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

Many enzymes require metals for activity, and, unfortunately for HPLC use, the presence of the metal can occasionally have significant effects on a separation. For an explanation of this problem, return to the reaction illustrated in Figure 4.1, the degradation of ATP to form AMP and PPj. The first HPLC method developed to assay this activity was carried out on a reversed-phase system with a mobile phase chosen for the exclusive separation of ATP from AMP (see Fig. 4.5). Since ADP was not involved, no thought was given to

Figure 4.5 HPLC analysis of enzymatic assay with ATP in free and metal-bound forms. Separations were carried out on a reversed-phase C18 column with a potassium phosphate mobile phase containing 10% methanol. The flow rate was 2 mL/min. Assay mixture of 100 jaL contained 8 mM Tris-HCl (pH 7.5), 2 mM ATP, and 2 mM MgCl2 and enzyme preparation containing ATP pyrophosphohydrolase (10 pig of protein). Chromatograms of 20 /xL samples illustrate incubation of (^4) zero time and (B)2 hours. (From Jahngen and Rossomando, 1983).

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its separation. Later, having decided to study the metal requirements of this reaction, we performed a series of experiments for this purpose.

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. 4.5B). 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.

Additional studies confirmed this possibility. For example, when the effect of metals on the chromatographic behavior of ATP was studied in more detail, the profiles illustrated in Figure 4.7 were obtained. Figure 4JA shows the profile at the lowest metal concentration used in the experiment. Two peaks of ATP, labeled I and II, are clearly resolved. Increasing the metal concentration (Fig. A.1B-F) led to an increase in the area of peak II and a corresponding decrease in peak I. This result suggests that the second peak was formed by the metal binding to the ATP.

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Figure 4.6 HPLC separation of ATP/Ca2+ mixture. Separations were carried out on a Ci8 (/¿.Bondapak) microcolumn (4.6 mm X 25 cm) with a mobile phase of 10 mM KH2P04 (pH 5.5) with 4% methanol; flow rate was 0.5 mL/min. The sample volume was 20 nL, and detection was at 254 nm. Dashed line, 40 nmol ATP; solid line, 40 nmol ATP plus 160 nmol CaCl2- (From Jahngen and Rossomando, 1983.)

SETTING UP THE ASSAY 73 C

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Figure 4.7 Effect of increasing amounts of Ca2+ in a mixture of this cation and ATP. Analysis by HPLC as described in Figure 4.6. Chromatograms are of 20 pL samples containing 40 nmol ATP and CaCl2 at (A) 60 nmol, (B) 100 nmol, (C) 120 nmol, (D) 160 nmol, (E) 200 nmol, and (F) 400 nmol. (From Jahngen and Rossomando, 1983.)

This conclusion was tested further by the addition of a radiolabeled metal, in this case calcium-45, together with the ATP in the reaction mixture. A sample was injected, and the two peaks were collected in separate fractions. Following an analysis of the fractions, the 45Ca elution profile was plotted on the chromatogram as shown in Figure 4.8. The calcium coelutes exclusively with the ATP in peak II (Fig. 4.8), confirming the conclusion that the second peak was indeed a metal-ATP.

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