liberated in the body is probably less than 1 part in a million. The

547.435-292: 577.153 KINETICS OF CHOLINE ESTERASE. By A. J. CLARK, J. RAVENT6S, E. STEDMAN, and ELLEN STEDMAN. From the Departments of Pharmacology and Medical Chemistry, University of Edinburgh. (Received for publication 27th April 1938.) THE kinetics of choline esterase have been analysed by Easson and Stedman [1936] and by Roepke [1937]. These estimations deal, however, with the action of the esterase on concentrations of Ac.Ch. between 0-1 and 1-0 per cent., whilst the concentration of Ac.Ch. liberated in the body is probably less than 1 part in a million. The following investigations were made to determine the action of esterase on Ac.Ch. present in physiological concentrations (i.e. parts per 1000 million). METHODS. The Ac.Ch. esterase used was a concentrated preparation prepared from horse serum by one of us (E. S.). Its activity was estimated by continuous titration at ph 7-4 [Stedman, Stedman, and White, 1933]. At room temperature (160 C.) 1 c.c. esterase hydrolysed 0-25 mg. Ac.Ch. in 100 sec. when the initial concentration of Ac.Ch. was 0-2 per cent. The rate of hydrolysis of butyryl choline at 300 C. showed a concentration of enzyme centres of 7 x 10-5 molar. The latter calculation was based on the known absolute activity of choline esterase as determined by Easson and Stedman [1936], who also found that under nearly optimal conditions at 30 C. each enzyme centre could hydrolyse 1500 molecules acetylcholine per sec. Fig. 1 shows that at a concentration of 1 in 10,000 the rate of hydrolysis of Ac.Ch. at 180 C. is two-thirds that at 370 C. It is probable that the temperature coefficient is higher with higher concentrations of substrate, but it may be assumed for the purposes of this paper that the theoretical maximum rate of destruction of Ac.Ch. at 180 C. is about 1000 mols./sec./enzyme centre. The rate of hydrolysis of Ac.Ch. by the stock esterase solution was determined independently by two methods, namely, chemical and biological. Chemical Estimation.-The chemical titration was carried out by the usual method with bicarbonate buffer at ph 7-4. It was found possible with this method to make measurements at concentrations 77

78 Clark, Raventos, Stedman, and Stedman of Ac.Ch. as low as 1 per 100,000, and fig. 1 shows the results thus obtained. Analysis of these curves shows that at concentrations between 5 and 10 parts per 10,000 the amount of Ac.Ch. hydrolysis per unit of time is constant. With lower concentrations (below 1 per 37 C 180C /0 0,30 *0 MIN. FiG. 1.-Hydrolysis of Ac.Ch. by 3 per cent. esterase; chemical estimation. Bicarbonate buffer; ph 7.4; 18 C. (except top curve). Ordinate: log. conc. Ac.Ch. unhydrolysed; abscissa: time. 10,000) the amount of Ac.Ch. destroyed per unit of time varies very nearly as the concentration since the relation between time and log. concentration of unaltered drug is nearly linear. The curves also show that at any concentration (e.g. 1 in 10,000) the proportion of Ac.Ch. hydrolysed per unit of time depends on the initial concentration employed. These points are of interest in that they confirm results obtained with the less accurate biological method. Biological Estimation.-The rate of hydrolysis of solutions of Ac.Ch. mixed with esterase was estimated on strips of isolated frog's auricle (R. esculenta Hung.) suspended in a bath of 5 c.c. capacity. This preparation has been described by Dale [1937]. The response of the auricle to a series of known concentrations of Ac.Ch. was measured at the beginning and end of each series of experiments. Standardisation of each auricle was essential because of the wide individual variation which has been noted by all who have studied the action of Ac.Ch. on the frog's heart. In the 20 auricles used in these experiments a 100-fold variation in sensitivity was observed; moreover, the sensitivity of an auricle usually increased 2- to 5-fold after a few hours' isolation. This biological method of estimation of Ac.Ch. has a relatively low degree of accuracy, but permits measurements over a huge range of

