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Studies of acetylcholinesterase utilizing automated methodology
Studies
of Acetylcholinesterase Automated Methodology
Utilizing
The lxocedure dcscribcd 11csre is l~sctl on :m :~utoinnt,ic, continiwu+ clialvsis, coloriinetric nietliotl wllicll inc;~surw the cluantit.y of acctit acid produced in ,z given t,inlc lwriocl. By utilizing it, for =1ChE assays, clata suitable for kinetic analysis cm bc gciierntctl. The precision anfl convenience of tlic s;vstc~irl make it c~stwiilcly awful for tlics tylws of stutliw.
482
hTE1.X
.\N 1) LIGWIS
and 0.5 ml/liter of a 3376 Rrij:@ 35 (At.las Cheniic:d Inclustrirs, Wilinington, Del.) surf&ant solution, adjusted to pH 8.2 with ?JaC)H. The reagent solutions were prot’ected from atmospheric CO, by storing and aspirating them from bottles fitted with Ascarite tubes. All of the rcnctions were carried out at 23.5”C. The individual solutions were lrlaintaincd at this temperature with the except’ion of enzyme solution, which was kept in an ice bath. The flow diagram used is shown in Fig. 1. The nlanifoltl was de~ignccl for :i variety of studies with AC&IS, :md is therefore ~ornvwl-hat more FLOWRATE ml/ml” ENZYME /
MEMBRANE
248
I 2
BUFFER
I310
BUFFER
0 247
SUBSTRATE COLOR
0
AIR
0 625
REAGENT
0 AIR
SALINE DILUENT
23
I 2 2 6
RECORDER
COLORIMETER 550 In&
FJG.
1, Flow diagram.
complex than was required to generate the data report,ed here. For the purpose of this paper, the effect of varying substrate concentrations on the quantity of a&c acid produred was determined. K, and otK, could t,hen be calculated. The arrows through the mixing coils “a” and ‘Lb” seen in the figure are used to signify varying incubation volumes, equivalent to cliffcrent incubation times. These were employed so that results could be obtained at tlivcrse cnzpmc preincubation and substrate-enzyme incubation t.imer. The former conditions were useful in studying the kinetics of specific, inhibit.or effect,s; these results will be reported elsewhere. Operat.ionally, a series of duplicate substrate samples were aspirated into the system at a sampling rate of 40/hr. The substrate was met by :L previously diluted and preincubated enzyme solution, the mixture was incubatc~l, and it, then pacscd into the dialyzer. A fraction of the acetic acid formed in tile reaction was picked up in the bottom color reagent stream. The loss in color, proportional to the acetic acid in the incubation mixture, WWHmeasured at, 550 rnbf,,,and recorded.
The response of the sy8tein to :kcet,ic acid standards is shown in Fig. 2. These tlata wr(’ obtained by acltling k~~ow\-n clunntitics to l)uffer, an<1 aspirating the resulting solutions 3 s mnplcs, with enzymeJ but no subArat,e, present. The response was linear to :~pp~osinlnt~ely 3.5 X 10. ’ .I/, or an absorbance of 0.38; at higher conccntrat~ions the wwitirit\of thcb assay decreased slight-ly.
The effect of incubat.ion time on the quantit,y of ncet~ic a&l ~~rocluct~l was tested by adjusting the enzyme-buffer ~wcincubnt~ion time, coil “a,” to zero, aspirating :L con&ant Streum of ACliI and XChE through thcx substrate and enzyme lines, respectively, rind wrying the incubation time, coil “h,” from 1 to approviinat~ely 17 min. The h(‘liE concentration wtd 3.2 units/ml and AC%1 was 1.88 X lo-:‘JI in the final reaction misturc~. The results obtained using alwortwlw wluw :ts a it~r:isu~c of actet~ic~ acid concent,ration are shown in Fig. 3. It van bc seen that the rate of hydrolysis is linear for approximately 2 min, nftcr which time the react,ion rate begins to decrensc. The quantities of acetic acid generated as a function of subst,rat,e concentrations at two conditions are listed in Table 1. In the first cspwiment the enzyme concentration was 3.2 units/ml, the preincubation time zero, and the incubation time 2.13 min. The second osperimcnt with
higher substrate concentrations was carried out with 3.9 units/d of AChE and an inruhation tmirne of 4.63 min. Each of the values shown is an average of two trials. The rtandard drvintion of thv rluplicntcs was 0.004 absorbance unit.
