4. Carrying out the Measurements
We ran the reactions in small 10ml measuring flasks, to which we added 5ml of the respective buffer solution ahead of time. Next came 0.5ml of the appropriate enzyme solution. We used a total of three different assays:
Next we added 0.5ml of the appropriate inhibitor solution (in the absence of inhibitor, 0.5ml of buffer), and lastly, after setting the temperature of these assays to 370C, 0.5ml of the radioactively labeled ACh solution, whereby we simultaneously started the stop watch.
When the reaction period was completed, which in most cases took an hour, but when determining the dependence of the reaction rate on substrate concentration only five minutes, we stopped the reaction by adding 2ml of 0.1M sodium tetraphenylborate solution. After a waiting period of one hour, which allowed for completion of precipitate formation, we filled the small flasks to the mark with twice distilled water.
We transferred 1.2ml of solution into a plastic centrifugation vessel and after centrifugation withdrew exactly 1ml of the supernatant, which was then measured directly in Bray's solution. The measured impulse rate yielded a value that was proportional to the amount of acetic acid released and thereby to the quantity of saponified ACh. We expressed the rate of reaction in impulses/reaction time, or, after dividing by the specific activity of the substrate stock solution, as µMol of released acetic acid/reaction time.
Each series of measurements consisted of:
The measured values were constantly corrected by subtracting the self-saponification rate. The following equation gives another overview of the derivation of the measured values (equation 22).
v = Rate of reaction (in µMol acetic acid/minute)
Ib = Total impulse rate (in impulses/minute)
IE = impulse rate of the self-saponification (in impulses/minute)
IU = impulse rate of the natural radioactivity (in impulses/minute)
A = specific activity of the ACh stock solution (in impulses/minute and µMol)
t = reaction time (in minutes)
For the measurement of the pH dependence of the inhibition, the value of vo as well as the self-saponification rate had to be determined separately for each pH, since both values are pH dependent. For the measurements that served to determine the reaction rate as a function of substrate concentration we used a different stock solution for each substrate concentration, whereby the radioactivity per unit of volume remained constant while the ACh concentration changed. In addition, in this case we added an excess of non-radioactively labeled ACh after the reaction was completed, to avoid falling short of the solubility product of the ACh-sodium tetraphenylborate compound.
a. AChE Inhibition by NaF
The following measurements where to be carried out in the concentration range equivalent to substrate saturation, within which the reaction rate is independent of the ACh-concentration. In order to determine this region we carried out a measurement in which we examined the reaction rate as a function of the ACh concentration. The course of the recordings is recreated in figure 7.
Figure 7 - Dependence of ACh Hydrolysis on ACh Concentration.
Phosphate-citrate buffer (following Mc.Ilvaine); pH = 7.7 ; T = 370C ; purified AChE from bovine erythrocytes with concentration: 0.0343mg/ml
Saturation was reached at about 2 x 10-3M ACh. A further increase in ACh concentration leads to a slight reduction in the reaction rate. This observation suggests that there is an inhibition occurring due to excess substrate, which cannot, however, be determined with certainty from this measurement. Since the reaction rate barely changes, even with significantly greater ACh concentrations, we could still carry out the measurement at 1.4 x 10-2M ACh. A measurement of the reaction rate as a function of time showed that the reaction rate remains constant over an hour. Applied to equation 2 this means that in V = k+2 • [ES], becomes independent of [S] (reaction of zeroth order). All of the enzyme is therefore present as ES.
Figure 8 shows a plot of the reaction rate vs. time. 25 µMol (=5 x 10-4M) of the AChE specific inhibitor Physostigmine were added after 30 minutes. The curve bends off, but continues to run linearly. The inhibition is 71.5%. The y-intercept (at t=0) represents the share of C-14 acetic acid in the stock solution.
Veronal/HCl buffer; pH=8.6; 370C; ACh concentration 1.43 x 10-2M. Enzyme as in fig 7.
