THROMBOSIS RESEARCH Vol. 13. PP. S-13. 0 Pngamon Press Ltd. 19% Printed inGreat Brimin.
0049-3W8/78iO701~
SOZ.oO/O
AMPLIFICATION OF COLOR YIELD OF CHROMOGENIC SUBSTRATES USING p-DIMETHYLAMINOCINNAMALDEHYDE Hau C. Kwaan, Robert B. Friedman and Marija Szczecinski Northwestern University Medical School
and
Veterans Administration Lakeside Hospital Chicago, Illinois 60611 USA
form 13.4.1978. (Received C;.2.1978: in revised Accepted by Editor >f.T. Barnhart)
ABSTRACT A method is described for amplifying the color of 4-nitroaniline which is produced during enzyme catalyzed amidolysis of synthetic chromogenic substrates. The liberated chromogen is directly coupled with p-dimethylaminocinnamaldehyde to yield a colored schiffbase compound. The color complex has an absorption maximum at 570 nm and, under the reaction conditions described, has a molar absorbancy of 9.1 x 104. The application of this method is presented for the enzyme urokinase using two different chromogenic substrates. A micromethod adaptation of this method is also described. This method can be used to amplify the color obtained in all enzyme assays where 4-nitroaniline is the liberated chromogen.
1NTR0DucTToN
Synthetic chromogenic substrates for measuring amidolytic activities in coagulation and fibrinolysis have recently been made available for a wide variety of enzyme assays.
Most
of
the commercially available substrates uti.
lize 4-nitroaniline (paranitroaniline, PNA) as chromogen,
Recently, we re-
ported (1) a method for improving the color yield of the chromogen by coupling it with a second chromogen subsequent to diazotization of the released PNA.
We now wish to report the development of an alternate method for color
amplification of PNA.
This procedure involves the direct coupling of PNA,
an aromatic amine, with an aldehyde, 4-dimethylaminocinnamaldehyde (DACA).
This reagent provides additional advantages over our previous method in that the intermediate diazotization step is eliminated.
In addition, the molar
absorbancy of the PNA-complex is higher than that of our earlier color am5
6
CNRO?1OGENTC
SLJSTRATE
ASSAYS
Vol.13,No.l
plification, but the utility of the original procedure is not changed. METHODS Substrates:
Substrate S-2227 (H-Gly-Glu-Arg-PNA, substrate for urokinase)
was obtained from AB Kabi, Molndal, Sweden.
Chromozym UK analog (substrate
for urokinase) was obtained from Pentapharm AG., Basle, Switzerland. Enzymes:
Urokinase (UK) (urinary) was obtained from Abbott Laboratories,
North Chicago, Il. (Lot # 2021-209). Reagents:
4-Dimethylaminocinnamaldehyde (WCA)
Chemical Co., Milwaukee, Wisconsin. fication.
was purchased from Aldrich
It was used without any further puri-
4-Nitroaniline (paranitroaniline, PNA) was purchased from East-
man Organic Chemicals, Rochester, N.Y.
It was recrystallized from water.
All other reagents were of reagent grade. Spectroscopy:
An Hitachi Perkin-Elmer Model 139 W-VIS
spectrophotometer
was used for measuring absorbancies. THE COUPLING REACTION:
Two methods could be employed, using hydrochloric
acid (HCl) or Trichloroacetic acid (TCA), respectively. is developed when TCA is used.
The strongest color
Precipitation of proteins would also occur
requiring remove1 by centrifugation.
This did not happen with HCl which,
however, produced a less intense color. Reaction with TCA:
A standard curve was generated by pipetting varying a-
mounts of PNA (a convenient range contained between 3 and 20 nmoles per tube) in 0.6 ml of water.
Aliquots of 40% trichloroacetic acid (TCA), 0.6 ml, were
added to each tube.
When preparing a standard curve to accompany an enzyme
assay, 0.3 ml of aqueous PNA solution was mixed with 0.3 ml of buffer. tubes were thoroughly mixed.
The
Aliquots of 1 ml each were transferred to a
second set oftubes and 0.5 ml of an alcoholic DACA solution (8 mg/ml) was added to each tube.