Kinetics of Choline Esterase concentrations; in particular it permits the estimation of high dilutions of Ac.Ch. since sensitive auricles gave a measurable response to concentrations as low as 1 in 109. The following methods were used: 1. Ac.Ch. was added to the bath, and after a full response had occurred esterase was added and the rate of recovery of the mechanical response was recorded. This method was satisfactory for weak concentrations of enzyme solution (10 per cent. or less). When higher concentrations were employed, the proteins, lipins, etc., in the esterase solution produced considerable changes in the auricular response and made the results uncertain. 2. Concentrations of enzyme solution between 10 and 33 per cent. were tested by first adding the esterase to the bath, allowing the auricular strip to attain equilibrium with the solution and then adding Ac.Ch. 3. The action of undiluted esterase could only be determined by mixing it with Ac.Ch. and adding samples of the mixture to the fluid around the auricle. This method could be used for all concentrations of esterase, but unlike the previous methods it did not provide a continuous record of the changes in the Ac.Ch. concentration; moreover, it was unsuitable for measuring the destruction of concentrations of Ac.Ch. less than 1 per million. The outstanding advantages of the frog's auricle method were its sensitivity and its speed. The auricle gave a full response in less than 5 minutes and was restored to equilibrium in a similar time after washing out. The auricle's response is therefore considerably more rapid than that of leech muscle. The chief objection to the method is that the auricle does not function satisfactorily in heavily buffered solutions. The Ringer's fluid was buffered with a small amount of phosphate (0-05 per cent. at ph 7-5), and it was difficult to maintain the ph constant when the Ac.Ch. concentration was high. The esterase is more sensitive to changes in reaction than is the mechanical response of the auricle; Easson and Stedman [1936] found that the esterase activity at ph 6*8 was two-thirds that at ph 8-0, and we found a similar result; Glick [1937] found that ph 8-4 was the optimum for choline esterase activity. The mechanical response of the auricle, on the other hand, is fairly rapidly depressed by ph 6-5, but is not immediately affected by ph 6-8. This sensitivity of the esterase to changes in ph accounts for the divergent estimates that have been made regarding the effect of changes in ph on the response of tissues to Ac.Ch. and to vagal stimulation. Changes in ph alter the activity of the tissue and the activity of the esterase, and hence two independent variables are present. The biological method was satisfactory for Ac.Ch. concentrations of 0 01 per cent. or less, but with higher concentrations the results were irregular, presumably owing to the acetic acid produced by hydrolysis. 79

80 Clark, Raventos, Stedman, and Stedman Since acidity can inhibit both the esterase activity and the response of the auricle such effects were particularly serious. The action on the auricle of the choline produced by hydrolysis was another source of error in the case of high concentrations of Ac.Ch. It was found that 0-1 per cent. choline produced about 50 per cent. inhibition of the frog's auricle, whilst 0*01 per cent. produced about 20 per cent. inhibition. Hence this source of error did not affect concentrations of Ac.Ch. below 0 01 per cent. The esterase when diluted with Ringer's fluid gradually lost its activity. For example, a dilution of 1 part in 10 was found to be completely inactivated after 3 hours. The phosphate may be responsible for this effect since two of the authors [E. Stedman and Ellen Stedman] have found that phosphates interfere with esterase activity. Dilutions of esterase were always freshly prepared, and chief attention was paid to effects produced within 20 min. of the dilution. The frog's auricle method was found to be most suitable for the study of the activity of the esterase on Ac.Ch. concentrations between 10 and 10,000 parts per 1000 million. Fortunately this covers the probable range of physiological concentrations. In order to reduce the chance of casual error the rate of destruction of Ac.Ch. was, when possible, measured by more than one method. The different methods were found to give concordant results provided the errors mentioned above were excluded. There are certain minor sources of error of which the most important is the fact that the addition of a serum extract, such as is the choline esterase solution, somewhat reduces the sensitivity of the auricle to Ac.Ch. The destruction of Ac.Ch. in the bath by the esterase present in the heart is not a serious source of error because the volume of the fluid, 5 c.c., is about 200 times greater than the volume of the auricle strip (average weight about 25 mg.). The heart esterase activity is unlikely to produce a measurable effect except in the case of the lowest dilutions (1 p.c.) of esterase studied. EXPERIMENTAL RESULTS. Fig. 2 shows examples of the tracings obtained. The changes in concentration of Ac.Ch. were estimated by measuring the amount of inhibition of the mechanical response from minute to minute and estimating the concentration of Ac.Ch. which corresponded to the amount of inhibition. Samples of the graphs plotted from such results are shown in fig. 3. These results prove clearly that with concentrations of Ac.Ch. of 0*01 per cent. and less the amount of Ac.Ch. destroyed per minute depends on the concentration. For example, with 10 per cent. esterase, and with Ac.Ch. concentrations of 100, 1, and 0 01 parts per million, the times of half hydrolysis were 1L8, 0 75, and 0-37 min.

