Stable Cholinesterase Preparations as Laboratory Standards of Activity JOSEPH ti. FLEISHER, S U S A N SPEAR, and E L E A N O R J. POPE Znzyme Chemistry Branch, Chemical Corps M e d i c a l Laboratories, A r m y Chemical Center,
activity can be applied to four of the most commonly used procedures for determining the activity of this enzyme.
Stable enzymatic standards for cholinesterase activity have been developed on filter paper disks using bovine red cell cholinesterase as the enzyme source. When stored in the refrigerator over desiccant, the disks retain their activity for at least 6 months. The enzyme activity may be eluted from these disks by solutions employed in any of the common procedures for the determination of cholinesterase activity. Since the cholinesterase activity found for the disks by any of these procedures varies by only 2 to 3%, the disks may be used as a reproducible standard source of cholinesterase activity for comparison between different analytical methods, or as a control on the reliability of a single method.
T
Md.
EXPERIMENTAL
H E use of various methods for the determination of cholinesterase activity has led t o some confusion as t o how activities measured by one procedure can be connected with or interpreted in terms of another. For example, cholinesterase activity has been measured in various laboratories, in terms of ApH per hour by the electrometric method ( 8 ) , microliters of carbon dioxide in a given time by the manometric method ( 2 ) , milliliters of standard alkali by the constant p H procedure (6), and micromoles of acetylcholine hydrolyzed in unit time by the colorimetric method ( 5 ) . The activity measured is generally not the same from one method to the other because the various procedures differ with respect to time, temperature, substrate, salt concentration, and other factors affecting enzyme activity. Variable results within any one method may be due to real changes in the enzyme activity or t o variability in the procedures applied for measuring that activity. A similar problem in usual analytical procedures is solved by the use of reproducible standards which can be made from the chemically pure substance and analyzed along with an experimental sample. Cholinesterase is not available as a chemically pure substance, and the enzyme has to be measured in terms of its activity. But if units of reproducible constant activity could be made, these could be used as standards to ascertain the source of variation in a given method for determining cholinesterase activity where variable results were being obtained. Moreover, the different methods of measuring enzyme activity could be compared with one another in terms of the activity of the cholinesterase standard. To make such a cholinesterase standard, it is necessary t o obtain a stable preparation which ran be stored for reasonable periods of time under conditions available to most laboratories. The factor of cost would militate against the use of lyophilized preparation every time a comparative study of activity was desired. Filter paper had been used for collecting blood specimens for cholinesterase determinations (3). However, quantitative recovery of the red blood cell cholinesterase was not obtained by the authors upon application of the methods outlined [Ainsworth and others ( 1 , 4 ) ] . Severtheless the use of filter paper would allow many identical unit sources of enzyme activity t o be made from small aliquots of a highly active source of cholinesterase commercially available in the form of a purified red blood cell cholinesterase. Because so many unit filter paper sources could be made from a few milliliters of the original enzyme solution, they would be relatively cheap. This report describes the preparation of such standards, which have been stable for as long as 6 months, and gives detailed directions by means of which these unit Eoiirces of cholinesterase
