. V., J. Smet, E. George, L. D. Meirleir, S. Seneca, J. V. Hove, G. Sebire, H. Verhelst, J. D. V. Vlem, P. Verloo and J. Leroy (2001). "Blue Native Polyacrylamide Gel Electrophoresis: A Powerful Tool in Diagnosis of Oxidative Phosphorylation Defects." Pediatric Research 50(5): 8.

Fernandez-Vizarra, E., V. Tiranti and M. Zeviani (2009). "Assembly of the oxidative phosphyration system in humans: What we have learned by studying its defects." Biochimica et Biophysica Acta 1793: 12.

McKenzie, M., M. Lazarou, D. R. Thorburn and M. T. Ryan (2007). "Analysis of mitochondrial subunit assembly into respiratory chain complexes using Blue Native polyacylamide gel electrophoresis." Analytical Biochemistry 364: 9.

Nijtmans, L. G. J., N. S. Henderson and I. J. Holt (2002). "Blue Native electrophoresis to study mitochondrial and other protein complexes." Methods 26: 8.

Reisinger, V. and L. A. Eichaker (2008). "Solubilization of membrane protein complexes for blue native PAGE." Journal of Proteomics 71: 7.

Zeviana, M. and S. D. Donato (2004). "Mitochondrial Disorder." Brain 127: 19.

(Coster, Smet et al. 2001, Nijtmans, Henderson et al. 2002, Zeviana and Donato 2004, McKenzie, Lazarou et al. 2007, Calvaruso, Smeitink et al. 2008, Reisinger and Eichaker 2008, Fernandez-Vizarra, Tiranti et al. 2009)

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