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Chromogenic substrates for cholinesterases: 1-[2-Thiazolylazo]-2-acetoxybenzene derivatives
+NALYTICAL
48, 33-44
BIOCHEMISTRY
Chromogenic
(1972)
Substrates
for
Cholinesterases:
l -[*2-Thiazolylazol-2-acetoxybenzene
c. Chemical
VAN
HOOIDONK, Laboratory
Derivatives
G. DE BORST, F. A. MITZKA, C. C. GROOS
RVO-TA\‘O, Received
Rijwijk August
(Z.H.),
The
ANIl
Netherlands
11, 1971
For the determination of cholinesterases it is convenient to have a direct spectrophotometric observation of the substrate hydrolysis. Acetylsalicylic acid (1) , benzoylcholine (21, a-naphthyl acetate (3)) indophenyl acetate (4)) 2,&dichloroindophenyl acetate (5)) and l-methylacetoxyquinolinium iodides (6) have been used as substrates in such procedures. We report here on a new t.ype of chromogenic substrate (I) derived from well-known heterocyclic azo dyestuffs t’hat have found a wide application in analytical chemistry for the spcctrophotometric determination of metal ions (7,8). A difference in color exists between the ester (I) and the alcohol (II) at the pH at, which the alcohol dissociates. This difference in color can be used for the spectrophotometric assay of cholinesterases. Ye CH /3
Ye CH /3
CH /3 o=c CHOLINESTERASE pH
+CH3CO0
7.0
5
II
I
S = .5-CH3,
Y = CHoSOl
S = 4-N(CH,)L,
Y = I (IB),
M.ATKR1AI.S
2-Aminothiazole KG (Germany).
(IA),
blue .AXI:,
(m.1~. S8.590°C’) 33
@ 1972 by
Academic
Press,
Inc.
light
yellow
-
blue
(IIAI
red (IIH)
METHODS
was obtainccl from EGA-Chemic~
e
2-Hydmxy-5-methylbenzene (p-cresol, m.p. 33-35°C) was obtained from BDH Chemicals (England). 2-Hydroxy-4-dimethylaminohenzene was purchased from Dr. Th. Schuchardt (Germany) and recrystallized from benzene, m.p. 85°C. Methyl iodide (redistilled hefore use, h.p. 43”C1, dimethyl sulfate (used without further purification), and acetic anhydride (AnalaR) were purchased from BDH Chemicals. Sodium nitrite was obtained from E. Merck (Germany). ,411 other chemicals were reagent grade. Principle
of Synthesis
I- [2-Thiazolylazo] -2-hydroxy-5-methylbenzene and I- [2-thiazolylazo] -2-hydroxy-4-dimethylaminobenzene were prepared in our lahoratory essentially according to the method described by *Jensen (9)) i.e., by reaction of the diazonium salt of 2-aminothiazole with the appropriate phenol. The acetates were obtained by rcfluxing with a large excess of acetic anhydride. The quaternization was accomplished with an excess of methyl iodide or dimethyl sulfate in benzene as solvent. Experimental Preparation of l- [d-thiazolylaxol-2-hydroxy-5-methylbenzene dirtbethyl sulfate. Sodium nitrite (3.5 gm I was dissolved in concentrated sulfuric acid (35 ml) at, a temperature below 60°C. After cooling in ice, acetic acid (40 ml) was added in a 10 min period. The mixture was cooled below 10” and a solution of 2-aminothiazolr (5 gm, 0.05 mole) in acetic acid (30 ml) was added dropwise. After addition, the mixture was stirred for 1 hr and t,hen poured into crushed ice (70 gm). This solution was added slowly to a well-cooled (0” ) solution of p-cresol (5.5 gm, 0.051 molej in ethanol (60 ml). After stirring for 45 min at O”, water (200 ml) was added. The precipitate was collected, dried, and recrystallized from ethanol/water. Yield: 6.5 gm (59%) of 1-I 2-thiazolylazol-2-hydroxy-5-methylbenzene, 1n.1). 130°C. The product (3.3 gm ) mentioned was dissolved in benzene (140 ml). Then dimethyl sulfate (5 ml) was added. The mixture was heated for 45 min at 50” and left overnight at room temperature. The precipitate was collected, dried, and recrystallized from ethanol/ether. Yield: 2.3 gm (44%‘i, m.p. 143°C. Calculated for C,,H,,O,N,S,: H 4.4, N 12.1, S 18.5.
Preparation
C 41.73,
of I- [%thiazolylazo]
H 4.38,
N
12.17,
S 18.57.
-2-acetoxy-5-methylbenzene
Found:
C 41.7,
dimethyl
CHROMOGER’IC
SUBSTRATES
FOR
35
CHOLIKESTERASES
sulfate. A mixture of l- [2-thiazolylazo] -2-hydroxy-5-methylbenzene (5 gm) and acetic anhydride (26 ml) was heated to reflux for 12 hr. After cooling, the mixture was poured into crushed ice (300 gm) and st’irred for 1 hr. The precipitate was filtered off, dried, and recrystallized twice from ethanol. Yield: 4.2 gm (70% I of l- [2-thiazolylazo]-2-acetoxy-5met’hylbenzene, m.p. 139°C. The product (4 gm) mentioned was dissolved in benzene (50 ml). Then acetic anhydride (1 ml) and dimethyl sulfate (4 ml) were added. After heating for 30 min at 35”, the mixture was left overnight. The formed crystals were filtered off, recrystallized from acetic anhydride, and dried ilz VUCUO.Yield: 2.1 gm (35%), m.p. 214-216°C (dec.‘). Calculated for C,,H,,O,N,,S,: H 4.4, N 10.9, S 16.6.
Preparation methiodide.
C 43.40,
H 4.42,
N
10.85.
S 16.55.
Found:
C 43.6.
of l-[2-thiazolylazo]-2-hydrox~-~-dimethylaminobenzenzene
2-Aminothiazole (10 gm, 0.10 mole) was dissolved in a mixture of concentrated sulfuric acid (18 ml) and water (80 ml 1. A solution of sodium nitrite (7 gm) in water (60 ml) was added dropwise under stirring, keeping the temperature at 3” by external cooling with ice. After the addition was complete, stirring was continued for 15 min. Then a solution of 2-hydroxy-4-dimethylaminohenzene ( 14 gm, 0.102 mole I in a mixture of concentrated sulfuric acid (6 ml ) and water (40 ml) was added during 10 mm, keeping the temperature at 5”. The precipitate was filtered off, suspended in water (500 ml ) , and neutralized with sodium carbonate (20 gm) The red precipitate wax filtered off and tlried in. VUCUO.Yield: 12 gm (48% ) of l- ]2-thiazolylazol-2-hydroxy-4dimethylaminobenzene, m.p. 127°C. Methyl iodide (5 ml ) was added to a solution of the product (2.5 gm) mentioned in benzene (100 ml). The mixture was refluxed for 1 hr and left overnight at room temperature. The formed crystals were filtered off, dried, and recrystallized from ethanol/ether. Yield: 1.2 gm (31% ) , m.p. 219°C. Calculated for C,zH,,ON,SI: C 36.93, C 37.1, H 3.9, I\T 14.4, S 8.3, I 32.3.
