Enzymes are proteins that act as catalysts to accelerate biochemical reactions.
Although many chemical reactions are thermodynamically feasible, they are often not spontaneous and require energy, termed “activation energy”, which is the minimum amount of energy that two molecules must have in order for them to start to react. In the laboratory, activation energy can be applied in the form of heat to increase the kinetic energy of the molecules to initiate the reaction. Clearly, thermal activation is not a feasible mode of initiating reactions within cells. So, the biological solution is to use enzymes as catalysts to lower the activation energy such that reactions can proceed at temperatures and pressures that are compatible with cellular processes.
One of the major objectives of a previous laboratory was to use centrifugation to separate and isolate different components within the cell. The success of the separation procedure is commonly assessed by monitoring the enzymatic activity of enzymes that are characteristic of components expected within each faction. For example, succinic dehydrogenase (SDH) activity is commonly used as an indicator of mitochondrial purity:
This inner mitochondrial membrane bound enzyme catalyzes the oxidation of succinate to fumarate within the Krebs cycle. SDH covalently binds the coenzyme FAD which accepts electrons during the oxidation of the succinate. In the cell, the reduced form of
FAD, FADH, normally becomes reoxidized by iron-sulfur proteins in the electron transport chain to complete the catalytic cycle. The activity of this electron cascade can be monitored in vitro by the addition of artificial electron acceptors of higher reduction potential than FAD that show changes in absorption characteristics upon reduction. One useful artificial electron acceptor is 2,6-dichlorophenol indophenol (DCPIP) which shows a decrease in absorption at 600nm upon reduction and can therefore be used to measure the rate of the enzyme catalyzed reaction.