latitude of experimental stutlies which could bc carried out in a very short time period. The & value of 5.3 x 10-’ dots not, :qqec with that of apl)rosimatcly 1.9 X lo-’ as determined by Myers (101. Since the intcrprctntion of the: ‘Lconstant” is at best difficult (9‘1, nttcmpts to cq)l:lin the diffrrcnce will not be made in this paper. The analytical syst,em :IS descrilbc~l has bwi USCII for the routine :~ssay of acctylcholinrst,crasc activity from a v:wit%y of ~ourcw. For these analyses t(he enzyme prcparaCons and apln+opriatc stantlardp wer(’ sampled through the enzyme line. The stock substrate concentrat,ion ww 7.33 X lo-” X. Since known enzyme activities coul~l by usc~l in c~onjunction with the samples, a standard curve could b(x constructwl and rwultS c:kulatecl without8 the use of clilut.ion fact,ors :I$ was rcquircd in the kinet.ic studies.
An automatic, continuous-flow tlinlysis system has been utilized to study the hydrolyai;; of ucetylcholine iodide by ncetSylcholinesterase; it can be used for both kinct’ic ptuclies and assays of the enzyme. The method is based on tlw calorimetric measurement of the ncet.ic acid produced. IMeasurements are made at a t,ime at which the acetic acid concentration is proportional to the initkl velocity of the reaction. A variety of experimental conditions wa. q t&ccl nncl it was determined that tbc method yields essentially the same IL and optimum substrate values as that obtained by classical procedures.
7. h‘ms.
At~trl. Bioc/r~r,l.. 13, 305 (196.5). H., AND BURKE, D.. J. Ant. Chenz. Sot. 9. wm, J. r,.. “Enzyme and Metnhnliv Inhibitors,” A~wlemic~ Press, Sew Stork. 1963.
8.
H. H.,
I,INE:WEAW:K.
56, 658 (1934). T’IBI. 1, 1111. 174-7.
127-8.
of Acetylcholinesterase Automated Methodology
Utilizing
The lxocedure dcscribcd 11csre is l~sctl on :m :~utoinnt,ic, continiwu+ clialvsis, coloriinetric nietliotl wllicll inc;~surw the cluantit.y of acctit acid produced in ,z given t,inlc lwriocl. By utilizing it, for =1ChE assays, clata suitable for kinetic analysis cm bc gciierntctl. The precision anfl convenience of tlic s;vstc~irl make it c~stwiilcly awful for tlics tylws of stutliw.
482
hTE1.X
.\N 1) LIGWIS
and 0.5 ml/liter of a 3376 Rrij:@ 35 (At.las Cheniic:d Inclustrirs, Wilinington, Del.) surf&ant solution, adjusted to pH 8.2 with ?JaC)H. The reagent solutions were prot’ected from atmospheric CO, by storing and aspirating them from bottles fitted with Ascarite tubes. All of the rcnctions were carried out at 23.5”C. The individual solutions were lrlaintaincd at this temperature with the except’ion of enzyme solution, which was kept in an ice bath. The flow diagram used is shown in Fig. 1. The nlanifoltl was de~ignccl for :i variety of studies with AC&IS, :md is therefore ~ornvwl-hat more FLOWRATE ml/ml” ENZYME /
MEMBRANE
248
I 2
BUFFER
I310
BUFFER
0 247
SUBSTRATE COLOR
0
AIR
0 625
REAGENT
0 AIR
SALINE DILUENT
23
I 2 2 6
RECORDER
COLORIMETER 550 In&
FJG.
