Spectrophotometric assays of enzymes

Having established a spectrophotometric assay for enzyme activity (see below), it is vital before setting off to do any detailed measurements of enzyme activity to check two factors. The first of these is that true initial rates are being measured. A plot of product (P) appearance (in this case some spectral change) against time (t) must be linear over the period of measurement to measure true initial rates (the slope of the [P]/t curve is, by definition the initial rate v). There are a number of reasons that such [P]/t curves may deviate from linearity  

The substrate has simply been used up. This can be checked by calculating the amount of product produced and determining whether a significant fraction of the substrate has been used. Furthermore, some reactions rapidly approach equilibrium (perhaps when only a small amount of substrate has been used up) and, if this is the case, the reaction will not proceed without some additional system for removing the product. 

A build up of inhibitoiy product may cause deviation from linearity. There are numerous examples of this phenomenon, which is hardly surprising as by definition most products have dissociated from the en25mie surface and are therefore capable of binding to the enzyme or one of the enzyme-substrate or enzyme-product complexes. 

The enzyme may be unstable under the conditions of assay. Selwyn (19) has devised a method to check this possibihty, where a plot of product concentration against the product of time and the enz5rme concentration (et) is made, and should give the same curve whatever initial concentration of enzyme is used. If, however, the enzyme is unstable, the concentration of active enzyme will be time dependent and in such cases the graphs of [P] versus et will give different curves for each initial concentration of enzyme. 

Another component of the assay other than the enzyme may be unstable. This may be checked by incubating the assay mixture without each of its components in turn for an appropriate period of time, and then to start the reaction by the addition of the missing component. If the initial rate is identical, irrespective of which component is added last, then instability can be ruled out. Conversely, if the initial rate is lower when one component is included in the pre-incubation but not when it is added last, this indicates an unstable component. 

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