H
Preparation methiodide.
I-2-acetoxy-4-dimethylaminobenzene
of l- Id-thiazolylazo
3.87,
S
14.36.
8 8.22,
I 32.52.
Found:
l- [2-Thiazolylazo] -2-hydroxy-4-dimethylaminobenzene (24 gm) was dissolved in acetic anhydride (50 ml). Pyridine (1 ml) was added and the mixture was heated to 120°C for 4 hr. After cooling, the mixture was poured into crushed ice (400 gm) and stirred for 1 hr. The precipitate was filtered off, dried, and recrystallized from acetone/water (1:2). Yield: ,&? gm (64% ) of l- [2-thiazolylazo] -2-acetoxy-4-dimethylaminobenzene, m.p. 156°C. The product (12 gm) mentioned was dissolved in a mixture of acetone
36
VAN
HOOIDONK
ET
AL.
(300 ml) and benzene (25 ml). Methyl iodide (25 ml) was added and the mixture was allowed to stand for 1 week at, room temperature. The desired product1 crystallized as violet-colored crystals, which were filtered off and washed several times with dry ether. Yield: 8 gm (45%), m.p. 232°C. Calculated for GJH1,OPNPSI: C 38.90, H 3.96, N 12.96, C 39.3, H 4.1, N 12.9, S 7.4, I 29.2.
S 7.42, I 29.36.
Found:
Enzymes Home serum butyrylcholinesterase (BuChE) (acylcholine acylhydrolase, EC 3.1.1.8‘) was purchased from Organon Ltd. Oss, The Netherlands, and had a specific activity of 3.5 pmole acetylcholine perchlorate (BDH Chemicals) hydrolyzed per minute per milligram protein (temperature 25°C; medium 20 ml 0.0005 M phosphate buffer, pH 7.5; substrate concentration 0.02 M) Protein concentration was 0.005 mg/ml. Acetylcholinestemse (AChE) (acetylcholine acetyl-hydrolase, EC 3.1.1.7) was a partially purified bovine erythrocyte enzyme from Winthrop Laboratories, Inc., New York, N. Y., and had a specific activity of 1.5 pmole acetylcholine perchlorate hydrolyzed per minute per milligram protein (temperature 25°C; medium 20 ml 0.0005M phosphate buffer, pH 7.5; substrate concentration 0.003 M) . Protein concent,ration was 0.01 mg/ml. Buffers Phosphate buffer (0.067 M, pH 7.0). 2.98 gm Na2HP0,*2H,0 (E. Merck) was dissolved in 250 ml distilled water (solution I), 2.28 gm KH,PO, (E. Merck) was dissolved in 250 ml distilled water (solution II). 150 ml solution I was titrated to pH 7.0 with 95 ml solution II. Phosphate buffer (0.05 M, pH 7.0) was made analogously. Spparatus All absorbance measurements were made with a Zeiss PM& II spectrophotometer fitted with a water-jacketed cell compartment. The temperature was controlled by a Haake ultrathermostat within O.l”C. All react,ions
were
carried
out, in Teflon-stoppered
1 cm silica
cells.
The
l The position of quaternization was proved by running NMR spectra on a JEOGC-6OH spectrometer, using dimethyl sulfoxide-ds as a solvent and tetracompound the proton methylsilane as internal reference. In the nonquaternized signals of the N(CHA2 group appeared at 3.11 ppm (singlet). In the quaternieed compound the proton signals of the N(CHsj2 group appeared at 3.32 ppm (doublet) and a new signal was observed at 4.07 ppm (singlet) which is characteristic for the N+CH, protons of the quaternized thiazole nucleus.
CHROMOGENIC
SUBSTRATES
FOR
3'7
CHOLINESTERASES
absorption spectra were recorded on a Zeiss RPQ 20A spectrophotometer. All pH measurements were performed on a Vibron pH meter, model 39A (Electronic Instruments, Ltd.) equipped with a glass electrode type GHS33 and a saturated potassium chloride-calomel reference electrode type RJ23. The specific activities of both enzymes were determined with a Radiometer Autotitrator (pH meter type TTTlc, scale expander type PHA630T, and syringe buret recorder type SBRB) equipped with a glass electrode type G202C and a saturated potassium chloride-calomel reference electrode type K401. The electrode compartment was thermostated by a Haake ultrathermostat, within O.l”C. The pH was effectively controlled within 0.02 pH unit. RESULTS
Spectrophotometric Measurements The spectral properties of the new substrates and their hydrolysis products were determined in a 0.05 M phosphate buffer, pH 7.0, at 25°C.
A
0.9
0.8
07
t v, Z u 0
0.6
0.5
0.2
0.1 0.0 CONCENTRATION
x
IO5
(M)
FIG. 1. Plot of optical density vs concentration of l-[2-thiaaolylazol-2-acetoxy& ,nethylbenzene dimethyl sulfate (01, 1-[2-thiazolylazol-2-hydroxy&methylbenzene dimethyl sulfate (W ), l-C2-thiazolylazol-2-acetoxy-4-dimethylaminobenzene methiodide (O), and l-~2-thiazolylazol-2-hydroxy-4dimethylaminobenzene methiodide (0) in 0.05 M phosphate buffer, pH 7.0, in 1 cm silica cells at wavelengths of maximum absorption (nm) at 25°C. Values in parentheses are molar extinction coefficients (mole-’ cm-‘).
38
VAN
0.9
700
HOOIDONK
(I
600
500
450
400
350
ET
AL.
A)
700 WAVELENGTH
650
600
550
500
450
Cnmi
FIG. 2. Sprctral changes involved in hut?-r?-lcholinesterasc-~atalq-zed hydrolysis of l-12-thiazol~-lazoI-2-aceto~~-5-~~e~h~lhenz~~n~~ dimethyl sulfate (IA) and 1-[2-thiazol~lnzol-2-ncetos~-4-dimeth~1:m~inobenzenr methiodide (IB) in 0.05 M phosphate huffrr. pH 7.0, nt 25°C. Time in minutes. Enzyme conrentrntion was 0.035 unit/ml rwction mistnrr.
