Biotechnology Assignment - Protein Enzymes

Biotechnology
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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 Technology Literature Reference
Spectrophotometric technology Spinazzi 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 technology Wu 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 technology Heinonsalo, 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-
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Electrode technology Aziz, 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 technology Shaner 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 by Spinazzi 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 affect nuclear DNA contributing 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, the enzymatic 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
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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 offered pertinent 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. The spectrophotometric 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