Next we studied the inhibition of two enzyme assays as a function of the NaF concentration at constant pH. Human blood was extracted from a slightly blocked arm vein, and coagulation was prevented by adding 10% isotonic citrate solution. We separated erythrocytes and serum by centrifugation and washed the blood cells three times with physiological NaCl solution (0.9%), after which the cells were suspended in an equal amount of "Ringer's solution" (Preparation 1). The serum was diluted with an equal amount of Ringer's solution and centrifuged away from the precipitated fibrin (Preparation 2). - The fibrin precipitates because the Ringer's solution contains Ca2+ which, due to the citrate present is no longer sufficiently complexed.– The Ringer's solution used in the subsequent reactions consisted of the following:
double-distilled water – 1,000ml
This solution, whose pH value was 7.4 and whose buffering capacity was relatively limited, was used to offset hemolysis of the erythrocytes, and to create the most natural conditions possible. We proceeded as was described in detail at the beginning of III,A,4. Figure 9 shows the plot of the inhibitory percentage as a function of NaF concentration.
Figure 9 – NaF Inhibition of AChE From Human Erythrocytes and PChE From Human Serum
ACh concentration 7.15 x 10-3M, Ringer's solution, T=370C.
The serum-cholinesterases are visibly more inhibited by the NaF than the AChE from the erythrocytes. The non-monotonic course of PChE inhibition at lower concentrations is probably the result of differing affinities of individual enzymes in the PChE mixture for the inhibitor. According to equations 16,17 and 18, independent of the type of inhibition, a straight line should arise when vo/v-1 is plotted against the inhibitor concentration, assuming that the number of binding sites on the enzyme for the inhibitor is the same as for the substrate. If several enzymes are simultaneously involved in the reaction, a linear dependence only develops when the affinities (reciprocal inhibitor constants) of the individual components for the inhibitor are equally large, which is rather unlikely given the number of PChEs. Figure 10 shows such a plot for the two curves from Figure 9.
Figure 10. Dependence of (vo/v) –1 on NaF Concentration
The course of the curves can, in the case of the serum preparation, be approximated by two straight lines with different slopes. This suggests that the reaction rate is considerably limited by just two components of the enzyme mixture, which have different affinities for the fluoride. The AChE of the erythrocytes yields a linear course, which suggests that the controlled variables of equations 16-18 are fulfilled here.
Next we determined the form of the inhibition from a plot in accordance with Lineweaver and Burk. Purified AChE from bovine erythrocytes (obtained from the company Serva in Heidelberg) again served as our enzyme specimen.
Figure 11 - Lineweaver-Burk Diagram of the Inhibition of AChE by NaF.
Curve 1: uninhibited reaction
Curve 2: 1.43 x 10-2M NaF measured in phosphate-citrate buffer, pH 7.7
The inhibition is competitive and the Michaelis constant of the uninhibited reaction is: KM = 4.2 x 10-4Mol/l. From equation 6 one calculates the inhibitor constant to be: KI = 6.26 x 10-3Mol/l. The affinity of the substrate for the enzyme is therefore, in this case, 15 times as great as that of the inhibitor. Using equation 16, the inhibitor constant can also be calculated from the slopes of the lines in figure 10. The following applies:
The letter “n” stands for the slope of the lines and is graphically derived from figure 10. We took the value for KM from the analysis of figure 11 (4.2 x 10-4M), and the substrate concentration had a value of 7.15 x 10-3M. By substituting these values into equation 23 we obtain, taking the value of the slope (n=1.08 x 102) into account, the inhibitor constant for the AChE of the erythrocytes: KI = 5.6 x 10-4Mol/l. This value, however, means that the dissociation constants of the enzyme/substrate complex (KM) and of the enzyme/inhibitor complex (KI) are roughly the same. A comparison with the constant (KI = 6.26 x 10-3M) derived from figure 11 shows that upon shifting to physiological conditions the enzyme is more strongly inhibited by the fluoride. Since a KM value for the PChE of the serum is not available to us, we can not analogously analyze the serum curve which, due to its non-monotonic course, seems of little purpose anyway. An inhibition of the AChE of the erythrocytes begins at fluoride concentrations > 5 x 10-4M ~ 9.5 mg/l. The serum-cholinesterases are already inhibited at concentrations > 7 x 10-5M ~1.3mg/l. These effects are not yet sufficient to lead to an explanation of a vagotonic effect, as is shown by inhibition of caries at physiological fluoride concentrations.