Figure 1 shows the relationship between optical density and the concentration of each species at the wavelength of maximum absorption. The rates of spont,aneoushydrolysis were determined in water and in 0.05 111phosphate buffer, pH 7.0; the half-lives are 123 and 10.3 hr for I - [2-thiazolylazo] -2-acctoxy-5-methylbenzene dimethyl sulfate and 134 and 12.9 hr for I- [2-thiasolylazo] -2-acetoxy-4-dimethylaminohenzene methiodide, respectively. Figure 2 shows the spectral changes during the butyrylcholinesterasecatalyzed liyclrolysis which are identical with the spectral changes observed during spontaneous hydrolysis. The spectra of the hydrolysis products are pH-dependent and are determined by the degree of ionization of the phenolic hydroxyl groups. A con\-entional potcntiometric titration (10) gave p&, values of 4.2 and 5.8 for l- [2-thiazolylazo] -2-hydroxy-5-methylbenzenc dimethyl sulfate and 1- 12- thiazolylazo] - 2 - hydroxy -4- dimethylaminobenzene methiodidc, respectively. This means that at pH 7.0 about 98% and 94r/rj, respectively, of the phenolic hydroxyl groups are present in the anionic form. The values in parentheses given in Fig. 1 for these compounds refer to the molar extinction coefficients of the phenolate ions. Enzyme
Activity
Measurements
Stock enzyme solutions (1 mg/ml) were prepared in 0.067 M phosphate buffer, pH 7.0. Stock substrate solutions were made in water (6 mg 1-[2-thiazolyl-
CHROMO(;EKI(‘
SI-RSTRATES
b’OR
(‘HOI,INlCSTERASI~:S
39
azoj -2-acetoxy-5-methylbenzene dimethyl sulfate and 7 mg I- [2thiazolylazo] -2-acetoxy-4-dimethylaminobenzene methiodide, reqwctively, in 20 nil). A number of enzyme solutions was prcparcd as follows: 0.1, 0.2, 0.4, solution were m:de ul) to 0.8, 1 .o, 2.0, and 3.0 ml of the stock cnzymc 12.5 ml with 0.067 JI phosphate buffer, pH 7.0. The assay solutions were prepared in 1 cm silica cells by mixing thoroughly 1.5 ml of the enzyme solution with 0.5 ml of the stock substrate solution. Both enzyme and stock substrate solutions were pre-eq”ilil)rat~,(l at 25°C. The final substrate concentration was 2 X lo--‘M and the fin:11 pH, 7.05. 15 set after mixing, the recording of the absorption rhanger was started and cont~inueclevery 15 wc ul) to 20% completion masimally. The hydrolysis of I- ]2-thiazolylazo I-2-acctoxy-5-Inctllylhcnzcnc climethyl sulfate was monitored at the wavelength of maximum absorption of the liberated phenolate ion (585 nm) ; because of the overlapping spectra of l- [2-thiazolylazo] -2-acetoxy-4-dimethylaminobenzene methiodide and its hydrolysis product we followed the hy(lrolysis at 485 nm. At both wavelengths the difference in absorbance between the substrate and product is maximal. Each run was corrected for the spontaneous hydrolysis. Plots of initial rates of hydrolysis vs enzyme concentrations are given in Fig. 3.
UNITS
OF
BvChE/ml
UNITS
OF
AChE/ml
FIG. 3. Plots of initial rate of hydrolysis (AOD/min) of I-[2-thiazolylazol-2acetoxy-5-methylbenzene dimethyl sulfate (IA. 0) and 1-[2-thiazolylazol-2-awtox.;4-dimethylaminobenzene methiodide (IB, 0) vs enzyme concentration (units of enzyme/ml reaction mixture) in 0.05 M phosphate buffer, pH 7.05, and 25°C. Substrate concentration 2 X 10.” M. Hydrolysis of IA was monitored at 555 nm. and of IB at 485 nm. Reactions were carried out in 1 cm silica cells.
40
VAN
HOOIDONK
Determination
ET
Kinetic
AL.
Parameters
As illustrated in Fig. 3, the init,ial rates of hydrolysis are linear with the enzyme concent,ration, as predicted by the Michaelis-Menten equation :
VnlaxlSl
(1)
’ = Km + WI where Vmax represent,s the maximal rate Menten constant, and [S] the substrate Evaluation of the values of K,, and measuring the initial rates of hydrolysis tration. Equation 1 can be rearranged
PI -=
of hydrolysis, K, the Michaelisconcentration. I’,;,, is usually accomplished by in relat’ion to substrate concento give:
-VKm + vl, max
V
ISI
(2)
A plot of ] S] /v vs ] S] yields a straight line, the slope equals l/V,I,,,, and the abscissa intercept equals -K,,. A typical example of such a plot is given in Fig. 4. The K,, and I’,,.,, values were calculated according to the weighted regression technique of Wilkinson (11). See Table 1. Inhibition
of ,Substrate
Hydrolysis
by Acetylcholine
To present evidence that the new subst’rates are hydrolyzed by cholinesteraee and not by any other esterase eventually present in the com-
.AL_/.-l-
-2-10
I2
-K
m
3
4
5
6
7
I 8 [S]
-II 9 x IO5
IO
II
I2
(M)
FIG. 4. Plot of [Sl/w vs [Yl for substrate reaction of 1-[2-thiazolylazol-2-acetoxy5-methylbenzene dimethyl sulfate with butyrylcholinesterase (0.21 unit/ml mixture) in 0.05 M phosphate buffer. pH 7.0, at 25°C.
reaction
CHROMOGENIC
SUBSTRATES
FOR
TABLE Michaelis-Menten
41
CHOLINESTERASES
1 Parameters
K, and V,,,,, values for the butyrylcholinesterase (BuChE)and acetylcholinesterase (AChE)-catalyzed hydrolysis of 1-[2-thiazolylazol-Z-acetoxy-Smethylbenzene dimethyl sulfate (IA), 1-[2-thiazolylazo]-2-acetoxy-4-dimethylami~~obenzene methiodide (IB), and acetylcholine. Conditions: for IA and IB, 0.05 fl!i phosphate buffer, pH 7.0; for acetylcholine, 0.005 41 phosphate buffer, pH 7.5. BuChE and AChE concentrations in all experiments 0.06 m&ml react,ion mixture. Temperature 25°C.
Enzyme
Substrate
BuChT?
I,4 IB Acelylcholine IA IB Acetylcholine
AChE
a From * From
literature literature
V mar (pmoleimin) (1.2 (1.X (3.1
f 0.2) + 0.2) 1.46 + 0.2) 2.68
x x X x
10-j 10-j 10-“a 10-5
X 10-4”
0.01 0.005 0.21 0.009 0.09
(12). (13).
