Some general points on assay design

The addition of acid is an effective precipitant of proteins but will not discriminate between the unwanted proteins and the coupling enzymes which will then be used, so it is vital that the acid is removed. TCA can be conveniently removed by extraction into ether or acetone, while perchloric acid can be neutralized with KOH which provides two functions the first is neutralization and the second the precipitation of insoluble potassium perchlorate. 

The components of the assay will normally be in a final volume of 2.5 ml although scaling down can normally be achieved for smaller cells or sample holders. Under these conditions it is usually desirable to dilute the sample extensively into the assay mixture so that any small effects on buffering capacity, ionic strength or potential inhibitors are minimized. 

It is important to drive the reaction essentially to completion. For many coupling enzymes that are linked to ATP (e.g. hexokinase) this is not a problem. 

Some reactions, however, are at thermodynamic equilibrium and need some thermodynamic assistance to be dragged over to completion. A classic example of this type of strategy is the addition of semicarbazide to trap ketones in the thermodynamic sink of the semicarbazone (see above). An alternative is to link a reversible reaction with an enzyme linked reaction that will provide an alternative thermodynamic sink. 

In addition to the above, many coupling enzymes require additional factors, such as Mg for most kinases and K for pyruvate kinase. 

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