Kinetics of Choline 'Esterase8.rIespectively. Hence the. amounts of Ac.Ch. destroyed per unit time at the three concentrations were in'the'ratio of 100, 2-5, and 0 05. SI81 FIG. 2.-Action of Ac.Ch. and esterase on isolated frog's auricle (10 c.c. bath). The tracings show: Ac.Ch. alone, log. conc. - 8'5, -8, - 7.5, and -7; esterase sol. 33 per cent. Ac.Ch., log. conc. - 6 and - 5; esterase sol. 10 per cent. Ac.Ch., log. conc. -6 and -5; esterase sol. 3 per cent. Ac.Ch., log. conc. - 6. Time in minutes. Fig. 3 shows that. in any single experiment the recovery curve follows a monomolecular course-that is to say, that the. proportion of drug still present that is, hydrolysed per unit of time remains constant.. 100 '34 8*..O -10 3 1-6-7 i4., ii i4 FIG. 3.-Hydrolysis of Ac.Ch. by esterase; biological estimation. Ordinates: log. onc. Ac.Ch. not hydrolysed. Abscissa: time in minutes. The numbers - n the curves show the per cent. concentration of the stock solution of esterase. 'The'curves show, however, that this proportion depends on the amount of drug orliginally introdu'ced. For exrample, the times required by -10Gpercent. esterase to raeduce the voacentration of Ac.Ch. from 1 per 2100 million to 01 per 100 million with. initial concentrations of 1, 100e, and 10,000 per million were respectively 1-2 2'2, and 6 min. The fact VOL. XXVIII., NO. 1.-1938. 6

82 Clark, Raventos, Stedman, and Stedman that the rate of hydrolysis at any particular concentration of Ac.Ch. is dependent on the initial concentration of Ac.Ch. is also shown in fig. 1, and this fact indicates that the hydrolysis is not an uncomplicated monomolecular reaction. The simplest explanation for this reduction of the rate of hydrolysis by increase of initial concentration of Ac.Ch. is to assume that the choline liberated competes with Ac.Ch. for occupation of the enzyme centres. Roepke [1937] concluded that the relative affinities of choline and Ac.Ch. were as 75 and 100. If this figure is correct the choline formed would be more than sufficient to account for the observed inhibition of hydrolysis. In the case of choline esterase containing 1 in 10,000 Ac.Ch., the relative concentrations of choline and Ac.Ch. would be 10,000 to 1 when the latter was reduced to 1 part in 100 million. This figure suggests the probability of a much greater inhibition by choline than that actually observed. Tests made on the rates of hydrolysis of Ac.Ch. alone and of Ac.Ch. in the presence of 1 in 10,000 choline showed, however, no measurable difference in the two conditions. Hence liberation of choline will not explain the retardation of hydrolysis with relatively high concentrations of Ac.Ch. which was found in our experiments. The time required to reduce the Ac.Ch. concentration to one half can be measured directly from curves of the type shown in figs. 1 and 3. Table I. shows the averages of the times thus obtained. From these TABLE I.-TimEs (SECS.) OF HALF HYDROLYSIS OF Ac.CH. HYDROCHLORIDE. Results obtained with biological method, except those in brackets which were obtained with chemical method. Log. conc. Ac.Ch... -3-4 -5-6 -7-8 Log. molar conc. Ac.Ch.. -23 3.3-4-3-5.3-6-3-7.3 Per cent. stock sol. Esterase conc, Calculated molar conc. x 105 100 7 66 21 13 4.5 33 2-3.. 26 14 12 11 20 1-4.. 76 33 27 19 14 10 0 7 110 44 39 28 21 3 0-21 (1920) (480) 420 150 80 54 1 0*07. 1000 240 140 82 figures the rates of hydrolysis (1/time half hydrolysis) can be determined. Fig. 4 shows the relation between enzyme concentration and rate of hydrolysis. This figure shows that, as a first approximation,