mercial enzyme preparations, we studied the enzyme-catalyzed hydrolysis of l- [Z-thiazolylnzo] -2-acetoxy-5-methylbenzene dimethyl sulfate in the presence of varying concentrations of acetylcholine. Treating acetylcholine as a competitive inhibitor, the initial rate of substrate hydrolysis is given by
L’,,,,,
VI+!&
(3)
1+I’l
[S]
(
K,
>
where V,,,aX represents the maximal rate of hydrolysis without added acetylcholine, [S] the substrate concentration, [I] the acetylcholine concentration, K, the Michaelis-Menten constant of the substrate, and iii the inhibition constant of acetylcholine. In the reciprocal form:
Thus, in the case of competitive inhibition, a plot of l/v vs [I] should yield a straight line with a slope of li,/V,,,,K~ IS] and an intercept of (1 + &/[S] )/VI,,,,. With butyrylcholinesterase (0.26 unit/ml reaction mixture, other experimental conditions identical as described above under Enzyme Activity Measurements), we observed AOD/min values (AE) of 0.210, 0.198, 0.162, 0.150, and 0.102 without, and in the presence of 2, 10, 20,
42
vAK HOOIDOSK El‘ AL
and 40 X lo-” M acctylcholine, respectively. A plot of IJAE vs [I] yields a straight line and gives a Ki value of 2.2 X lo-” M. With acetylcholinesterase (0.10 unit/ml reaction mixture 1, we observed LIE values of 0.210, 0.138, 0.120, 0.090, 0.072, and 0.060 without and in the presence of 2.5, 5, 10, 15, and 20 X 10-l M acetylcholine, respectively. The plot of l/!& vs [I] gives a fii value of 1.4 X lo-’ M. The calculated Ki values are of the same order as the K,,, values of acctylcholine reported in the literature (see Table 1). This indicates that the new substrates and acet~ylcholine are hydrolyzed by the same cnzymc. Norcover, the enzyme-catalyzed hydrolysis of acctylcholine is also inhibited by addition of the new substrates. For example, with an acctylcholinesterase preparation (0.5 mg in 20 ml 0.0005 M phosphate buffer, pH 7.5, and at 25°C) the hydrolysis of acetylcholine (,lO-” MI1 was fount1 to produce 1.07 pmole acid/mm and that of l- [2-thiazolylazo] -2-acetosy-5-methylbenzene dimethyl sulfate t 5 X lo-’ 1M 1, 0.24 pmole acid/min, while that of a mixture of the two was 0.40 pmole acid/ min. When two different enzymes were operative, we should have observed the sum, namely, 1.31 qlole acid/mm. DISCUSSION It is interesting to compare the kinetic parameters of the thiazolylazo esters with those of acetylcholine. The most striking feature is the high affinity, characterized by the K,, value s, of l- ]2-thiazolylazo] -2-acetoxy5-nlethylbcnzenc dimethyl sulfate for both cholinesterases and 1- ] 2thiazolylazo ] -2-acctoxy-4-dimcthylaminobenzene methiodide for butyrylcholinestcrasr. The latter compound turned out to be a poor substrate for acetylcholinesterase (see Fig. 3 1 ; therefore no attempts were made to evaluate the kin&c, parameters. In order to obtain a better understanding of the stereochemical requircmcnts of the new substrates, we studied their space-filling Courtauld atomic models. These moclcls reveal a distance of approximately 5A between the quaternary nitrogen atom and the carbonyl-carbon atom. For ncetylcholine, this distance is about 4.7 A. These values agree well with the value of 4.3-4.7 A reported by Hollingworth et nl. (141 for the clistnncc in bovine crythrocyte cholinesterasc bet.wecn the anionic site and the reactive serine residue which forms a covalent. bond with the carbonyl-carbon atom of the ester. Wilson and Quan (15 I reported earlier a value of approximately 5 B for the same distance. In view of these data the excellent fit of the thiazolylazo esters on the enzymes seems understandable. On the other hand, the Ii,,,ax values illustrate that acetylcholine is by far the better substrate.
CHROMOGEKIC
SVBSTRATES
FOR
43
CHOLIKESTERASES
As the enzymic ester hydrolysis comprises a three-step reactioncomplexation, acetylation, and deacctylation-the rate-limiting step for the thiazolylazo esters and acetylcholine may he different. The poor substrate properties of I - [2-thiazolylazo] -2-acetosy-4-dimethylamino~wnzene methiodirlc for aretylclioline~tcrase may hc unclcrstantl:rl~lc in the same terms. The pwsent investigations produc~~(l practical data only ; ~norc dctailed studies on the rntw of acetylation and tlcacctplation will proT’ide more basic information. In conclusion we may state that the new substrates may hc urcd for the assay of cholinesterases. The highly colored hydrolysis products absorb at long wavelengths where tlistur~):tncc by protein absorption is (xxcludctl. The relatively high molecular extinction cocfficicnt~s ancl the favorable Ii,,, values permit the use of low conwntrations. The rccommended concentration is 2 X lOmA31. Enzyme quantities :IS low as 0.015 unit of ~~utyrylcholinesterase anrl 0.005 unit of ace~ylcliolincstcrnrC can he tlctcrminctl. The initial rates of hydrol;c 7s.q .-L at constant suhatrntc concentration are linear with the enzyme concentration. The whole prorcdure is simple and rapid am1 does not require addition of extra reagents or coni~~licatecl manipulations. Although azo com~~ou~~ls might he light >cnGtivcL, WV found no indication that on csposure to daylight the new suhstratcs arc dccomposccl 01 converted into isomeric products. Nevertheless we recommend storing the new substrates in wll-stoppered brown bottles and excluding n moist environment to prevent spontaneous hydrolysis.
For the spectrophotometric assay of hutyrylcholinestcrasc ( BuChE ) (EC 3.1.1.81 and acetylcholinesttrase I ACrhE 1 (EC 3.1.1.7) n new type of chromogenic substrates has heen dcvelopecl. The synthesis and the BuChEand AChE-catalyzed hydrolysis of I- [2-thiazolylazo] -2-aretosy-5-methylbcnzcIlc tlimethyl sulfate and l- [2-thiazolylazo] -2-acetoxy-4-dimethylltmi~~inobenzene methiotlide arc described. ACKNOWLEDGMENT The
aut.hors
XC indebted
to Mr.
N. Kramer
for
thr
&nwntnl
nnalyscs.
REFERENCES 1. HOFSTEE, 2. KALOW,
3. 4.
GOMORI. &IAMRR,
B. J. H.. Science TV., AND
LINDSAY.
G., J. Lab. D.
s..
AND
Clip. ~AMSOS,
114, 128 (1951) H. A., (‘ntl. J. &ocher?a. dfrci. 42, 445 (JR”?). R. M.. A/lul. (‘/rent.
Physiol.
33, 568 (1955)
30, 251 (1958).
44 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
VAN
HOOIIX~NK
ET
AL.
DE HORST. C., HOOGE, F. N.. AND ARKENBOUT, Cr. J., Nature 182, 1017 (1958). PRINCE, A. K., Arch. Biochem. Biophys. 113, 195 (1966). ANDERSON. R. G.. .4x0 NICKLESS, G., Analyst 92, 207 (1967). K.~w.~sE, A., Japan Annlyst 11, 621 (1962). JENSEN, B. S., Acta Chem. Scaud. 14, 927 (1960). Constants of Acids and Bases.” ALBERT, 8.. AXD SERJEAST. E. P., “Ionization Mcthum. London. 1962. WILKINSON, G. S., Biochem. J. 80, 324 (1961). YAKOVLEV. V. A., AND AGABEKYAN. R. S.. Biokhi,miyn 32, 293 (1967). &XJPKA. R. M.. Biochemistry 3, 1749 (1964). HOLLINGWORTH. R. M.. F~K~To, T. R.. AND METCALF. R. L.. J. Agr. Food Chem. 15, 235 (1967). WILSON. I. H., AND QUAN, C., Arch. B&hem. Biophys. 73, 131 (1958).