Materials and Equipment. COLORIMETRIC lIETr$oD. Phosphate buffer, 0.131-11, pH 7.2, rras prepared by mixing 7 parts by volume of a solution of 23.8 grams of disodium hydrogen phosphate dihydrate per liter and 3 parts of a solution of 18.2 grams of potassium dihydrogen phosphate per liter, pH being adjusted to 7.2 if necessary. Acetylcholine, 0.04M, 0.7266 gram of acetylcholine chloride (Merck) in 100 ml. of 0.00151 acetate buffer, pH 4.5: stable indefinitely in the cold. Acetylcholine, '0.004X. Acetvlcholine, 'O.O04X, 0.04M solution diluted with ith 9 volumes of phosphate buffer. Made freshly in quantity required for the analvses. H$droxylamine hydrochloride 2M, 27.8 grams dissolved in distilled water to 200 ml. Above solutions are kept refrigerated \Then not in use. Sodium hydroxide, 3.5M, 28 grams dissolved in distilled 1%-ater to 200 ml. Alkaline hydroxylamine, equal volumes o hydrochloride and 3.5M sodium hydroxide use in a quantity required for the samples being analyzed. Made u p freshly for each set of samples run. Hydrochloric acid, concentrated acid (specific gravity, 1.18) diluted with 2 volumes of water. Ferric chloride, 0.37M, 10 grams of ferric chloride hexahydrate dissolved t o 100 ml. in 0.lM hydrochloric acid. Potassium chloride, 0.3X, 22.4 grams of potassium chloride dissolved in 1 liter of distilled water. Klett-Summerson photoelectric colorimeter with green filter s o . 54. Water bath, thermostatically controlled at 25' C. ELECTROMETRIC METHOD. llichel's red cell buffer ( 8 ) ,0.02N sodium barbital (4.1236 grams), 0,004M potassium orthophosphate (monobasic) (0.5446 gram), and 0.60Jf potassium chloride (44.730 grams). For 1 liter of buffer, the reagents are dissolved in 900 ml. of distilled water; 28.0 ml. of 0.lM hydrochloric acid are added while shaking the solution, and the volume is then made t o mark. The pH should be 8.10 a t 25' C. Acetylcholine, 0.1lM, 2.00 grams of acetylcholine chloride in 100 ml. of distilled water. A few drops of toluene are added t o the above solutions as a preservative, and they are kept refrigerated when not in use. Beckman pH meter Model G. Water bath, thermostatically controlled a t 25' C. MANOMETRIC METHOD. Bicarbonate buffer, 0.025M containing 0.03M magnesium chloride. Dissolve 2.1 grams of sodium bicarbonate in 500 ml. of water. Add 6.1 grams of magnesium chloride hexahydrate. Make to 1 liter with additional distilled water. Equilibrate with a rapid stream of 5% carbon dioxide in nitrogen for 10 minutes, and keep stoppered nhen not in use. Acetylcholine 0.11M (see electrometric method). Warburg manometric apparatus. TITRIMETRIC ( CONSTAWT PH) METHOD. Potassium chloride, 0.6.W, dissolve 44.8 grams of potassium chloride in 1 liter of distilled water. Sodium barbital, 0.0231, dissolve 4.1236 grams in 800 ml. of distilled water. Add 0.1S hydrochloric mid to pH 7.4; then make t o 1 liter with additional vater. Acetylcholine, 0.11M. (See electrometric method.) Sodium hydroxide, standardized, approximately 0 . 0 2 s . Beckman, Model G p H meter with shielded glass electrode. Magnetic stirrer. Titration vessel, 8- t o 10-ml. capacity, thermostated a t 25" C. The pH meter, stirrer, and titration vessel should be grounded ( t o minimize electrical disturbances of the glass electrode). Microburet, graduated in 0.001 ml. I n addition, each of the above methods requires purified bovine red cell cholinesterase ( Winthrop-Steams) obtained as a nearly white lyophilized powder readily soluble in aqueous solution and general laboratory equipment and other chemicals as indicated in the text. Procedure and Results. PREPARATIOX O F FILTERPAPER
1080
1081
V O L U M E 27, NO. 7, J U L Y 1 9 5 5 Table I. Effect of Stabilization Medium on Initial Recovery of Enzyme iictivity from Disks Stabilization Medium 0.3 -1f KC1
+ 0.1% gelatin
+ 0.5g6 gelatin
Std.
+ 1% gelatin + 2 7 , bovinealbumin
S t d . .\I. Std. A I . S t d . 31.