The tubes were mixed thoroughly and left at room tem-
perature for 15 minutes. Reaction with HCl: solution.
A volume of 0.5 ml was utilized for the PNA-containing
It was mixed with 0.5 ml of 0.2 g HCl.
Ethanolic (or methanolic)
DACA solution (8 mg/ml) was added directly to the tubes and the tubes were mixed thoroughly. The color was measured at 570 nm.
Under these conditions, a sample con-
taining 5 mnoles/ml should have an optical density of approximately 0.28 when using
TCA.
A standard curve should accompany every enzyme assay.
Vol.?g,No.l
CHRO~IOGESTC
ERZYMEASSAY:
TCA method:
numbered tubes
in an ice
genic
substrate,
SC'RSTRATE
Aliquots
bath.
of 0.6
ASSAYS
ml of K% TCA were placed
A 5 mmolar aqueous
the urokinase
solution
soluizion
(100 I.U./ml),
of
and the buffer
NaCl, 0.15 E ea., pH 8.4) were pre-incubated at 37'.
in
the chromo(Tris-
Two sets of controls
were arranged for monitoring the reagents during the digestion in the incubator and in the TCA tubes.
Pre-digest controls consisted of aliquots of
0.24 ml urokinase, 0.3 ml buffer, and 0.06 ml substrate solution. was added to bring the sample volume to 0.6 ml. says are
to be performed,
the controls
enzyme must be added to the acid digest
mixture
contained
substrate solution.
first
can be combined to avoid
Water
When a larger number of asinto
spuriously
one tube. high
The
blanks.
The
2.4 ml buffer, 2;O ml urokinase solution, and 0.5 ml
The concentrations in the digest were then 40 Xl/ml
for the urokinase and 0.5 mnolar for the substrate. UK could be employed.
Lower concentrations of
Using the micromethod described below, enzyme digests
containing as little as 2 W/ml
could safely be measured with accuracy.
Aliquots of 0.6 ml were removed from the digestion tube at intervals of 1, 2, 3, 4, 6, 8, and 10 minutes and added immediately to the appropriate TCA tube in the ice bath. After completion of the digest, another set of substrate,,enzyme, and buffer blanks was prepared to serve as post-digest controls.
The TCA tubes
were mixed at room temperature and centrifuged (550 x g, 10 min.).
Ali-
quots of 1 ml were rembved and the DACA coupling reaction was performed as described above. HCl Method:
Aliquots from the enzyme digest of 0.5 ml were pipetted into
numbered test tubes containing 0.5 ml
0.2 E HC1 in an ice bath.
Pre- and
post-incubation controls were also prepared as described above, except that volumes of 0.5 ml were withdrawn instead.
After completion of the sampling
the tubes were removed from the ice bath and 0.5 ml of the WCA
solution
was added. Micromethod:
The DACA-TCA assay could be scaled down to the microlevel.
The outline of the reaction procedure is the same except that the volumes are substantially reduced.
Buffer, 0.625 ml, was mixed with 0.5 ml of en-
zyme solution followed by 0.125 ml of substrate solution.
Aliquots of 0.25
ml were removed at 1, 2, 4, and 6 minutes and added to 0.25 ml of cold 40% TCA.
After mixing and centrifugation, 0.4 ml was removed and mixed with 0.2
ml of the DACA reagent. using
microcurvettes.
The color was measured in the spectrophotometer
8
CHROWOGE?iTC
STBSTRATE
ASSAYS
v01.13,s0.1
OPTW LL DENSIT Y 0.7
a6
0.5
0.4
0.3 0.2 0.1
/
500
520
540
560
WAVEL E NGTH. FIGURE
580
600
nm
1
Spectra of MCA-PNA complexes: -in the presence of TCA (PNA concentration 11.1 nmoles/ml);. -in the presence of HCl (PNA concentration 13.3 blank. nmoles/ml);-reagent
RESULTS Calorimetry: The spectrum of the DACA-PNA complex is shown in Figure 1. Little difference was observed between the colored material obtained in the presence of TCA and that obtained in the presence of HCl. tion was at565-570 MI. 555 nm.