1, Flow diagram.
complex than was required to generate the data report,ed here. For the purpose of this paper, the effect of varying substrate concentrations on the quantity of a&c acid produred was determined. K, and otK, could t,hen be calculated. The arrows through the mixing coils “a” and ‘Lb” seen in the figure are used to signify varying incubation volumes, equivalent to cliffcrent incubation times. These were employed so that results could be obtained at tlivcrse cnzpmc preincubation and substrate-enzyme incubation t.imer. The former conditions were useful in studying the kinetics of specific, inhibit.or effect,s; these results will be reported elsewhere. Operat.ionally, a series of duplicate substrate samples were aspirated into the system at a sampling rate of 40/hr. The substrate was met by :L previously diluted and preincubated enzyme solution, the mixture was incubatc~l, and it, then pacscd into the dialyzer. A fraction of the acetic acid formed in tile reaction was picked up in the bottom color reagent stream. The loss in color, proportional to the acetic acid in the incubation mixture, WWHmeasured at, 550 rnbf,,,and recorded.
The response of the sy8tein to :kcet,ic acid standards is shown in Fig. 2. These tlata wr(’ obtained by acltling k~~ow\-n clunntitics to l)uffer, an<1 aspirating the resulting solutions 3 s mnplcs, with enzymeJ but no subArat,e, present. The response was linear to :~pp~osinlnt~ely 3.5 X 10. ’ .I/, or an absorbance of 0.38; at higher conccntrat~ions the wwitirit\of thcb assay decreased slight-ly.
The effect of incubat.ion time on the quantit,y of ncet~ic a&l ~~rocluct~l was tested by adjusting the enzyme-buffer ~wcincubnt~ion time, coil “a,” to zero, aspirating :L con&ant Streum of ACliI and XChE through thcx substrate and enzyme lines, respectively, rind wrying the incubation time, coil “h,” from 1 to approviinat~ely 17 min. The h(‘liE concentration wtd 3.2 units/ml and AC%1 was 1.88 X lo-:‘JI in the final reaction misturc~. The results obtained using alwortwlw wluw :ts a it~r:isu~c of actet~ic~ acid concent,ration are shown in Fig. 3. It van bc seen that the rate of hydrolysis is linear for approximately 2 min, nftcr which time the react,ion rate begins to decrensc. The quantities of acetic acid generated as a function of subst,rat,e concentrations at two conditions are listed in Table 1. In the first cspwiment the enzyme concentration was 3.2 units/ml, the preincubation time zero, and the incubation time 2.13 min. The second osperimcnt with
higher substrate concentrations was carried out with 3.9 units/d of AChE and an inruhation tmirne of 4.63 min. Each of the values shown is an average of two trials. The rtandard drvintion of thv rluplicntcs was 0.004 absorbance unit.
latitude of experimental stutlies which could bc carried out in a very short time period. The & value of 5.3 x 10-’ dots not, :qqec with that of apl)rosimatcly 1.9 X lo-’ as determined by Myers (101. Since the intcrprctntion of the: ‘Lconstant” is at best difficult (9‘1, nttcmpts to cq)l:lin the diffrrcnce will not be made in this paper. The analytical syst,em :IS descrilbc~l has bwi USCII for the routine :~ssay of acctylcholinrst,crasc activity from a v:wit%y of ~ourcw. For these analyses t(he enzyme prcparaCons and apln+opriatc stantlardp wer(’ sampled through the enzyme line. The stock substrate concentrat,ion ww 7.33 X lo-” X. Since known enzyme activities coul~l by usc~l in c~onjunction with the samples, a standard curve could b(x constructwl and rwultS c:kulatecl without8 the use of clilut.ion fact,ors :I$ was rcquircd in the kinet.ic studies.
An automatic, continuous-flow tlinlysis system has been utilized to study the hydrolyai;; of ucetylcholine iodide by ncetSylcholinesterase; it can be used for both kinct’ic ptuclies and assays of the enzyme. The method is based on tlw calorimetric measurement of the ncet.ic acid produced. IMeasurements are made at a t,ime at which the acetic acid concentration is proportional to the initkl velocity of the reaction. A variety of experimental conditions wa. q t&ccl nncl it was determined that tbc method yields essentially the same IL and optimum substrate values as that obtained by classical procedures.
7. h‘ms.
At~trl. Bioc/r~r,l.. 13, 305 (196.5). H., AND BURKE, D.. J. Ant. Chenz. Sot. 9. wm, J. r,.. “Enzyme and Metnhnliv Inhibitors,” A~wlemic~ Press, Sew Stork. 1963.
8.
H. H.,
I,INE:WEAW:K.
56, 658 (1934). T’IBI. 1, 1111. 174-7.
127-8.