By Vladimir Feld, Ph.D.

St. Petersburg, Russia

INTRODUCTION
Determination of the catalytic activity of cholinesterases inevitably involves storing and transporting the blood samples (plasma, serum, erythrocytes). Since the location of sampling and that of analyzing are not likely to be located together, the samples must be transported, during which the samples may exposed to various environmental conditions. One of the most reliable ways to conserve the samples is to freeze them.

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 cases^2,3 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.

EXPERIMENTAL SECTION

Reagents and materials:
Lyophilized preparations of cholinesterases were used: acetylcholinesterase of human blood erythrocytes (AChE) with an activity of 2.3 U/mg and equine blood serum butyrylcholinesterase (BuChE) with an activity of 0.3 U/mg. Acetylcholine iodide (ACh) and butyrylcholine iodide (BuCh) were used as sources of the cholines. The cholinesterase inhibitor selected was O-isobutyl-S-diethylamino ethylmethylthiophosphonate (IDAM).

Method:
The catalytic activity was measured using Ellman’s method⁴, in which the optical density of the stained product in 0.01 M phosphate buffer, pH 7.4, at 37çC and a wavelength of ž_max = 412 nm was measured.

RESULTS
The effects of various factors (temperature, pH of the solution, ionic background) on the rate of enzyme reactivation and on the rate of the IDAM decomposition were studied. For this purpose, the inhibitor (IDAM) at a final concentration of 1x10^-6 mg/mL was added to the solution of the enzyme (AChE or BuChE) in 0.01 M phosphate buffer, pH 7.5, and incubated for 1 hour at 37çC, until the catalytic activity was completely inhibited. Then either a solution containing varying concentrations of KCl, or a phosphate buffer at varying pH, or ethanol was added. Both the catalytic activity of human AChE in the examined mixture and the content of the free inhibitor (IDAM) in the solution were monitored. In the latter case, the examined solution, containing the enzyme and IDAM, was added to the native enzyme. The decrease in activity of the native enzyme was used to determine the concentration of free IDAM. After analyzing the activity of the enzyme and determining the content of free IDAM, the solution was poured into tubes and stored at various temperatures. The content of free IDAM and the catalytic activity were monitored at certain intervals. In a parallel, water was added to human AChE instead of IDAM. Then the solution that contained KCl, or the phosphate buffer at various pHs or ethanol were stored under the same conditions as the inactivated enzyme. The various solutions (but not the ethanol) and conditions are given in Table 1.

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 +4çC is observed for those solutions containing KCl. The most significant reactivation is observed for those solutions held at - 18çC 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 çC and -18 çC. The most significant restoration of the catalytic activity of the enzyme (up to 60%) is observed at - 18 çC in KCl solutions. The comparison is with control solutions containing no IDAM. The concentration of free IDAM drops both at +4 çC and at -18 çC , however, at -18 çC, this process is more rapid. It is of interest to note that at +4 çC, 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 çC, 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 çC is about the same as that at -20 çC (cf.-18 çC). However, the decomposition proceeds more rapidly at -8 çC, and at -70 çC 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 çC and decreases with further cooling. At -70 çC, 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 çC. 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 çC). 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 çC 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 çC, in a frozen state; it is also observed when the samples are supercooled at -8 çC rather than frozen. No restoration of the enzyme’s catalytic activity was observed. A slight restoration of the catalytic activity at +4 çC begins only after complete disappearance of the inhibitor during a long storage of the enzyme.

DISCUSSION
Abnormal variations of physical and chemical properties of water, for example, those of dielectric permittivity in the perisurface layer are well known⁵. It is also known^6,7 that there is a liquid microphase in ice, particularly in the presence of electrolytes⁸, and the motility of water molecules there can be very high as that of the protons in ice⁹. For example, the theory of the "proton breakdown" that accounts for the damage effect of ionizing radiations¹⁰. A frozen solution not only permits diffusion processes in it, but in certain cases it creates more favorable conditions to accelerate them.¹¹

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.

CONCLUSION
As a result of these experiments, we have been found that the spontaneous ChE reactivation process, inactivated by certain bifunctional organophosphorus inhibitors, is characterized by a negative temperature coefficient and its rate becomes maximal at -8 çC. The spontaneous reactivation process at negative temperatures is accompanied by a complete decomposition of the excess free inhibitor present in a frozen solution. This decomposition of the inhibitor is an enzymatic process, because it ceases completely if the enzyme is denatured. Reactivation of the enzyme in a frozen state completely stops when inhibitors of the radical reactions are introduced, e.g, ethanol or hydroquinone.

REFERENCES

1. Braid P.E. and Nix M., Stability of esterases in stored blood reactions. Am. Ind. Hyg. Assoc. J. 1973, Vol.43, P.360-366.
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3. Szinicz L. et al. Development of a standard operation procedure for determination of acetylcholinesterase (AChE) activity in the blood of organophosphate poisoned patients. mini-CB MTS / PMMA, 1997.
4. Ellman G.L. A new and rapid colorimetric determination of acetilcholinesterase activity // Biochem. Pharm.- 1961.- Vol. 7, P. 88-89.
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7. Condition and role of water in biological objects.// Collected articles. Ed. By Dr. L.P.Kayushin. Moscow. Nauka.-1967.-155pp.
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Table 1
Variations of the activity of inhibited human AChE under various conditions of storage

Table 2
Time history of the catalytic activity of human AChE, of inactivated IDAM and the concentrations of free inhibitor present in the solution of the enzyme at various temperatures and compositions of the solution

Table 3
Effect of temperature on the time history of the catalytic activity of human AChE inactivated by IDAM and on the change of the concentration of free IDAM,[I], present in the solution.