Kinetics of Choline Esterase there is a linear relation between the concentration of enzyme and the rate of hydrolysis. That is to say the amount of Ac.Ch. destroyed per unit of time per unit of enzyme is approximately constant. This 83 0108 /0 20 30 FIG. 4.-Ordinate: Rate of hydrolysis = 1/time in sees. half hydrolysis. Abscissa: + per cent. of stock esterase solution. The curves show results with concentrations of Ac.Ch. from 1 in 10,000 to 1 in 100 million. conclusion was tested by calculating these qualities. Table II. shows that the amounts of Ac.Ch. hydrolysed per unit of enzyme per unit of time at any concentration of Ac.Ch. are fairly constant for concentrations of enzyme from 3 to 33 per cent. TABLE II.-RATE OF DESTRUCTION OF AC.CH. /ig./sec./c.c. UNDILUTED ESTERASE. (Calculated from Table I.) Range of log. concentration to to to to6 t7 8 o- c.h. 4-3 - 53-6-3-7.3-8-3 jug. per c.c. removed by 1 500 50 5 0 5 0 05 0*005 half hydrolysis. Concentration of esterase as per cent. of stock solution. 100 7-6 2-4 0-38 0-11 33.. 5-8 1 1 0-12 0-014 20.. 3.3 0-76 0 093 0-013 0-0018 10.. 4-5 1-14 0-128 0-018 0-0024 3 (12.7) (4.7) 0 39 0-11 0-021 0-0031 1.... 05 0-21 0-036 0-0061 Averages 10-2 4-1 0 7 0-13 0-02 0-0034

,84 Clark, Ravent6s, Stedman, and Stedman The averages of the figures for each consideration of Ac.Ch. are plotted in fig. 5 Which shows a fairly exact linear relation between log. cqnc. Ac.Ch. and log, amount- hydrolysed (,ug./sec./c.c. undilutted FIG. 5.-Rate of hydrolysis of Ac.Ch, Vertical lines: biological measurements; circles: chemical meoeurements. Ordinate: log. concentration Ac.Ch.; Abscissa: 1og. amount Ac.Ci. hydrolysed,.g./sec./c.c. stock esterase solution. esterase) with concentrations of Ac.Ch. less than 1 in l04. The relation can be expressed by the following formula: log. K + 0-83'log. x =log. y (x =conc. Ac.Ch. and y = amount hydrolysed). Hence K(x)0'83 =y. One of the authors [Clark, 1926] calculated the rate of destruction of Ac.Ch. by the frog's ventricle and concluded that over a range of concentrations from 1 per 100 million to 1 per 1000 the destruction could be expressed by exactly the same formula as that given above. DISCUSSION. Our results show that the hydrolysis of Ac.Ch. by esterase follows a course which is common in enzyme actions. When the concentration of drug is excessive in relation to the concentration of enzyme centres the amount hydrolysed per unit of time is constant, but when the concentration falls below a certain figure the proportion of drug present which is hydrolysed per unit of time is nearly constant and hence the

