Experimental Techniques Of Classical Biochemistry

The brief history of classical biochemistry (i.e., pre-molecular biology) related above highlights the progression of the science towards the elucidation of mechanisms (and their constituents) of increasingly smaller sizes from the micro-scale to the molecular. As a number of the "classical" techniques employed in the aforementioned experiments continue to find use alongside the largely molecular techniques in the experimental approaches of modern biochemistry, we will now shift to a brief explanation of the techniques most relevant to the application of biochemistry for food biotechnology. Since the period of time from the 1970s to the present has seen largely the development of experimental techniques relevant to molecular biology, these advances will be covered in the subsequent subsection of this chapter which will discuss relevant molecular biology techniques.

A main preoccupation of biochemistry is the study of enzyme activity (enzymology). For the better part of a century, the primary ways to determine the activity of an enzyme have been (1) to detect the presence or change in concentration of a predicted (or known) reaction product, (2) to follow the incorporation of a radioactively-labeled molecule (such as 14C) in products of a metabolic pathway, or (3) to measure the change in concentration of a required co-factor (such as NADP+) by detecting its absorbance at a certain wavelength of light using a spectrophotometer.

Key to the study of enzyme activity is the isolation of the reaction components (i.e., the enzyme, its substrate(s), and any required co-factors). A quick review of the history of biochemistry will reveal that our understanding of biological processes has increased concomitant to our ability to purify compounds related to or involved in such processes. Once the required reaction components have been isolated, the reaction can be reconstituted in vitro and the reaction kinetics investigated. We will introduce and describe two major biochemical approaches that have been (and continue to be) used to purify enzymes (proteins) and other compounds or molecules from crude solutions: chromatography and isoelectric focusing. Both approaches are typically employed subsequent to a salt precipitation procedure to concentrate the protein in a crude sample (with the most common salt used for this procedure being ammonium sulfate). Afterwards, we will discuss spectrophotom-etry, its history, and its relevance to enzymology.

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