Reactions 6 Components of the Reaction Mixtures Can

6 Components of the Reaction Mixtures Can Cause Problems Effects of Metals on Separation 

A reaction mixture was prepared that contained a metal at concentrations in excess of that of ATP. The reaction was started by the addition of the enzyme, and samples were taken and analyzed by the HPLC method. Surprisingly, the chromatograms for the experiments that included metals were different from the ones obtained earlier. In the original experiments, only two peaks were present, those representing ATP and AMP. However, in the experiment that included the metal calcium, the chromatograms showed the elution of an additional peak jsut after the ATP emerged (Fig. A.5B). Further studies showed that the new peak had a retention time identical to that of ADP, and therefore we assumed that this second peak was ADP. From these findings, we speculated that the metal had stimulated the activity of an enzyme that catalyzed the formation of ADP. 

Additional studies were performed. We analyzed samples of the reaction mixture before we added the enzyme and very soon afterward, before any enzymatic reaction could have taken place. Interestingly, these chromatograms (Fig. 4.6) also showed two peaks, with peak II identical in retention time to the presumptive ADP peak. In the absence of any metal, a single ATP peak (peak I) was observed, suggesting that the metal had altered the chromatographic properties of ATP. 

In designing an assay for an enzyme, it is often necessary to introduce a termination step into the protocol (see Chapter 1). This is often done when protein is present in the incubation mixture at a concentration that would dog the column. There are a variety of ways to accomplish this termination process. Ideally, it would not be necessary to add reagents that might otherwise clog the column or alter its performance. For example, consider the changes that occur in the incubation mixture when the reaction is terminated by add. The addition of trichloroacetic acid (TCA) will reduce the pH of the incubation 

A method that offers an alternative to the use of adds and bases is the addition of chelators such as EDTA. This technique is suitable only for reactions in which the enzymatic activities have an absolute requirement for a metal whose removal will terminate the activities. 

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