Kinetics of Choline Esterase amount hydrolysed is nearly proportional to the concentration. [Amount hydrolysed varies as (conc.)0;83.] Analysis of fig. 1 shows that with a concentration of enzyme centres of 2 x 10-6 molar the amount of Ac.Ch. hydrolysed per unit of time is nearly constant when the concentrations of Ac.Ch. is higher than - 3 molar, whilst the amount destroyed becomes nearly proportional to the concentration when the drug concentration falls below - 4 molar. The biological method was unfavourable for the study of high concentrations of drug, but the results obtained agreed with the conclusion that the amount of Ac.Ch. hydrolysed became nearly constant when the molar drug concentration was 1000 times or more the molar, concentration of enzyme centres. The results with high concentrations of Ac.Ch. agree with those obtained by Stedman and Stedman [1935] and by Roepke [1937]. The results with Ac.Ch. in low concentrations are of importance because the probable physiological range of Ac.Ch. concentrations lies between 1 and 1000 parts per 1000 million. In this range of concentration the amount of Ac.Ch. hydrolysed per unit of time varies as (conc.)0-83, and this introduces an error into various estimates that have been made of the rate of hydrolysis of Ac.Ch. in muscle. For example, Marnay and Nachmansohn [1938] found that 100 mg. muscle containing nerve endings split 0 4 to 0-8 mg. Ac.Ch. in 60 min. when the Ac.Ch. concentration was 1 per 1000. They concluded that probably 0-265 mg. Ac.Ch. were hydrolysed p.h. at the nerve endings in 100 mg. of muscle, and that therefore 0-001 u.g. Ac.Ch. could be hydrolysed in a frog's sartorius (300 mg.) in 5 m./sec. They estimated the nerve endings as less than 1/1000 of the muscle volume and a concentration of 0.001,u.g. Ac.Ch. in 300/1000 cmm. is 1 part in 300,000. Fig. 4 shows that if 12 parts Ac.Ch. are hydrolysed per unit of time at a concentration of 1 per 1000 then about 0 5 part will be hydrolysed per unit of time when the concentration is 1 in 300,000. If 265,.g. Ac.Ch. are hydrolysed per hour at a concentration of 1 per 1000, then 265/60 x 60 =0 074 u.g. are hydrolysed per sec., and at a concentration of 1 in 300,000 the hydrolysis will be 0-003,u.g. per sec., instead of 0-2,t.g./sec. as calculated by Marnay and Nachmansohn [1938]. It is, moreover, interesting to note that if the Ac.Ch. release were only one-thirtieth of the amount assumed above (i.e. 0 000,033,u.g.) and the local concentration were thus reduced to 1 part in 10,000,000, then the hydrolysis of this amount would still require about 0-2 sec. Fig. 4 shows that, as a first approximation, the amount of Ac.Ch. destroyed per unit time varies as the esterase concentration. The results are definitely against the assumption that khe hydrolysis increases as some power of the esterase concentration. Our results therefore 85

86 Kinetics of Choline Esterase suggest that with any probable concentration of esterase the hydrolysis of even minute quantities of Ac.Ch. is likely to take at least 0-1 sec. The results show that the number of Ac.Ch. molecules hydrolysed per sec. per enzyme centre is surprisingly small. The molar concentration of undiluted esterase is 7 x 10-6, and Table I. shows that such a solution hydrolyses half the Ac.Ch. present at the same molar concentration in about 15 sec. Hence 30 molecules of esterase only hydrolyse 1 molecule of Ac.Ch. per sec., and the rates of hydrolysis at lower concentrations of Ac.Ch. are still smaller. SUMMARY. 1. The rate of hydrolysis of Ac.Ch. by esterase has been measured by chemical and by biological methods and the results obtained show a good agreement. 2. With high concentrations of Ac.Ch. (more than 0.01 per cent.) the amount hydrolysed per sec. is constant. 3. With low concentrations of Ac.Ch. (less than 0.001 per cent. or 5 x 10-5 molar) the amount of Ac.Ch. hydrolysed per sec. varies as (conc. Ac.Ch.)0.83. 4. The amount of Ac.Ch. hydrolysed per sec. is approximately proportional to the esterase concentration. 5. These results indicate that with the undiluted stock esterase solution the time for half hydrolysis of low concentrations of Ac.Ch. (1 in 10 million) is about 0.2 sec. The expenses of this research were defrayed by a grant from the Moray Fund of the University of Edinburgh, for which the authors desire to tender their thanks. REFERENCES. CLARK, A. J. (1926). J. Phy8iol. 64, 123. DALE, A. S. (1937). Ibid. 89, 316..GUCK, D. (1937). Biochem. J. 81, 521. MARNAY, A., and NACHMANSOHN, D. (1938). J. Phy8iol. 92, 37. ROEPKE, M. H. (1937). J. Pharmacol. Baltimore, 59, 264. STEDMAN, E., STEDMAN, ELLEN, and WHITE, A. C. (1933). Biochem. J. 27, 1055. STEDMAN, E., and STEDMAN, ELLEN (1935). Ibid. 29, 2107. STEDMAN, E., and EASSON, L. H. (1936). Proc. Roy. Soc., B, 121, 142.