+ 4r:
bovine albumin
Concentration of Enzyme Preparation,
5%
i: 1 1 0.2
1 0 2 1
Assay Method Titrimetric Electrometric Titrimetric Electrometric Manometric Electrometric Manometric Electrometric llanometric t
% of y e t Activity Recovered 29 25 92 82
81
80 81 92 91
0.2 S t d . 11. 8% bo\-inealbumin 0.2 Manometric 32 Std. 11. (Standard medium): 0.3.U potassium chloride, 0.5qo bovine Iiemoglobin, 0.008.11 phosphate buffer: adjusted t o pH 7.4 after addition of stabilizer couiponent a n d before addition of enzyme preparation.
+
STAZIDARDS. A stabilizing medium is first prepared containing 0.3J1potassium chloride, o.5y0bovine hemoglobin, 470 bovine albumin, and 0.008M phosphate buffer. The p H is adjusted to i . 4 . Then the purified bovine red blood cell cholinesterase is added to the stabilizing medium to make a 1 solution of enzyme for use in the colorimetric, constant pH, and electrometric method; and a 0.2% solution of enzyme for the manometric method. Aliquots of 0.02 ml. of the freshly made enzyme solution are placed on Whatman No. 31 filter paper with a microburet. The area occupied by the enzyme solution is delineated by the hemoglobin present in the stabilizing medium. After drying for 20 to 30 minutes, the filter paper standards (disks) are cut out and stored over desiccant in the cold. ELUTION. For the colorimetric, electrometric, and titrimetric methods. A disk prepared with the 1% enzyme solution is placed in a 25-ml. Erlenmeyer flask and covered with 5 ml. of a leaching solution (the composition of which is given in each of the assay methods above). The flask is rotated to wet the disk and then placed in the refrigerator. Maximal elution is obtained nithin 1 hour. For the manometric method. S o separate elution is necessary. 1Iaximal elution is obtained within the 15-minute period required for temperature equilibration and gassing. (The procedure is briefly outlined below.)
ing volume of a standard alkali added to time expresses the enzyme activity and may be recalculated in terms of micromoles of alkali added per minute, a figure numerically equal to the micromoles of acetylcholine hydrolyzed per minute. Manometric Method ( 2 ) . A disk prepared with the 0.2% enzyme is cut into several thin ribbons which are placed in the main section of a Warburg vessel and covered with 2 ml. of 0.025.11 bicarbonate buffer, pH 7.4, containing 0.03M magnesium chloride. The side arms receive 0.2 ml. of 0.11M acetylcholine. The vessels are attached to their manometers, which are then transferred to the water bath a t 25' and gassed with 5 % carbon dioxide-95% nitrogen for 15 minutes while shaking. The remainder of the procedure is carried out in the usual manner. The carbon dioxide output in microliters was plotted against time, and found to be linear throughout the BO-minute interval studied.
EFFECT O F STABILIZATION MEDIUJIo s RECOVERY OF EXZYME ACTIVITYOF DISKS. The effect of different stabilizing media used for the preparation of the disks on the subsequent recovery of activity was measured by making the enzyme stock solution in the various media shown in Table I. The activity of suitable aliquots along with controls of the enzyme solution was then determined by the methods indicated in the tahle.