48, 33-44
BIOCHEMISTRY
Chromogenic
(1972)
Substrates
for
Cholinesterases:
l -[*2-Thiazolylazol-2-acetoxybenzene
c. Chemical
VAN
HOOIDONK, Laboratory
Derivatives
G. DE BORST, F. A. MITZKA, C. C. GROOS
RVO-TA\‘O, Received
Rijwijk August
(Z.H.),
The
ANIl
Netherlands
11, 1971
For the determination of cholinesterases it is convenient to have a direct spectrophotometric observation of the substrate hydrolysis. Acetylsalicylic acid (1) , benzoylcholine (21, a-naphthyl acetate (3)) indophenyl acetate (4)) 2,&dichloroindophenyl acetate (5)) and l-methylacetoxyquinolinium iodides (6) have been used as substrates in such procedures. We report here on a new t.ype of chromogenic substrate (I) derived from well-known heterocyclic azo dyestuffs t’hat have found a wide application in analytical chemistry for the spcctrophotometric determination of metal ions (7,8). A difference in color exists between the ester (I) and the alcohol (II) at the pH at, which the alcohol dissociates. This difference in color can be used for the spectrophotometric assay of cholinesterases. Ye CH /3
Ye CH /3
CH /3 o=c CHOLINESTERASE pH
+CH3CO0
7.0
5
II
I
S = .5-CH3,
Y = CHoSOl
S = 4-N(CH,)L,
Y = I (IB),
M.ATKR1AI.S
2-Aminothiazole KG (Germany).
(IA),
blue .AXI:,
(m.1~. S8.590°C’) 33
@ 1972 by
Academic
Press,
Inc.
light
yellow
-
blue
(IIAI
red (IIH)
METHODS
was obtainccl from EGA-Chemic~
e
2-Hydmxy-5-methylbenzene (p-cresol, m.p. 33-35°C) was obtained from BDH Chemicals (England). 2-Hydroxy-4-dimethylaminohenzene was purchased from Dr. Th. Schuchardt (Germany) and recrystallized from benzene, m.p. 85°C. Methyl iodide (redistilled hefore use, h.p. 43”C1, dimethyl sulfate (used without further purification), and acetic anhydride (AnalaR) were purchased from BDH Chemicals. Sodium nitrite was obtained from E. Merck (Germany). ,411 other chemicals were reagent grade. Principle
of Synthesis
I- [2-Thiazolylazo] -2-hydroxy-5-methylbenzene and I- [2-thiazolylazo] -2-hydroxy-4-dimethylaminobenzene were prepared in our lahoratory essentially according to the method described by *Jensen (9)) i.e., by reaction of the diazonium salt of 2-aminothiazole with the appropriate phenol. The acetates were obtained by rcfluxing with a large excess of acetic anhydride. The quaternization was accomplished with an excess of methyl iodide or dimethyl sulfate in benzene as solvent. Experimental Preparation of l- [d-thiazolylaxol-2-hydroxy-5-methylbenzene dirtbethyl sulfate. Sodium nitrite (3.5 gm I was dissolved in concentrated sulfuric acid (35 ml) at, a temperature below 60°C. After cooling in ice, acetic acid (40 ml) was added in a 10 min period. The mixture was cooled below 10” and a solution of 2-aminothiazolr (5 gm, 0.05 mole) in acetic acid (30 ml) was added dropwise. After addition, the mixture was stirred for 1 hr and t,hen poured into crushed ice (70 gm). This solution was added slowly to a well-cooled (0” ) solution of p-cresol (5.5 gm, 0.051 molej in ethanol (60 ml). After stirring for 45 min at O”, water (200 ml) was added. The precipitate was collected, dried, and recrystallized from ethanol/water. Yield: 6.5 gm (59%) of 1-I 2-thiazolylazol-2-hydroxy-5-methylbenzene, 1n.1). 130°C. The product (3.3 gm ) mentioned was dissolved in benzene (140 ml). Then dimethyl sulfate (5 ml) was added. The mixture was heated for 45 min at 50” and left overnight at room temperature. The precipitate was collected, dried, and recrystallized from ethanol/ether. Yield: 2.3 gm (44%‘i, m.p. 143°C. Calculated for C,,H,,O,N,S,: H 4.4, N 12.1, S 18.5.
Preparation
C 41.73,
of I- [%thiazolylazo]
H 4.38,
N
12.17,
S 18.57.
-2-acetoxy-5-methylbenzene
Found:
C 41.7,
dimethyl
CHROMOGER’IC
SUBSTRATES
FOR
35
CHOLIKESTERASES
sulfate. A mixture of l- [2-thiazolylazo] -2-hydroxy-5-methylbenzene (5 gm) and acetic anhydride (26 ml) was heated to reflux for 12 hr. After cooling, the mixture was poured into crushed ice (300 gm) and st’irred for 1 hr. The precipitate was filtered off, dried, and recrystallized twice from ethanol. Yield: 4.2 gm (70% I of l- [2-thiazolylazo]-2-acetoxy-5met’hylbenzene, m.p. 139°C. The product (4 gm) mentioned was dissolved in benzene (50 ml). Then acetic anhydride (1 ml) and dimethyl sulfate (4 ml) were added. After heating for 30 min at 35”, the mixture was left overnight. The formed crystals were filtered off, recrystallized from acetic anhydride, and dried ilz VUCUO.Yield: 2.1 gm (35%), m.p. 214-216°C (dec.‘). Calculated for C,,H,,O,N,,S,: H 4.4, N 10.9, S 16.6.
Preparation methiodide.
C 43.40,
H 4.42,
N
10.85.
S 16.55.
Found:
C 43.6.
of l-[2-thiazolylazo]-2-hydrox~-~-dimethylaminobenzenzene
2-Aminothiazole (10 gm, 0.10 mole) was dissolved in a mixture of concentrated sulfuric acid (18 ml) and water (80 ml 1. A solution of sodium nitrite (7 gm) in water (60 ml) was added dropwise under stirring, keeping the temperature at 3” by external cooling with ice. After the addition was complete, stirring was continued for 15 min. Then a solution of 2-hydroxy-4-dimethylaminohenzene ( 14 gm, 0.102 mole I in a mixture of concentrated sulfuric acid (6 ml ) and water (40 ml) was added during 10 mm, keeping the temperature at 5”. The precipitate was filtered off, suspended in water (500 ml ) , and neutralized with sodium carbonate (20 gm) The red precipitate wax filtered off and tlried in. VUCUO.Yield: 12 gm (48% ) of l- ]2-thiazolylazol-2-hydroxy-4dimethylaminobenzene, m.p. 127°C. Methyl iodide (5 ml ) was added to a solution of the product (2.5 gm) mentioned in benzene (100 ml). The mixture was refluxed for 1 hr and left overnight at room temperature. The formed crystals were filtered off, dried, and recrystallized from ethanol/ether. Yield: 1.2 gm (31% ) , m.p. 219°C. Calculated for C,zH,,ON,SI: C 36.93, C 37.1, H 3.9, I\T 14.4, S 8.3, I 32.3.
