Figure 1 shows the kinetics of alkaline phosphatase activity of the holo-enzyme with different quantities of alkaline phosphatase.  Linear kinetics was observed during the initial portion of the reaction which exhibited high r2 values by linear regression (Figure 2).  The values were used to generate a linear standard curve (y = 0.0068x + 0.0661 r2 = 0.994) that correlated enzymatic activity (dA/dt) with concentration or mass of alkaline phosphatase (Figure 3).  The incubation of alkaline phosphatase with Chelex 100 resin caused a marked change in the absorbance characteristics of the holo-protein as determined by diode array spectroscopy (Figure 4).  These changes can be clearly observed in Figure 5 which provides an overlay of alkaline phosphatase (trace in blue) and the apo-protein (trace in red) at 254nm.
       Data obtained from the elemental analysis of the profiles shown in Figure 6 by coupled ICPMS showed the presence of Zn primarily in peaks eluting at 10.733 and 15.5 minutes.  These peaks occurred in both the holo and the apo enzyme (Figure 7 and 8) respectively.  No major signals for Zn were obtained from any of the other peaks.  Enzymatic assays of fractions collected from the two peaks indicated that activity was present only in the peak eluting at 10.733 minutes which concurred with the apparent molecular weight of the enzyme of 150 kDa.  Comparisons of the enzyme activity of the protein normalized samples for this peak gave values of 0.2059 activity/ug and 0.0548 activity/ug for the holo and apo enzyme respectively.  These values corresponded to a 73% reduction in activity.  Quantification of the zinc associated with the active peak by FIA gave values of 2.779 ng of Zn/ug protein for alkaline phosphatase and 0.718 ng of Zn/ug protein for apo-alkaline phosphatase indicating a 75% loss of metal which corresponded closely to the observed decrease in enzymatic activity.
        It is concluded that Chelex 100 incubation can sequester Zn from alkaline phosphatase in a manner that is directly proportional to the observed loss in biological activity.  However, further refinement of the chelation process is required to increase Zn removal if this procedure is to be used for apo-substrate generation.