I
a
3.51
2 3.0 = I
/
METHODSFOR DETERWSISG ACTLVITY OF DISK CHOLINESSTANDARDS. After elution cholinesterase activity in
TERASE
the eluate may be measured by any of the commonly employed procedures To illustrate assay procedures applicable to measuring the activity in the eluates of the disks, brief descriptions of four commonly used methods are given
Figure 1. Relation between cholinesterase activity and concentration of enzyme on disks
Colorimetric Method. Each disk is eluted in 5 ml. of 0.3M potassium chloride. One-milliliter aliquots of eluate are then incubated with 1 ml. of 0.004M acetylcholine-phosphate solution for 10 minutes a t 25" C. The residual acetvlcholine is determined colorimetrically by the Hestrin ( 7 ) procedure. The quantity of acetylcholine hydrolyzed is obtained by subtracting the residual quantity measured from the 4 micromoles originally added, since nonenzymatic hydrolysis is nil under these conditions. Details have been described (6). Electrometric Method (8). Each disk is eluted in 5 nil. of a solution of 1 volume of LIichel's red cell buffer diluted with an equal volume of water, followed by removal of 2-ml. aliquots of eluate. .4t known intervals, 0.2 ml. of O.llM acetylcholine is Jdded with mixing and the initial p H is read I minute later with the Beckman pH meter. The vessels containing the mixture of eluted enzyme and substrate are incubated a t 25" C. followed by leading of the pH value exactly 60 minutes after the initial pH waq read. Readings are corrected for nonenzymatic hrdrolysis. Titrimetric (Constant pH) Method (6). Each disk is eluted i n 5 ml. of 0.6M potassium chloride-0.170 gelatin solution at pH 7.4. -42-ml. aliquot of eluate is transferred to the titration vessel and is folloived by 1.5 ml. of additional 0.6M potassium chlorideO.lyOgelatin solution and 1.0 ml. of 0.02.11 sodium barbital. The pH of the mixture is adjusted to p H 7.4 and 0.5 ml. of O.llM acetylcholine is added with mechanical stirring. Small volumes of standard sodium hydroxide are then added at frpquent intervals so as to maintain the p H a t 7.4. The volume of sodium hydroxide an! the time a t which the galvanometer needle of the pH meter registers null a t pH 7.4 are recorded. The slope of the plot relat-
Media buffered in the pH range of optimal stability of the enzyme lead to increased recovery from the dried disks. Increasing the protein content of the stabilizer up to a certain optimum also serves to increase the recovery from the disks. Above this optimum, as in the instance of the increase in bovine albumin from 4 to 8%, there is a precipitous decrease in the recovery, which is probably due to the slower drying of the heavy pellicle deposited by the enzyme in the 8% albumin leading to possible partial denaturation RELATION B E T W E E S E S Z Y \ f E ,kCTIVITY ATD QUANTITYO F CSZYMEOS DISKS. The relation between cholinesterase activity and the quantity of enzyme on the disks was studied by dissolving the lyophilized preparation in the stabilizing medium containing 0.5% gelatin so as to make a 5% stock solution. Portions of the 5% preparation ~i ere then further diluted n ith additional stabilizer to yield 1, 2, 3, and 4y0 solutions. Disks Rere made, and eluted in 5 ml. of potassium chloride-gelatin solution at pH 7.4 followed by determination of the activity of a suitable aliquot of the eluate by the titrimetric method. Results are shown in Figure 1. The activity recovered from the dried disks is proportional to the amount of the enzyme solution originally placed on the disks.
0
I 2 3 4 5 PERCENT CONCENTRATION OF ENZYME APPLIED TO PAPER
1082
A N A L Y T I C A L CHEMISTRY
Table 11. Reproducibility of Methods for Determining Cholinesterase Activity of Disk Standards Disk
Colorimetric
1 2 3 4 5 6 7 8 9 Mean S.D. % variation
Electrometric
Acetylcholine hydrolyzed, micromoles 1.44 1.49 1.49 1.40 1.45 1.42
l,45 &0.034 2.3
Titrimetric
Manometric
R a t e of addition of 0.01755M Carbon dioxide sodium hydroxo u t p u t in 30 min., pl. ide, ml./min. 0.0178 79 0.0182 81 0.0181 78 0.0178 87 0.0186 78 0.0186 79 81 82 85 0,0182 81 0 0,00036 3 10 2.0 3.6
(ApH), (ApH/hr.) 1.43 1.42 1.41 1.44 1.40 1 42 1.39 1.43 1.40 1.42 10.017 1.2
*
6.4
Therefore A = 1.45 X 5 t = 10 m = 0.02 ml. of a 1% solution = 0.2 mg. Substituting:
U
=
725 1 X - = 3.63 micromoles/minute/mg. 10 0 2
Electrometric Method. The results obtained in this method, which are in terms of ApH units, may be converted to micromoles of acetylcholine hydrolyzed by titrating hlichel's red cell buffer with a standard acid, noting the successive changes in pH produced during the titration by given increments of acid. The data may be plotted on a curve as shown in Figure 2 and the micromoles of acetylcholine hydrolyzed corresponding to a given change of p H may be read off the curve. According to the results of Table 11, 2 ml. of a 5-ml. eluate produced a ApH change of 1.42 pH units per hour equivalent to 12.8 micromoles of acetylcholine hydrolyzed (Figure 2). Therefore : A = 12.8 X 2.5 t = 60 minutes m = 0.2 mg.