H
Preparation methiodide.
I-2-acetoxy-4-dimethylaminobenzene
of l- Id-thiazolylazo
3.87,
S
14.36.
8 8.22,
I 32.52.
Found:
l- [2-Thiazolylazo] -2-hydroxy-4-dimethylaminobenzene (24 gm) was dissolved in acetic anhydride (50 ml). Pyridine (1 ml) was added and the mixture was heated to 120°C for 4 hr. After cooling, the mixture was poured into crushed ice (400 gm) and stirred for 1 hr. The precipitate was filtered off, dried, and recrystallized from acetone/water (1:2). Yield: ,&? gm (64% ) of l- [2-thiazolylazo] -2-acetoxy-4-dimethylaminobenzene, m.p. 156°C. The product (12 gm) mentioned was dissolved in a mixture of acetone
36
VAN
HOOIDONK
ET
AL.
(300 ml) and benzene (25 ml). Methyl iodide (25 ml) was added and the mixture was allowed to stand for 1 week at, room temperature. The desired product1 crystallized as violet-colored crystals, which were filtered off and washed several times with dry ether. Yield: 8 gm (45%), m.p. 232°C. Calculated for GJH1,OPNPSI: C 38.90, H 3.96, N 12.96, C 39.3, H 4.1, N 12.9, S 7.4, I 29.2.
S 7.42, I 29.36.
Found:
Enzymes Home serum butyrylcholinesterase (BuChE) (acylcholine acylhydrolase, EC 3.1.1.8‘) was purchased from Organon Ltd. Oss, The Netherlands, and had a specific activity of 3.5 pmole acetylcholine perchlorate (BDH Chemicals) hydrolyzed per minute per milligram protein (temperature 25°C; medium 20 ml 0.0005 M phosphate buffer, pH 7.5; substrate concentration 0.02 M) Protein concentration was 0.005 mg/ml. Acetylcholinestemse (AChE) (acetylcholine acetyl-hydrolase, EC 3.1.1.7) was a partially purified bovine erythrocyte enzyme from Winthrop Laboratories, Inc., New York, N. Y., and had a specific activity of 1.5 pmole acetylcholine perchlorate hydrolyzed per minute per milligram protein (temperature 25°C; medium 20 ml 0.0005M phosphate buffer, pH 7.5; substrate concentration 0.003 M) . Protein concent,ration was 0.01 mg/ml. Buffers Phosphate buffer (0.067 M, pH 7.0). 2.98 gm Na2HP0,*2H,0 (E. Merck) was dissolved in 250 ml distilled water (solution I), 2.28 gm KH,PO, (E. Merck) was dissolved in 250 ml distilled water (solution II). 150 ml solution I was titrated to pH 7.0 with 95 ml solution II. Phosphate buffer (0.05 M, pH 7.0) was made analogously. Spparatus All absorbance measurements were made with a Zeiss PM& II spectrophotometer fitted with a water-jacketed cell compartment. The temperature was controlled by a Haake ultrathermostat within O.l”C. All react,ions
were
carried
out, in Teflon-stoppered
1 cm silica
cells.
The
l The position of quaternization was proved by running NMR spectra on a JEOGC-6OH spectrometer, using dimethyl sulfoxide-ds as a solvent and tetracompound the proton methylsilane as internal reference. In the nonquaternized signals of the N(CHA2 group appeared at 3.11 ppm (singlet). In the quaternieed compound the proton signals of the N(CHsj2 group appeared at 3.32 ppm (doublet) and a new signal was observed at 4.07 ppm (singlet) which is characteristic for the N+CH, protons of the quaternized thiazole nucleus.
CHROMOGENIC
SUBSTRATES
FOR
3'7
CHOLINESTERASES
absorption spectra were recorded on a Zeiss RPQ 20A spectrophotometer. All pH measurements were performed on a Vibron pH meter, model 39A (Electronic Instruments, Ltd.) equipped with a glass electrode type GHS33 and a saturated potassium chloride-calomel reference electrode type RJ23. The specific activities of both enzymes were determined with a Radiometer Autotitrator (pH meter type TTTlc, scale expander type PHA630T, and syringe buret recorder type SBRB) equipped with a glass electrode type G202C and a saturated potassium chloride-calomel reference electrode type K401. The electrode compartment was thermostated by a Haake ultrathermostat, within O.l”C. The pH was effectively controlled within 0.02 pH unit. RESULTS
Spectrophotometric Measurements The spectral properties of the new substrates and their hydrolysis products were determined in a 0.05 M phosphate buffer, pH 7.0, at 25°C.
A
0.9
0.8
07
t v, Z u 0
0.6
0.5
0.2
0.1 0.0 CONCENTRATION
x
IO5
(M)
FIG. 1. Plot of optical density vs concentration of l-[2-thiaaolylazol-2-acetoxy& ,nethylbenzene dimethyl sulfate (01, 1-[2-thiazolylazol-2-hydroxy&methylbenzene dimethyl sulfate (W ), l-C2-thiazolylazol-2-acetoxy-4-dimethylaminobenzene methiodide (O), and l-~2-thiazolylazol-2-hydroxy-4dimethylaminobenzene methiodide (0) in 0.05 M phosphate buffer, pH 7.0, in 1 cm silica cells at wavelengths of maximum absorption (nm) at 25°C. Values in parentheses are molar extinction coefficients (mole-’ cm-‘).
38
VAN
0.9
700
HOOIDONK
(I
600
500
450
400
350
ET
AL.
A)
700 WAVELENGTH
650
600
550
500
450
Cnmi
FIG. 2. Sprctral changes involved in hut?-r?-lcholinesterasc-~atalq-zed hydrolysis of l-12-thiazol~-lazoI-2-aceto~~-5-~~e~h~lhenz~~n~~ dimethyl sulfate (IA) and 1-[2-thiazol~lnzol-2-ncetos~-4-dimeth~1:m~inobenzenr methiodide (IB) in 0.05 M phosphate huffrr. pH 7.0, nt 25°C. Time in minutes. Enzyme conrentrntion was 0.035 unit/ml rwction mistnrr.
