Assay of Activities in the Extracellular

Enzymatic activities will often be found in the extracellular fluid surrounding oigans, tissues in biological fluids, or the medium supporting the growth of mammalian cells, bacteria, yeast, or fungi. The enzymatic composition of these fluids can vary considerably, and the assay of enzymatic activities in such fluids presents several major problems. 

The first is the presence of proteolytic activities, which must be inhibited early in the procedure to prevent the degradation of other enzyme proteins. Second, the amount of protein present in these fluids is usually in excess of what an HPLC analytical column can handle without becoming clogged. And finally, these fluids often contain many low molecular weight compounds, either those added as nutrients or those present as a result of cellular metabolism. Since such compounds may resemble either the substrate or product, or both, of the enzymatic reaction under study, their presence in the reaction mixture could interfere with the assay. At the very least, such compounds will pass through the analytical column and appear on a chromatogram, confusing the experimental results. 

Low molecular weight compounds may be removed, and a variety of methods are available, including dialysis and gel filtration chromatography. The removal of excess protein may be more complicated. It can be dealt with before the assay by further purification of the sample. Alternatively, the excess can remain during the incubation and be removed after the assay by introducing a termination step to precipitate all proteins, which are then removed by filtration. And finally, proteolytic activities can be eliminated by the addition of the inhibitory cocktail mentioned below. 

A word of caution Growth media that contain serum contain serum-associated enzymes. The presence of such endogenous activities must be considered in any purification scheme, since they can produce confusing results. 

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