tc
Substituting: 1 12" 2'5 X - = 2.67 micromoles/minute/mg. U = 60 0 2
6.8 6.6
PH 7.0
Colorimetric Method. One fifth of the eluate hydrolyzed 1.45 micromoles in 10 minutes.
I
#
Titrimetric Method. TWO milliliters of a 5-ml. eluate required 0.0182 ml. of 0.01755M sodium hydroxide per minute (see Table 11). 0.0182 ml. of 0.01755M sodium hydroxide = 0.319 micromole
76v,, Therefore :
-4 = 0.139 X 2.5 t
m
= 1 = 0.2 mg.
Substituting:
70 8 8 0
2
4
6
8
,
,
IO
12
MICROMOLES HCI
Figure 2.
,
14
,
jy
= 0.319
1
16
ADDED
Titration curve of Ilichel's red cell buffer
APPLICABILITY O F DISK STAXD.4RDS TO VARIOES LIETHODS USED FOR ~IEASURISG CHOLISESTERASE ACTIVITY. The appli-
cability of the disk standards to various methods used for measuring cholinesterase activity was established by analyzing six or more disks prepared from a single source by each of the methods described, at 25" C. The activities measured in the units peculiar to each method are given in Table 11. The reproducibility obtainable by each method is also presented. CALCULATIOX O F SPECIFIC -4CTIVITY FOR EACH;\lETHOD O F DETERJIISISG CHOLIXESTERASE AiCTIVITY O F DISKS. In order to have some measure of comparison for the methods described in
the previous section, the activity measured by each method in units peculiar to it must be converted into a common denominator of measurement. The unit of activity chosen (specific activity) has been micromoles of acetylcholine hydrolyzed per minute per milligram of enzyme source. The manner of converting the activities measured by each method is given below. The general formula used is: A C, specific activity = -
1 t X m
where A = activity of total eluate from 1 disk t = time in minutes used for the measurement of enzymatic activity m = weight in milligrams of enzyme preparation applied to the disk
2.5
1 - = 3.99 micromoles/minute/mg. 0.2
x
Manometric Method. Mean activity (Table 11),81 microliters of carbon dioxide:
81 A = __ micromoles of acetylcholine hydrolyzed in 30 minutes 22 4 t = 30
Table 111. Specific Activity of Disk Standards by Various .Methods of Analysis s o . of
Method Colorimetric Electrometric Titrimetric Alanometric
Disks 6 9 6 9
Snecific Activitv. Micromoles Acetylcholine Hvdrolyzed/JIin. M g . 3 63 1 67 3 99 3 01
S.D.
=to. 11
Table IV. Effect of Storage Conditions and Stabilization Media upon Retention of Initial Activity of Disk Standards Stahiiization Media Std. albumin
+
4% bovine
Time of Storage ' 22 days
1' ii ~
+ +
22 days 6 months
Ternperature, C. 3 23 25 25 3
Relative Humidity,
70
Ob 05
50 100
Ob
70 of Initial Activity 100 94 87 62 99
Std. AI. 0.5% gelatin 6 months 3 Ob 78 Std. AI. 1 % gelatin 6 months 3 Ob 75 0.3.11 potassium chloride, 0.5% ,bovine 0 Std. M . (standard medium): hemoglobin, 0.008.M phosphate buffer: adjusted t o p H 7.4 after addition of stabilizer component a n d before addition of enzyme preparation. b Stored over anhydrous calcium chloride.