Figure 1 shows the relationship between optical density and the concentration of each species at the wavelength of maximum absorption. The rates of spont,aneoushydrolysis were determined in water and in 0.05 111phosphate buffer, pH 7.0; the half-lives are 123 and 10.3 hr for I - [2-thiazolylazo] -2-acctoxy-5-methylbenzene dimethyl sulfate and 134 and 12.9 hr for I- [2-thiasolylazo] -2-acetoxy-4-dimethylaminohenzene methiodide, respectively. Figure 2 shows the spectral changes during the butyrylcholinesterasecatalyzed liyclrolysis which are identical with the spectral changes observed during spontaneous hydrolysis. The spectra of the hydrolysis products are pH-dependent and are determined by the degree of ionization of the phenolic hydroxyl groups. A con\-entional potcntiometric titration (10) gave p&, values of 4.2 and 5.8 for l- [2-thiazolylazo] -2-hydroxy-5-methylbenzenc dimethyl sulfate and 1- 12- thiazolylazo] - 2 - hydroxy -4- dimethylaminobenzene methiodidc, respectively. This means that at pH 7.0 about 98% and 94r/rj, respectively, of the phenolic hydroxyl groups are present in the anionic form. The values in parentheses given in Fig. 1 for these compounds refer to the molar extinction coefficients of the phenolate ions. Enzyme
Activity
Measurements
Stock enzyme solutions (1 mg/ml) were prepared in 0.067 M phosphate buffer, pH 7.0. Stock substrate solutions were made in water (6 mg 1-[2-thiazolyl-
CHROMO(;EKI(‘
SI-RSTRATES
b’OR
(‘HOI,INlCSTERASI~:S
39
azoj -2-acetoxy-5-methylbenzene dimethyl sulfate and 7 mg I- [2thiazolylazo] -2-acetoxy-4-dimethylaminobenzene methiodide, reqwctively, in 20 nil). A number of enzyme solutions was prcparcd as follows: 0.1, 0.2, 0.4, solution were m:de ul) to 0.8, 1 .o, 2.0, and 3.0 ml of the stock cnzymc 12.5 ml with 0.067 JI phosphate buffer, pH 7.0. The assay solutions were prepared in 1 cm silica cells by mixing thoroughly 1.5 ml of the enzyme solution with 0.5 ml of the stock substrate solution. Both enzyme and stock substrate solutions were pre-eq”ilil)rat~,(l at 25°C. The final substrate concentration was 2 X lo--‘M and the fin:11 pH, 7.05. 15 set after mixing, the recording of the absorption rhanger was started and cont~inueclevery 15 wc ul) to 20% completion masimally. The hydrolysis of I- ]2-thiazolylazo I-2-acctoxy-5-Inctllylhcnzcnc climethyl sulfate was monitored at the wavelength of maximum absorption of the liberated phenolate ion (585 nm) ; because of the overlapping spectra of l- [2-thiazolylazo] -2-acetoxy-4-dimethylaminobenzene methiodide and its hydrolysis product we followed the hy(lrolysis at 485 nm. At both wavelengths the difference in absorbance between the substrate and product is maximal. Each run was corrected for the spontaneous hydrolysis. Plots of initial rates of hydrolysis vs enzyme concentrations are given in Fig. 3.
UNITS
OF
BvChE/ml
UNITS
OF
AChE/ml
FIG. 3. Plots of initial rate of hydrolysis (AOD/min) of I-[2-thiazolylazol-2acetoxy-5-methylbenzene dimethyl sulfate (IA. 0) and 1-[2-thiazolylazol-2-awtox.;4-dimethylaminobenzene methiodide (IB, 0) vs enzyme concentration (units of enzyme/ml reaction mixture) in 0.05 M phosphate buffer, pH 7.05, and 25°C. Substrate concentration 2 X 10.” M. Hydrolysis of IA was monitored at 555 nm. and of IB at 485 nm. Reactions were carried out in 1 cm silica cells.
40
VAN
HOOIDONK
Determination
ET
Kinetic
AL.
Parameters
As illustrated in Fig. 3, the init,ial rates of hydrolysis are linear with the enzyme concent,ration, as predicted by the Michaelis-Menten equation :
VnlaxlSl
(1)
’ = Km + WI where Vmax represent,s the maximal rate Menten constant, and [S] the substrate Evaluation of the values of K,, and measuring the initial rates of hydrolysis tration. Equation 1 can be rearranged
PI -=
of hydrolysis, K, the Michaelisconcentration. I’,;,, is usually accomplished by in relat’ion to substrate concento give:
-VKm + vl, max
V
ISI
(2)
A plot of ] S] /v vs ] S] yields a straight line, the slope equals l/V,I,,,, and the abscissa intercept equals -K,,. A typical example of such a plot is given in Fig. 4. The K,, and I’,,.,, values were calculated according to the weighted regression technique of Wilkinson (11). See Table 1. Inhibition
of ,Substrate
Hydrolysis
by Acetylcholine
To present evidence that the new subst’rates are hydrolyzed by cholinesteraee and not by any other esterase eventually present in the com-
.AL_/.-l-
-2-10
I2
-K
m
3
4
5
6
7
I 8 [S]
-II 9 x IO5
IO
II
I2
(M)
FIG. 4. Plot of [Sl/w vs [Yl for substrate reaction of 1-[2-thiazolylazol-2-acetoxy5-methylbenzene dimethyl sulfate with butyrylcholinesterase (0.21 unit/ml mixture) in 0.05 M phosphate buffer. pH 7.0, at 25°C.
reaction
CHROMOGENIC
SUBSTRATES
FOR
TABLE Michaelis-Menten
41
CHOLINESTERASES
1 Parameters
K, and V,,,,, values for the butyrylcholinesterase (BuChE)and acetylcholinesterase (AChE)-catalyzed hydrolysis of 1-[2-thiazolylazol-Z-acetoxy-Smethylbenzene dimethyl sulfate (IA), 1-[2-thiazolylazo]-2-acetoxy-4-dimethylami~~obenzene methiodide (IB), and acetylcholine. Conditions: for IA and IB, 0.05 fl!i phosphate buffer, pH 7.0; for acetylcholine, 0.005 41 phosphate buffer, pH 7.5. BuChE and AChE concentrations in all experiments 0.06 m&ml react,ion mixture. Temperature 25°C.
Enzyme
Substrate
BuChT?
I,4 IB Acelylcholine IA IB Acetylcholine
AChE
a From * From
literature literature
V mar (pmoleimin) (1.2 (1.X (3.1
f 0.2) + 0.2) 1.46 + 0.2) 2.68
x x X x
10-j 10-j 10-“a 10-5
X 10-4”
0.01 0.005 0.21 0.009 0.09
(12). (13).