Maximum absorp-
This maximum shifted, however, after 20 hours to
The molar absorbance obtained under the conditions described is
9.1 x lo4 with TCA and 3.7 x lo4 in the presence of HCl. tion obeys Beer's law in the
The color produc-
concentration range of 5-50 micromolar PNA.
optimal conditions for the reaction have been determined.
One of our main
concerns was the relatively high blanks obtained when the DACA was acidified. The blank obtained with the HCl was greater than when the reagent was mixed with TCA.
Thus, a specific concentration of &WA
minimal blank value was used in the present study.
which would produce a As acidifying agents,
TCA and HCl gave comparable results, though the color with the TCA was greater.
TCA has the advantage of precipitating proteins if their presence in-
terferes with the assay.
As is the case of our previous method (l), the
concentration of TCA is not too critical (between 30-40%) as long as the pH is below 1.4.
In the case of the HCl-mediated reaction, however, an optimal
Vol.
CHROMOGENIC
13,No.l
SUBSTRATE
concentration of 0.2 N, HCl was observed. proceed.
ASSAYS
9
Above pH 2, the reaction did not
Higher concentrations of HCl significantly reduced the color yield.
Buffers in the concentrations specified for the enzyme digest did not interfere with color production though an increased salt concentration did reduce the color production by 11%.
The color yield rose as a function of the
concentration of DACA (Figure 2).
A working concentration of 8.0 mg/ml was
found to provide a satisfactory balance between higher color yield and excessive background. the DACA.
Either methanol or ethanol could be used as solvent for
An acidic aqueous solution could also be used, but the color de-
velopment was not as satisfactory.
Though WCA
reagent was prepared fresh
daily, there was no significant deterioration of the reagent on standing for prolonged periods.
The color was stable between 15 minutes and 3 hours.
Storage of the reacted samples in the light or the dark had no effect on the color produced, and similarly, maintaining the reacted samples at 37' produced no difference from those kept at ambient temperatures. Enzymology:
The Lineweaver-Burke plot for determining the kinetic values of
urokinase as measured using substrate S-2227 is shown in Figure 3. The KR 4 value using the conditions described is 3.3 x 10 molar, which follows close0 PTICAL DENSITY 570 nm 03
0.2
0.1
2
4
6
a
FIGURE 2 Color development of DACA-PNA complex as a function of the concentration of WCA in the alcoholic reagent.-DACA-PNA;s reagent blank.
St-rBSTRdTE
CHROHOGEWTC
10
ASSAYS
1 -‘S -1
1
2
1’5
FIGURE 3
FIGURE 4
Lineweaver-Burke plot of the action of UK on substrate S-2227. Reaction conditions are described in the text.
Lineweaver-Burke plot of the action of UK on chromozym UK analog. Reaction conditions are described in the text.
ly, the values obtained in our earlier study. min.
The Vtix is 16.7 nmoles/ml/
This substrate has residual free PNA (approx. 2-3 mole %) and, similar-
ly, the Chromozym UK analog contained a percent of inactive material but no The latter substrate has a yellow color which could possibly in-
free PNA.
terfere with a direct spectrophotometric assay. has its color maximum in another region.
Tbe DACA complex, however,
Figure 4 shows the Lineweaver-
Burke plot for urokinase as measured using the substrate Chromozym UK ana'Thes
log.
for the enzyme, under the conditions described using this sub-
strate is 8.91 x lo6 Molar and its V
MELX
is 1.55 nmole/ml/min.
The reaction
rates are linearly proportional to the enzyme concentrations in the ranges measured.
No differences were observed between the pre- and post-digest
controls.
Figure 5 shows the relationship between activity and enzyme con-
centration using the micromethod.
Enzyme activities of 2 Ill/mlcan be meas-
ured. DISCUSSION Aldehydes have long been used as calorimetric reagents for aromatic amines and vice versa (2,3).
Of these, p-dimethylaminocinnamaldehyde has been
proven to be effective (4,s). It has also been applied as a detecting re-
Vol. 13,rjo.l
CHROMOGEXIC
STjBSTRATE
11
ASSAYS
Reaction Velocity
I.U.
per
ml Digest
FIGURE 5 Reaction velocity of urokinase on substrate S-2227 as measured using the micromethod described in the text.
