of TEMPERATURE on CHOLINESTERASE STORAGE
By Vladimir Feld, Ph.D.
St. Petersburg, Russia
Frozen samples of plasma or erythrocytes can be held for a long time with their enzymatic catalytic activity practically unchanged1. Less is known about how freezing affects the enzyme activity of blood, especially after the blood is treated with enzyme inhibitors. Although, there have been reports about the long storage of frozen plasma inactivated by sarin2 or frozen erythrocytes inactivated by organophosphorus inhibitors3. In such cases2,3 the catalytic activity during storage suffered no changes.
Actually the problem of conserving frozen samples of inactivated enzymes is not as simple as it may seem. During freezing, the rates of many processes may increase rather than decrease, as is conventionally expected, and this may lead, in principle, to a certain degree of restoration of the catalytic activity.
In this connection, the storage of inactivated acetylcholinesterase (AChE) under various conditions was studied. When cholinesterases are completely inactivated by an irreversible inhibitor, a certain amount of free inhibitor that is unbound with the enzyme may be present in the solution. Therefore, both the catalytic activity of the enzyme and the concentration of the free inhibitor were determined at various times during the samples storage.
Reagents and materials:
Table 1 shows the effect of the composition of the solution on the catalytic activity of human AChE, with the value of the activity expressed in percentage of the control. As shown in the table, the catalytic activity of the inactivated enzyme at pH 7.5 and 8.1 remains unchanged, i.e., no restoration of it occurs. Some restoration of the activity at +4ÁC is observed for those solutions containing KCl. The most significant reactivation is observed for those solutions held at - 18ÁC for 72 hours. The reactivation is greatest for solutions containing KCl in acidic buffered solutions. It should be noted that in an acidic buffer, the dilute solutions of IDAM in the frozen state (in the absence of the enzyme) remain unchanged for many months.
Table 2 shows the change in catalytic activity of human AChE (inactivated by IDAM) and the concentration of the free inhibitor (IDAM) present in the enzyme solution as a function of storage time of KCl and in the phosphate buffer at pH 7.5, at 4 ÁC and -18 ÁC. The most significant restoration of the catalytic activity of the enzyme (up to 60%) is observed at - 18 ÁC in KCl solutions. The comparison is with control solutions containing no IDAM. The concentration of free IDAM drops both at +4 ÁC and at -18 ÁC , however, at -18 ÁC, this process is more rapid. It is of interest to note that at +4 ÁC, only when the concentration of free IDAM drops to 0 (exactly below than 3x10-11 M), the reactivation process starts (after 4 days), whereas at the frozen state in KCl solutions at -20 ÁC, the restoration of the activity of the enzyme begins in a day with free IDAM still present.
The most interesting results are those examining the effect of temperature on the rates of enzyme reactivation and inhibitor decomposition. These results are summarized in Table 3 and show that at positive temperatures, the catalytic activity was not restored during the observation period.
The concentration of the free inhibitor in the enzyme solution decreases with time at all temperatures; the rate of IDAM decomposition at +20 ÁC is about the same as that at -20 ÁC (cf.-18 ÁC). However, the decomposition proceeds more rapidly at -8 ÁC, and at -70 ÁC it terminates completely. As for the restoration of the catalytic activity, the rate of this process increases sharply on freezing (under the conditions of the experiment at positive temperatures the reactivation process proceeds very slowly). The fastest reactivation is at -8 ÁC and decreases with further cooling. At -70 ÁC, neither decomposition of IDAM nor reactivation of AChE occurs.
The addition of 0.1 M ethanol to the solution sharply lowers the decomposition rate, and no restoration of the catalytic activity is observed even at -18 ÁC. The introduction of 0.10 M hydroquinone ( a free radical scavenger) prevents both the reactivation of AChE and the decomposition of free IDAM. The same effect is observed with the introduction of oxymethacyl (5-oxy-methyluracil), which, at concentrations as low as 10-5 M, sharply decreases the reactivation rate on freezing. In control experiments, we determined the decomposition rate of IDAM in the absence of the enzyme, in solutions of KCl and at various pHs. We also measured the decomposition of IDAM after the addition of human AChE (denatured by heating in a solution to 100 ÁC). There was no spontaneous decomposition within the observation period (30 days), and the denatured AChE had no effect on the conservation of the inhibitor in the solution.
