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Biotechnology Biotechnology Name Affiliation
Biotechnology Introduction Proteins are polymers constituting of amino acid monomers. Of the known types of proteins, the current paper reviews enzymes. Enzymes are remarkably similar in structure to the other proteins but their distinct functionality is always fascinating. Various methods have proved vital in the study of different enzymes with respect to their composition, functionality and enzyme kinetics and enzyme assay. This review focuses on the different technologies or methods that have been used to study enzyme assay. Summary of Protein Technologies Protein TechnologyLiterature Reference Spectrophotometric technologySpinazzi M, Casarin A, Pertegato V, Salviati L, Angelini C. Assessment of mitochondrial respiratory chain enzymatic activities on tissues and cultured cells. Nature protocols. 2012 Jun 1;7(6):1235. Polarimetric technologyWu SH, Nee SF, Yang DM, Chiou A, Nee TW. Polarimetric signature imaging of anisotropic bio-medical tissues. Biomed. Eng. Res.. 2013 Mar;2:20-9. Sampling technologyHeinonsalo, J., Kabiersch, G., Niemi, R. M., Simpanen, S., Ilvesniemi, H., Hofrichter, M., ... & Steffen, K. T. (2012). Filter centrifugation as a sampling method for miniaturization of extracellular fungal enzyme activity measurements in solid media.Fungal Ecology,5(2), 261- 269.
Biotechnology Electrode technologyAziz, M.A., Patra, S. and Yang, H., 2008. A facile method of achieving low surface coverage of Au nanoparticles on an indium tin oxide electrode and its application to protein detection.Chemical Communications, (38), pp.4607-4609. Fluorescence technologyShaner NC, Patterson GH, Davidson MW. Advances in fluorescent protein technology. Journal of cell science. 2007 Dec 15;120(24):4247-60. Spectrophotometric Technology The study conducted bySpinazzi and his team noted that mitochondrial dysfunction significantly contributes to disorders in humans including mitochondrial abnormalities. Some of these conditions affect the mitochondrial itself while some affectnuclear DNAcontributing to ailments such aging, neurodegenerative conditions (Parkinson’s disease and Alzheimer’s disease)and diabetes, to mention a few1. A clear understanding of mitochondrial activities is necessary to avert undesirable outcomes. Various technological approaches have been used to study mitochondrial respiratory chain (RC) during enzymatic activities. In the current study,theenzymatic activities of RC complexes I–IV were assayed using spectrophotometric techniques2. Like in many other studies, the results were standardized to the aggregate muscle protein composition and to the activity of citrate synthase to mirror mitochondrial matrix enzyme. However, the technique posed one glaring problem of the assays: at all times, the enzymatic reactions are assessed using in vitro environments3. These 1 2 3
Biotechnology conditions are hardly physiological with respect to pH, substrate concentrations, osmolarity and cellular context and disallow the estimation of respiratory pairing. Despite that challenge, spectrophotometric technology offeredpertinent quantitative data regarding the top catalytic reactions of the RC complexes. In addition the procedure are easy to duplicate and can be undertaken by use of iced up tissue and cellular samples. An acceptable into mitochondrial enzymatic activities ought to encompass a blend of other assays including determination of polarographical oxygen utilizations in intact insulated mitochondria, ATP synthesis and permeabilized cells or tissues. Assessment of the mitochondrial membrane potential is also necessary to determine any dysfunction. Nonetheless, the aforementioned techniques have certain shortcomings in that investigators require fresh samples and are time consuming due to the complexity of the procedures involved. As such, spectrophotometric technology remains a first-line method both for research investigations on mitochondrial ailments and for diagnostics. Studies from recent years indicate that most published protocols for spectrophotometric assays were dissatisfactory in that there were complex biochemical hindrances that led to enzymatic inhibition or to unsatisfactory linearity of the kinetics with regards to protein concentration. Such analytical shortcomings have been known to severely prejudice sensitivity and precision, bringing about analytical discrepancies and considerable disagreements of results from various laboratories. The protocols in the current study were designed to overpower or negate most of these challenges, in an attempt to improve the sensitivity, specificity and precision with little adjustments to the framework of the procedure. Thespectrophotometric technology depicted herein specify the steps for mitochondrial RC enzyme reaction analysis in which small quantities of muscle tissue from mammals and cultured skin fibroblasts were used compared to previously
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