N”2 &NH (JH
$02”
0
‘F “2’2 ‘F”24 H,N-C-C+-C-C-V-C-C-NA6HHGHk6A
0
-NO2
S-2227 Urokinase
+
0
H2N- 0
+ Peptide -NO2
PNA
+ (CH3)2N-
0 0
-::;.c”,O
(CH3j2N-
DACA
0 0
Schiff
-?=!-!=N-O-N,
Base
FIGURE 6 Reaction scheme for the amplification of the PNA liberated from a chromogenic substrate by urokinase.
CHROMOGENIC
12
agent in chromatography (6,7).
SL'BSTHATE
ASSAYS
Vol. 13,No.l
The present study utilizes this aldehyde re-
agent for amplifying the color developed during the enzyme catalyzed hydrolysis of synthetic substrates.
Most of the commercially available synthetic
peptide substrates for coagulation and fibrinolytic enzymes use PNA as the chromogen.
The color yield from this chromogen can be unsatisfactory when
the enzyme activity is low.
This is especially apparent in the case of uro-
kinase, where the color yield may be in the area of 0.005 O.D. units per min. or less.
We have successfully amplified the color (1) by diazotizing the
liberated PNA and then coupling it with N-(1-naphthyl)-ethylenediamine (NED). This procedure, however, requires several intermediary steps.
The aldehyde
reagnet 4-dimethylaminocinnamaldehyde (D&CA), on the other hand , permits a one-step process for amplifying the color or PNA. advantage in that the color yield of the MCA-PNA than that of the NED reagent.
It provides an additional complex is even greater
The reaction scheme is depicted in Figure 6.
The color obtained is stable and the method is applicable under a variety of enzyme digest conditions.
The method may be used in any enzyme as-
say in which PNA is the liberated chromogen, as is the case with our earlier method (1).
When HCl is used as the acid, the procedure is simplified fur-
ther since a centrifugation step is eliminated.
This suggests that the DACA
method using HCl should be particularly attractive for adaptation to automated systems of analysis.
DACA has been used for the assay of para-amino-
hippuric acid in kidney function tests in automated systems (8.9).
This
should greatly facilitate the automation of these hematological assays for which synthetic substrates are available. ACKNOWLEDGEMENTS We gratefully thank Dr. Lars Mellstam of AB Kabi for the S-2227 and Dr. Kurt Stocker of Pentapharm for the Chromozym UK-analog. REFERENCES 1.
FRIEMN, R.B., KWAAN, H.C., and SZCZECINSKI, M. Improved sensitivity of chromogenic substrate assays for urokinase and plasmin. Thromb. Res. (in press) 1978.
2.
BRUN, C. A rapid method for the determination of para-aminohippuric acid in kidney function tests. 3. Lab. Clin. Med. 37, 995, 1951.
3.
SAWIC'KI, E., STANLEY, T.W., and JOHNSON, H. Comparison of spectrophotometric and spectrophluorometric methods for the determination of malonaldehyde. Anal. Chem. 35, 199, 1963.
4.
PESEZ, M., and BARTOS, J. Sur la colorimetrie des arylamines primairer et des derives indoliques a laide du p-dimethylaminocinnamaldehyde.
Vol.'3.~0.'
CHRWlOGE?;IC
Bul. Sot. Chem. France.
ST,XiSTRXTE
ASS_AYS
13
3082, 1966.
5.
STRELL, M., and JOHNSON, S. Neue Polymethinfarbstoffe durch Umsetzung von p-Dimethylamino-zimtaldehyd mit primaren aromatischen Aminen, verschiedenen Heterocyclen und Sulfonamiden. Arch. Pharm. 293, 984, 1960.
6.
HARLN-MASON, J., and ARCHER, A.A.P.G. Use of p-dimethylaminocinnamaldehyde as a spray reagent for indole derivatives on paper chromatograms. Biochem. J. 69, 60, 1958.
7.
BAUMANN,P. Quantitative determination of indolic compounds in the rat brain using p-dimethylaminocinnamaldehyde as reagent. J. Chromatog. 109, 313, 1975.
