Continuous versus discontinuous enzyme assays

An enzyme assay tells you how active an enzyme is, meaning how fast it turns substrate (the molecule it acts on) into product. There are two types, split by whether the reaction is followed live or sampled.

In a continuous assay, a signal (something that changes as the reaction proceeds) is read again and again from one reaction mixture. Spectrophotometry measures absorbance, the amount of light the solution absorbs. When the reaction produces a change that can be detected, directly or through a linked reaction (a second reaction sometimes added because the product gives no useful change by itself), the absorbance changes as product forms or substrate is used up. Chemiluminescence is another signal: the reaction gives off light, and its amount is measured. The slope of the signal against time (how steeply it changes) gives the reaction rate. One run is enough, so continuous assays are described as the most convenient type.

In a discontinuous assay, samples are taken from the reaction and measured separately at set times. Radioactivity (a way of measuring product in samples that have been taken) and chromatography (a method that separates the parts of a mixture) are examples. This takes more effort but can be more sensitive, meaning it can detect smaller amounts of product. Samples can be measured with a sensitive method after product has built up, so a faint reaction becomes big enough to measure.

Both types measure what the enzyme does, not how much enzyme is present. That is the job of an immunoassay, which uses antibodies to detect a specific protein. A continuous assay also suits studying an enzyme inhibitor (a molecule that slows an enzyme), because the rate is followed in real time from one mixture, so the slope can be compared with and without the inhibitor.