b. pH Dependence of the Inhibition
Heilbronn(18) and Krupka(19) already determined that, at constant fluoride concentrations, the inhibition of AChE by fluoride rises with falling pH value. Because of the low HF concentrations (10-6 - 10-8M) they did not, however, attribute this effect to the activity of the HF molecule, which along with F- is always present in aqueous solution. We therefore undertook the task of determining if the inhibition is always proportional to the given HF concentration, which can be calculated from the dissociation equation for HF. This dissociation equation approximately follows (replacing the activities by concentrations) the relationship:
In a buffered system [H+] is constant. The HF concentration therefore depends on the pH value of the buffer as well as the fluoride concentration. The concentration of NaF used in the experiment can be used in place of [F-] in this equation, since its decrease due to HF formation can be ignored. Since both the enzymatic activity as well as the self-saponification rate of the ACh are pH dependent they must be separately determined for each pH value used. The inhibition is then calculated, after subtraction of the self-saponification, by relating the reaction rate at one pH value with the uninhibited reaction rate at the same pH value. The strength of the pH dependence of the self-saponification becomes apparent in figure 12.
Figure 12- pH Dependence of the Self-Saponification of ACh in Phosphate-Citrate Buffer
A straight line arises when log v is plotted against the pH value. According to that line the hydrolysis is catalyzed by OH-, which is understandable. A negatively charged intermediate condition arises upon alkaline saponification of an ester.
This intermediate condition is stabilized by the positive charge on the quaternary nitrogen atom in the ACh, since the molecule is now outwardly neutral. The saponification catalyzed by H+ would, however, yield a positive intermediate condition, which in the case of ACh is impractical because of the double positive charge. Due to this condition, the balance should lie almost entirely on the left side here.
The change in enzymatic activity as a function of the pH value emerges in figure 13. The pH optimum lies at 7.5 and thereby roughly corresponds to that of the blood.
Figure 13 - pH Dependence of the Enzymatic Activity
Purified AChE from bovine erythrocytes, phosphate-citrate buffer.
Next we carried out a series of measurements to determine, at a constant pH value each time, the dependence of the AChE inhibition on the NaF concentration. Purified AChE from bovine erythrocytes once again served as the enzyme. In addition we used a phosphate-citrate buffer (following Mc.Ilvaine), whose pH value can be varied between pH 8 - 2.2 by mixing 0.1 M citric acid with 0.2M Na2HPO4. We used the region from pH 8 - 6.5. Figure 14 reproduces the course of the inhibition of the enzyme by NaF in the described pH region.
Figure 14 - Enzymatic Inhibition vs. NaF Concentration at Different pH Values
pH = a) 8 , b) 7.5 , c) 7 , d) 6.5.
If the inhibition is caused by the HF molecule, then regions of equal inhibition on the curves should correspond to regions of equal HF concentration. We therefore calculated the HF concentrations for each measured point using equation 24 and compared them to the inhibition, whereby we could determine an agreement, which can be seen in the following table:
Table 1 - F- Inhibition of AChE* at Different pH Values and Equivalent HF Concentration. *purified preparation from the company Serva
Key to abbreviations: Conc. = Concentrations; Int. = Intersections; Calc. = Calculated.
|Parameters of the Segment ||NaF Conc. at the Int. ||Calc. HF Conc. |
Based on the table, it is likely that the inhibition occurs by way of the HF molecule binding to the reactive center of the AChE. The following model could illustrate this fact.
According to a hypothesis posited by Barlow(25), the ester group of the ACh is fixed to the N atom of a histidine residue and to the OH group of a serine residue by way of a dipole bond.
Hypothetical Binding Mechanism of the ACh to the AChE According to Barlow
A strong dipole like the HF molecule should be able to block the acceptor site in question by forming a strong hydrogen bridge. Since the binding is reversible, a competitive inhibition of the AChE should result.
Hypothetical Binding Mechanism of the HF Molecule to the Reactive Center of the AChE.
If one relates the inhibitor constant of the fluoride (KI = 6.26 x 10-3M) to the concentration of free HF one gets a value of KI = 3.2 x 10-7M, which demonstrates the great affinity of the HF molecule for AChE. By decreasing the pH value it is possible, as we have seen, to achieve a meaningful increase in the inhibitory effect of the fluoride on AChE. If the pH value sinks below 7.4 anywhere in the organism, which is often the case, it can result in a stronger inhibition of AChE by fluoride (by way of HF) than in other places with the same fluoride concentration but a pH value of 7.4. The region of the fluoride's effect thereby expands to include smaller concentrations, so that physiological concentrations could also possibly lead to an effect in this direction.