8.
PAREKH, C.K., KIRPAN, J., PETERSON, A., RASSERT, G.L., and MURPHY, B.F. Automated simultaneous determination of p-aminohippurate and creatinine in plasma or urine. Clin. Chem. 20, 348, 1974.
9.
YATZIDIS, H. Simplified determination of plasma p-aminohippurate in kidney function tests. Nephron 15, 78, 1975.
0049-3W8/78iO701~
SOZ.oO/O
AMPLIFICATION OF COLOR YIELD OF CHROMOGENIC SUBSTRATES USING p-DIMETHYLAMINOCINNAMALDEHYDE Hau C. Kwaan, Robert B. Friedman and Marija Szczecinski Northwestern University Medical School
and
Veterans Administration Lakeside Hospital Chicago, Illinois 60611 USA
form 13.4.1978. (Received C;.2.1978: in revised Accepted by Editor >f.T. Barnhart)
ABSTRACT A method is described for amplifying the color of 4-nitroaniline which is produced during enzyme catalyzed amidolysis of synthetic chromogenic substrates. The liberated chromogen is directly coupled with p-dimethylaminocinnamaldehyde to yield a colored schiffbase compound. The color complex has an absorption maximum at 570 nm and, under the reaction conditions described, has a molar absorbancy of 9.1 x 104. The application of this method is presented for the enzyme urokinase using two different chromogenic substrates. A micromethod adaptation of this method is also described. This method can be used to amplify the color obtained in all enzyme assays where 4-nitroaniline is the liberated chromogen.
1NTR0DucTToN
Synthetic chromogenic substrates for measuring amidolytic activities in coagulation and fibrinolysis have recently been made available for a wide variety of enzyme assays.
Most
of
the commercially available substrates uti.
lize 4-nitroaniline (paranitroaniline, PNA) as chromogen,
Recently, we re-
ported (1) a method for improving the color yield of the chromogen by coupling it with a second chromogen subsequent to diazotization of the released PNA.
We now wish to report the development of an alternate method for color
amplification of PNA.
This procedure involves the direct coupling of PNA,
an aromatic amine, with an aldehyde, 4-dimethylaminocinnamaldehyde (DACA).
This reagent provides additional advantages over our previous method in that the intermediate diazotization step is eliminated.
In addition, the molar
absorbancy of the PNA-complex is higher than that of our earlier color am5
6
CNRO?1OGENTC
SLJSTRATE
ASSAYS
Vol.13,No.l
plification, but the utility of the original procedure is not changed. METHODS Substrates:
Substrate S-2227 (H-Gly-Glu-Arg-PNA, substrate for urokinase)
was obtained from AB Kabi, Molndal, Sweden.
Chromozym UK analog (substrate
for urokinase) was obtained from Pentapharm AG., Basle, Switzerland. Enzymes:
Urokinase (UK) (urinary) was obtained from Abbott Laboratories,
North Chicago, Il. (Lot # 2021-209). Reagents:
4-Dimethylaminocinnamaldehyde (WCA)
Chemical Co., Milwaukee, Wisconsin. fication.
was purchased from Aldrich
It was used without any further puri-
4-Nitroaniline (paranitroaniline, PNA) was purchased from East-
man Organic Chemicals, Rochester, N.Y.
It was recrystallized from water.
All other reagents were of reagent grade. Spectroscopy:
An Hitachi Perkin-Elmer Model 139 W-VIS
spectrophotometer
was used for measuring absorbancies. THE COUPLING REACTION:
Two methods could be employed, using hydrochloric
acid (HCl) or Trichloroacetic acid (TCA), respectively. is developed when TCA is used.
The strongest color
Precipitation of proteins would also occur
requiring remove1 by centrifugation.
This did not happen with HCl which,
however, produced a less intense color. Reaction with TCA:
A standard curve was generated by pipetting varying a-
mounts of PNA (a convenient range contained between 3 and 20 nmoles per tube) in 0.6 ml of water.
Aliquots of 40% trichloroacetic acid (TCA), 0.6 ml, were
added to each tube.