Using BuChE in place of human AChE, we also succeeded in observing the process of decomposition of alkylaminophosphonate in the presence of BuChE. As with AChE, denaturing BuChE by heating it to 100 ÁC in solution prevents the BuChE from accelerating the decomposition of such inhibitors.
As a result of these experiments, it has been found that when freezing enzyme solutions containing an excess of the inhibitors of the IDAM type, the inhibitors gradually decompose and the catalytic activity of the enzyme is restored. The rate of these processes depends on many factors. The reactivation process is observed only below 0 ÁC, in a frozen state; it is also observed when the samples are supercooled at -8 ÁC rather than frozen. No restoration of the enzymes catalytic activity was observed. A slight restoration of the catalytic activity at +4 ÁC begins only after complete disappearance of the inhibitor during a long storage of the enzyme.
In our experiments, for example, reactivation in a frozen state occurs only when there is no excess inhibitor. This is explained in our view by assuming the molecules of an organophosphorus inhibitor are in a free state in the solution, localize in the liquid microphase during freezing, and gradually diffuse to the active sites during storage. Localization of dissolved substances in the liquid microphase has been proven by the nuclear magnetic resonance and electron paramagnetic resonance methods, e.g., the Menshutkin reaction in frozen solutions8.
However, even when the excess inhibitor disappears completely, the reactivation process at positive temperatures is extremely slow. This suggests that during freezing of the solution, either the concentration of compounds contributing to reactivation or their lifespan increases. Such compounds may be free radicals. It is known that the lifespan of the free radicals in frozen solutions increases12. The addition of ethanol decreases the reactivation rate, which can be explained in the context of the free radical reaction. In addition, ethanol destroys the structure of water13 thus hindering the "translational and rotary motion"14 of chemicals, which can slow reaction rates.
The free radical mechanism of reactivation is supported by the fact that in a solid state (frozen or absorbed on the surface) the active sites are localized and cannot migrate, hence, reactivation can occur only when migrating particles can move freely and reach an active site. The rate of diffusion of a free inhibitor to active sites in a frozen state may be slower than the rate of the movement of radicals within the frozen solution. This may explain why the process of reactivation is ahead of that of decomposition.
In addition to these studies with the alkylaminothiophosphonate of IDAM, similar experiments were conducted with other inhibitors: the iodomethylate and fluoro (O-isobutylmethylfluorophosphonate) analogues of IDAM, sarin, soman, DDVP and paraoxon. Under similar conditions of freezing, the rates of reactivation of human AChE inactivated by IDAM, its iodomethylate and O-isobutylmethylfluorophosphonate were the same. Human AChE inactivated by sarin, soman, DDVP and paraoxon was not reactivated even when the excess of these inhibitors was removed by dialysis.
Since the interaction of human AChE with alkylaminothiophosphonate results in the breaking of the P-S bond, while the interaction with O-isobutylmethylfluoro-phosphonate causes the breaking of the P-F bond, the products obtained during the interaction of human AChE with IDAM, its iodomethylate and O-isobutylmethylfluorophosphonate are identical. This affirms that the capacity for reactivation is governed by the strength of the bonds of the phosphoryl residue of the inhibitor on the surface of the active site.
The inhibitor decomposition process in a frozen state depends on the concentration of the enzyme and it ceases if the enzyme is denatured. This suggests that inhibitor decomposition observed during freezing of the solutions of human AChE is an enzymatic process, which most probably involves the same active sites that hydrolyze the substrate and react with organophosphorus inhibitors. Alkylaminophosphonate (IDAM) and alkylfluorophosphonate (soman) are most likely to react with the same active site of the enzyme. The strength of the enzyme-inhibitor complex is determined by the nature of the groups bound to the site of esterase rather than to that of anion. The rate of the reactivation process increases with decreasing pH. However, the optimal enzyme reactivation (pH 7.5) is related to the reactions of the native enzyme. In the native enzyme, the anionic site seems to play a more important role than in those reactions of the inactivated enzyme, where the rate increases with decreasing pH, much like the rate of aging.