Utilization of Electrophoretic Methods to Examine Mitochondrial Respiratory Chain Complex Defects

A well-liked technique for spectrophotometric and polarographic evaluation of mitochondrial respiratory chain complexes is called Blue Native Gel Electrophoresis (BNGE). For instance, the mitochondria's inner membrane is home to a number of complexes that are part of the oxidative phosphorylation (OXPHOS) system. As a result, they have an impact on aerobic ATP synthesis. Additionally, mitochondrial disorders are caused by nuclear and mitochondrial DNA mutations that affect the action of the enzymes (Leary 2012, p.1). Specifically, the BNGE is typically used in addition to the respiratory chain's enzymology. Additionally, the foundation of BNGE is the electrophoretic separation of protein complexes based on their inherent molecular weight in the Coomassie blue dye. The extraction of protein complexes occur from membrane with a minor detergent (Strecker et al 2010, p.3). Consequently, the dyes are exposed to the complexes, which generate the negative charge that is needed for separation using electrophoretic process without changing their innate conformation (Jha, Wang and Auwerx 2016, p.1).

The various oxidative phosphorylation complexes of the mitochondria can be analyzed by immunoblotting, by protein staining with particular antibodies. Similarly, analyses can also be done using in gel action stain assays (Jha, Wang and Auwerx 2016, p.3). The process requires materials from GE health care such as high molecular weight innate markers, which are utilized to monitor the movement of protein complexes in the gel (Díaz, et al 2009, p.1). Instead, mitochondrial arrangements and materials can be obtained from bovine heart or rat heart, which are then preserved with lauryl maltoside. Moreover, the preparations are utilized as respiratory complexes (Leary 2012, p.2). In case adequate protein is used, the various complexes can be identified while moving via the gel.

Analysis of respiratory complexes can be performed using silver staining or Coomassie blue. Some of the material used in this process include 10% acetic acid, methanol, Coomassie R250, and 0.1% Serva G. The solution is then filtered using a filter paper to eliminate insoluble components (Díaz, et al 2009, p.3). Coomassie stain can identify protein bands of approximately 0.5 µg. However, a more sensitive approach can be applied via silver staining. Following the separation of protein complexes using electrophoretic, the respiratory complexes preserve enzymatic action (Strecker et al 2010, p.3).

Blue native gel electrophoresis permits for the protein separation between 100-10,000 kDa based on the concentration level of used acrylamide gradient. The OXPHOS complexes separation in an acrylamide gradient of 4-13 per cent tolerates for the identification of distinct bands on the blue native gel conforming to each complex (Ohtake, et al 2014, p.5). The molecular weights of the bands are different for every complex. For instance, complex I has an approximate weight of 1,000 kDa, complex V has 1,600 kDa, complex III has 500 kDA, while complex IV and II have 200 kDa and 130 kDA respectively (Leary 2012, p.4). Moreover, the analysis of defects in mitochondrial respiratory complexes samples by Blue Native Gel Electrophoresis allows screening of deficiencies on many complexes at a single period (Karkucinska-Wieckowska, et al 2015, p.3). Meanwhile the respiratory sequence can be arranged in super complexes or super molecular systems.

Less distinct defects on the oxidative phosphorylation (OXPHOS) complexes can be identified in the samples analyzed through in gel operation stain (Jha, Wang and Auwerx 2016, p.5). When this method is used, the sample has lower enzymatic action in every respiratory complex when matched to different samples (Díaz, et al 2009, p.3). Therefore, in order to acquire more elaborate deficiencies on the OXPHOS, more analysis should be conducted using western blot which offer more details on whether defects on the enzyme is caused by lower activity on the enzyme or lower stable state levels of the respiratory complex (Strecker et al 2010, p.3).

Conclusion

The Blue native gel electrophoresis provides an opportunity to analyse oxidative phosphorylation (OXPHOS) implanted in the inner membrane of the mitochondria. The Coomassie dye enables analysis of defects of mitochondrial respiratory complexes through electrophoretic distinction (Jha, Wang and Auwerx 2016, p.5). The technique uses gel electrophoresis, which is normally done wing electroblotting to give extra information concerning a particular protein.



References

Díaz, F., Barrientos, A. and Fontanesi, F., 2009. Evaluation of the mitochondrial respiratory chain and oxidative phosphorylation system using blue native gel electrophoresis. Current Protocols in Human Genetics, pp.19-4.

Jha, P., Wang, X. and Auwerx, J., 2016. Analysis of Mitochondrial Respiratory Chain Supercomplexes Using Blue Native Polyacrylamide Gel Electrophoresis (BN‐PAGE). Current protocols in mouse biology, pp.1-14.

Karkucinska-Wieckowska, A., Pronicki, M. and Wieckowski, M.R., 2015. Histoenzymatic Methods for Visualization of the Activity of Individual Mitochondrial Respiratory Chain Complexes in the Muscle Biopsies from Patients with Mitochondrial Defects. Mitochondrial Regulation: Methods and Protocols, pp.85-93.

Leary, S.C., 2012. Blue native polyacrylamide gel electrophoresis: a powerful diagnostic tool for the detection of assembly defects in the enzyme complexes of oxidative phosphorylation. Mitochondrial Disorders: Biochemical and Molecular Analysis, pp.195-206.

Ohtake, A., Murayama, K., Mori, M., Harashima, H., Yamazaki, T., Tamaru, S., Yamashita, Y., Kishita, Y., Nakachi, Y., Kohda, M. and Tokuzawa, Y., 2014. Diagnosis and molecular basis of mitochondrial respiratory chain disorders: exome sequencing for disease gene identification. Biochimica et Biophysica Acta (BBA)-General Subjects, 1840(4), pp.1355-1359.

Silvestri, E., Lombardi, A., Cioffi, F. and Goglia, F., 2015. BN-PAGE-based approach to study thyroid hormones and mitochondrial function. Mitochondrial Regulation: Methods and Protocols, pp.111-122.

Strecker, V., Wumaier, Z., Wittig, I. and Schägger, H., 2010. Large pore gels to separate mega protein complexes larger than 10 MDa by blue native electrophoresis: isolation of putative respiratory strings or patches. Proteomics, 10(18), pp.3379-3387.