When preparing a standard curve to accompany an enzyme
assay, 0.3 ml of aqueous PNA solution was mixed with 0.3 ml of buffer. tubes were thoroughly mixed.
The
Aliquots of 1 ml each were transferred to a
second set oftubes and 0.5 ml of an alcoholic DACA solution (8 mg/ml) was added to each tube.
The tubes were mixed thoroughly and left at room tem-
perature for 15 minutes. Reaction with HCl: solution.
A volume of 0.5 ml was utilized for the PNA-containing
It was mixed with 0.5 ml of 0.2 g HCl.
Ethanolic (or methanolic)
DACA solution (8 mg/ml) was added directly to the tubes and the tubes were mixed thoroughly. The color was measured at 570 nm.
Under these conditions, a sample con-
taining 5 mnoles/ml should have an optical density of approximately 0.28 when using
TCA.
A standard curve should accompany every enzyme assay.
Vol.?g,No.l
CHRO~IOGESTC
ERZYMEASSAY:
TCA method:
numbered tubes
in an ice
genic
substrate,
SC'RSTRATE
Aliquots
bath.
of 0.6
ASSAYS
ml of K% TCA were placed
A 5 mmolar aqueous
the urokinase
solution
soluizion
(100 I.U./ml),
of
and the buffer
NaCl, 0.15 E ea., pH 8.4) were pre-incubated at 37'.
in
the chromo(Tris-
Two sets of controls
were arranged for monitoring the reagents during the digestion in the incubator and in the TCA tubes.
Pre-digest controls consisted of aliquots of
0.24 ml urokinase, 0.3 ml buffer, and 0.06 ml substrate solution. was added to bring the sample volume to 0.6 ml. says are
to be performed,
the controls
enzyme must be added to the acid digest
mixture
contained
substrate solution.
first
can be combined to avoid
Water
When a larger number of asinto
spuriously
one tube. high
The
blanks.
The
2.4 ml buffer, 2;O ml urokinase solution, and 0.5 ml
The concentrations in the digest were then 40 Xl/ml
for the urokinase and 0.5 mnolar for the substrate. UK could be employed.
Lower concentrations of
Using the micromethod described below, enzyme digests
containing as little as 2 W/ml
could safely be measured with accuracy.
Aliquots of 0.6 ml were removed from the digestion tube at intervals of 1, 2, 3, 4, 6, 8, and 10 minutes and added immediately to the appropriate TCA tube in the ice bath. After completion of the digest, another set of substrate,,enzyme, and buffer blanks was prepared to serve as post-digest controls.
The TCA tubes
were mixed at room temperature and centrifuged (550 x g, 10 min.).
Ali-
quots of 1 ml were rembved and the DACA coupling reaction was performed as described above. HCl Method:
Aliquots from the enzyme digest of 0.5 ml were pipetted into
numbered test tubes containing 0.5 ml
0.2 E HC1 in an ice bath.
Pre- and
post-incubation controls were also prepared as described above, except that volumes of 0.5 ml were withdrawn instead.
After completion of the sampling
the tubes were removed from the ice bath and 0.5 ml of the WCA
solution
was added. Micromethod:
The DACA-TCA assay could be scaled down to the microlevel.
The outline of the reaction procedure is the same except that the volumes are substantially reduced.
Buffer, 0.625 ml, was mixed with 0.5 ml of en-
zyme solution followed by 0.125 ml of substrate solution.
Aliquots of 0.25
ml were removed at 1, 2, 4, and 6 minutes and added to 0.25 ml of cold 40% TCA.
After mixing and centrifugation, 0.4 ml was removed and mixed with 0.2
ml of the DACA reagent. using
microcurvettes.
The color was measured in the spectrophotometer
8
CHROWOGE?iTC
STBSTRATE
ASSAYS
v01.13,s0.1
OPTW LL DENSIT Y 0.7
a6
0.5
0.4
0.3 0.2 0.1
/
500
520
540
560
WAVEL E NGTH. FIGURE
580
600
nm
1
Spectra of MCA-PNA complexes: -in the presence of TCA (PNA concentration 11.1 nmoles/ml);. -in the presence of HCl (PNA concentration 13.3 blank. nmoles/ml);-reagent
RESULTS Calorimetry: The spectrum of the DACA-PNA complex is shown in Figure 1. Little difference was observed between the colored material obtained in the presence of TCA and that obtained in the presence of HCl. tion was at565-570 MI. 555 nm.