V O L U M E 2 7 , NO. 7, J U L Y 1 9 5 5 = 0.02 ml. of a 0.2% solution = 0.04 mg. Substit,uting:
VL
'I
=
81 1 1 X - X - = 3.01 micromoles/minute/mg. 22 4 30 0 04
Table I11 summarizes the specific activities as calculated for each of the methods above together with the standard deviation to be expected for each met,hod of assay. The variability found for each method as calculated from the ratio of the standard deviations t o the mean specific activit,y is wit'hin a few per cent in every instance. STABILITY O F CHOLISEBTER.4SE DISKS UPON STOR.4GE. The stabilit,y of the disks prepared in the various media was studied by determining the activity of freshly made disks by one cr more of the methods previously described; t'hen st,oring similar diska under the conditions and for the time noted in Table IV followed by a det,ermination of their activity. Inspection of Table I T T shorn that optimal retention of initial activity i.5 achieved by storing in the cold over a desiccant. Disks decrease t o 62% of t,heir initial act,ivity in 22 days when exposed t o an atmosphere of 100% humidity a t room temperature. The effect of the stabilization inediuni is shown by a coniparison of the disks prepared in the bovine albumin n-ith those prepared in the gelatin medium and stored under identical conditions. The disks prepared in the 47, bovine albumin had lost virtually no activity in 6 months, whereas those prepared in the gelatin medium had lost one fourt,h of t,heir initial activity. DISCUSSION
The conditions used in the four methods for measuring the cholinesterase activity of the disks differ in salt concentration, presence of protein stabilizer, substrate concentration, and period of time used in measurement,. Because these factors all affect the stability and activity of the enzyme, it is to be expected that the specific cholinesterase act,ivit)- of a given preparation, meaeured by different procedures but expressed in the same units, will vary from one method to the nest. However, the variability of 2 to 3% found for the activity of the disks by any of the procedures described here permits their use in calibrating the activity measured bj- one met,hod in terms of another, provided that a given set of conditions is consistently followed. I n addition, the disks may serve to point out sources of experimental variations
1083 where these are significantly greater than the 2 to 3% variation attributable to the disks themselves. As the degree of purity of the original enzyme preparation ultimately determines the activity of the disks, preparations other than the one used in this study will yield different specific activities. Standardization of different techniques may nevertheless be achieved with any such preparation. Under the conditions specified in this report the titrimetric procedure gave the highest specific activity, followed in turn by the colorimetric, manometric, and electrometric procedures. The last named method has the advantage of requiring the fewest reagents, and permitting inany samples t o be measured with a minimum of manual operations, thus minimizing the tediousnem involved in the careful performance of the titrimetric procedure when the latter is performed manually. The colorimetric method, while requiring more reagents than the other procedures, is the fastest, and compares favorably in precision and sensitivity, with any of t,he methods studied. If eluates of filter paper standards are used for inhibition studies a t inhibitor concentrations yielding a lo^ level of cholinesterase activity, t'hen the colorimetric method offers t'he additional advantage of determining the amount of acetylcholine remaining after exposure to the residual enzyme in the eluate, rather than the small amount of acetic acid formed in excess of the blank determination. .4CKNOWLEDGMENT
The authors wish to thank Bernard J. Jandorf for helpful criticism on the manuscript. LITERATURE CITED
(1) Ainsmorth. A I . , Davies, D. R., and Rutland, J. P., to be published. ( 2 ) Ammon, R , Arch. g e s P h y s i o l (Pflugers). 233, 486-91 (1933). (3) Augustinsson, K B , and Heimburger, G , d c t a Physiol Scand., 30 (41.45-54 11953). . ~~~, (1) Berry;