mercial enzyme preparations, we studied the enzyme-catalyzed hydrolysis of l- [Z-thiazolylnzo] -2-acetoxy-5-methylbenzene dimethyl sulfate in the presence of varying concentrations of acetylcholine. Treating acetylcholine as a competitive inhibitor, the initial rate of substrate hydrolysis is given by
L’,,,,,
VI+!&
(3)
1+I’l
[S]
(
K,
>
where V,,,aX represents the maximal rate of hydrolysis without added acetylcholine, [S] the substrate concentration, [I] the acetylcholine concentration, K, the Michaelis-Menten constant of the substrate, and iii the inhibition constant of acetylcholine. In the reciprocal form:
Thus, in the case of competitive inhibition, a plot of l/v vs [I] should yield a straight line with a slope of li,/V,,,,K~ IS] and an intercept of (1 + &/[S] )/VI,,,,. With butyrylcholinesterase (0.26 unit/ml reaction mixture, other experimental conditions identical as described above under Enzyme Activity Measurements), we observed AOD/min values (AE) of 0.210, 0.198, 0.162, 0.150, and 0.102 without, and in the presence of 2, 10, 20,
42
vAK HOOIDOSK El‘ AL
and 40 X lo-” M acctylcholine, respectively. A plot of IJAE vs [I] yields a straight line and gives a Ki value of 2.2 X lo-” M. With acetylcholinesterase (0.10 unit/ml reaction mixture 1, we observed LIE values of 0.210, 0.138, 0.120, 0.090, 0.072, and 0.060 without and in the presence of 2.5, 5, 10, 15, and 20 X 10-l M acetylcholine, respectively. The plot of l/!& vs [I] gives a fii value of 1.4 X lo-’ M. The calculated Ki values are of the same order as the K,,, values of acctylcholine reported in the literature (see Table 1). This indicates that the new substrates and acet~ylcholine are hydrolyzed by the same cnzymc. Norcover, the enzyme-catalyzed hydrolysis of acctylcholine is also inhibited by addition of the new substrates. For example, with an acctylcholinesterase preparation (0.5 mg in 20 ml 0.0005 M phosphate buffer, pH 7.5, and at 25°C) the hydrolysis of acetylcholine (,lO-” MI1 was fount1 to produce 1.07 pmole acid/mm and that of l- [2-thiazolylazo] -2-acetosy-5-methylbenzene dimethyl sulfate t 5 X lo-’ 1M 1, 0.24 pmole acid/min, while that of a mixture of the two was 0.40 pmole acid/ min. When two different enzymes were operative, we should have observed the sum, namely, 1.31 qlole acid/mm. DISCUSSION It is interesting to compare the kinetic parameters of the thiazolylazo esters with those of acetylcholine. The most striking feature is the high affinity, characterized by the K,, value s, of l- ]2-thiazolylazo] -2-acetoxy5-nlethylbcnzenc dimethyl sulfate for both cholinesterases and 1- ] 2thiazolylazo ] -2-acctoxy-4-dimcthylaminobenzene methiodide for butyrylcholinestcrasr. The latter compound turned out to be a poor substrate for acetylcholinesterase (see Fig. 3 1 ; therefore no attempts were made to evaluate the kin&c, parameters. In order to obtain a better understanding of the stereochemical requircmcnts of the new substrates, we studied their space-filling Courtauld atomic models. These moclcls reveal a distance of approximately 5A between the quaternary nitrogen atom and the carbonyl-carbon atom. For ncetylcholine, this distance is about 4.7 A. These values agree well with the value of 4.3-4.7 A reported by Hollingworth et nl. (141 for the clistnncc in bovine crythrocyte cholinesterasc bet.wecn the anionic site and the reactive serine residue which forms a covalent. bond with the carbonyl-carbon atom of the ester. Wilson and Quan (15 I reported earlier a value of approximately 5 B for the same distance. In view of these data the excellent fit of the thiazolylazo esters on the enzymes seems understandable. On the other hand, the Ii,,,ax values illustrate that acetylcholine is by far the better substrate.
CHROMOGEKIC
SVBSTRATES
FOR
43
CHOLIKESTERASES
As the enzymic ester hydrolysis comprises a three-step reactioncomplexation, acetylation, and deacctylation-the rate-limiting step for the thiazolylazo esters and acetylcholine may he different. The poor substrate properties of I - [2-thiazolylazo] -2-acetosy-4-dimethylamino~wnzene methiodirlc for aretylclioline~tcrase may hc unclcrstantl:rl~lc in the same terms. The pwsent investigations produc~~(l practical data only ; ~norc dctailed studies on the rntw of acetylation and tlcacctplation will proT’ide more basic information. In conclusion we may state that the new substrates may hc urcd for the assay of cholinesterases. The highly colored hydrolysis products absorb at long wavelengths where tlistur~):tncc by protein absorption is (xxcludctl. The relatively high molecular extinction cocfficicnt~s ancl the favorable Ii,,, values permit the use of low conwntrations. The rccommended concentration is 2 X lOmA31. Enzyme quantities :IS low as 0.015 unit of ~~utyrylcholinesterase anrl 0.005 unit of ace~ylcliolincstcrnrC can he tlctcrminctl. The initial rates of hydrol;c 7s.q .-L at constant suhatrntc concentration are linear with the enzyme concentration. The whole prorcdure is simple and rapid am1 does not require addition of extra reagents or coni~~licatecl manipulations. Although azo com~~ou~~ls might he light >cnGtivcL, WV found no indication that on csposure to daylight the new suhstratcs arc dccomposccl 01 converted into isomeric products. Nevertheless we recommend storing the new substrates in wll-stoppered brown bottles and excluding n moist environment to prevent spontaneous hydrolysis.
For the spectrophotometric assay of hutyrylcholinestcrasc ( BuChE ) (EC 3.1.1.81 and acetylcholinesttrase I ACrhE 1 (EC 3.1.1.7) n new type of chromogenic substrates has heen dcvelopecl. The synthesis and the BuChEand AChE-catalyzed hydrolysis of I- [2-thiazolylazo] -2-aretosy-5-methylbcnzcIlc tlimethyl sulfate and l- [2-thiazolylazo] -2-acetoxy-4-dimethylltmi~~inobenzene methiotlide arc described. ACKNOWLEDGMENT The
aut.hors
XC indebted
to Mr.
N. Kramer
for
thr
&nwntnl
nnalyscs.
REFERENCES 1. HOFSTEE, 2. KALOW,
3. 4.
GOMORI. &IAMRR,
B. J. H.. Science TV., AND
LINDSAY.
G., J. Lab. D.
s..
AND
Clip. ~AMSOS,
114, 128 (1951) H. A., (‘ntl. J. &ocher?a. dfrci. 42, 445 (JR”?). R. M.. A/lul. (‘/rent.
Physiol.
33, 568 (1955)
30, 251 (1958).
44 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
VAN
HOOIIX~NK
ET
AL.
DE HORST. C., HOOGE, F. N.. AND ARKENBOUT, Cr. J., Nature 182, 1017 (1958). PRINCE, A. K., Arch. Biochem. Biophys. 113, 195 (1966). ANDERSON. R. G.. .4x0 NICKLESS, G., Analyst 92, 207 (1967). K.~w.~sE, A., Japan Annlyst 11, 621 (1962). JENSEN, B. S., Acta Chem. Scaud. 14, 927 (1960). Constants of Acids and Bases.” ALBERT, 8.. AXD SERJEAST. E. P., “Ionization Mcthum. London. 1962. WILKINSON, G. S., Biochem. J. 80, 324 (1961). YAKOVLEV. V. A., AND AGABEKYAN. R. S.. Biokhi,miyn 32, 293 (1967). &XJPKA. R. M.. Biochemistry 3, 1749 (1964). HOLLINGWORTH. R. M.. F~K~To, T. R.. AND METCALF. R. L.. J. Agr. Food Chem. 15, 235 (1967). WILSON. I. H., AND QUAN, C., Arch. B&hem. Biophys. 73, 131 (1958).