Maximum absorp-
This maximum shifted, however, after 20 hours to
The molar absorbance obtained under the conditions described is
9.1 x lo4 with TCA and 3.7 x lo4 in the presence of HCl. tion obeys Beer's law in the
The color produc-
concentration range of 5-50 micromolar PNA.
optimal conditions for the reaction have been determined.
One of our main
concerns was the relatively high blanks obtained when the DACA was acidified. The blank obtained with the HCl was greater than when the reagent was mixed with TCA.
Thus, a specific concentration of &WA
minimal blank value was used in the present study.
which would produce a As acidifying agents,
TCA and HCl gave comparable results, though the color with the TCA was greater.
TCA has the advantage of precipitating proteins if their presence in-
terferes with the assay.
As is the case of our previous method (l), the
concentration of TCA is not too critical (between 30-40%) as long as the pH is below 1.4.
In the case of the HCl-mediated reaction, however, an optimal
Vol.
CHROMOGENIC
13,No.l
SUBSTRATE
concentration of 0.2 N, HCl was observed. proceed.
ASSAYS
9
Above pH 2, the reaction did not
Higher concentrations of HCl significantly reduced the color yield.
Buffers in the concentrations specified for the enzyme digest did not interfere with color production though an increased salt concentration did reduce the color production by 11%.
The color yield rose as a function of the
concentration of DACA (Figure 2).
A working concentration of 8.0 mg/ml was
found to provide a satisfactory balance between higher color yield and excessive background. the DACA.
Either methanol or ethanol could be used as solvent for
An acidic aqueous solution could also be used, but the color de-
velopment was not as satisfactory.
Though WCA
reagent was prepared fresh
daily, there was no significant deterioration of the reagent on standing for prolonged periods.
The color was stable between 15 minutes and 3 hours.
Storage of the reacted samples in the light or the dark had no effect on the color produced, and similarly, maintaining the reacted samples at 37' produced no difference from those kept at ambient temperatures. Enzymology:
The Lineweaver-Burke plot for determining the kinetic values of
urokinase as measured using substrate S-2227 is shown in Figure 3. The KR 4 value using the conditions described is 3.3 x 10 molar, which follows close0 PTICAL DENSITY 570 nm 03
0.2
0.1
2
4
6
a
FIGURE 2 Color development of DACA-PNA complex as a function of the concentration of WCA in the alcoholic reagent.-DACA-PNA;s reagent blank.
St-rBSTRdTE
CHROHOGEWTC
10
ASSAYS
1 -‘S -1
1
2
1’5
FIGURE 3
FIGURE 4
Lineweaver-Burke plot of the action of UK on substrate S-2227. Reaction conditions are described in the text.
Lineweaver-Burke plot of the action of UK on chromozym UK analog. Reaction conditions are described in the text.
ly, the values obtained in our earlier study. min.
The Vtix is 16.7 nmoles/ml/
This substrate has residual free PNA (approx. 2-3 mole %) and, similar-
ly, the Chromozym UK analog contained a percent of inactive material but no The latter substrate has a yellow color which could possibly in-
free PNA.
terfere with a direct spectrophotometric assay. has its color maximum in another region.
Tbe DACA complex, however,
Figure 4 shows the Lineweaver-
Burke plot for urokinase as measured using the substrate Chromozym UK ana'Thes
log.
for the enzyme, under the conditions described using this sub-
strate is 8.91 x lo6 Molar and its V
MELX
is 1.55 nmole/ml/min.
The reaction
rates are linearly proportional to the enzyme concentrations in the ranges measured.
No differences were observed between the pre- and post-digest
controls.
Figure 5 shows the relationship between activity and enzyme con-
centration using the micromethod.
Enzyme activities of 2 Ill/mlcan be meas-
ured. DISCUSSION Aldehydes have long been used as calorimetric reagents for aromatic amines and vice versa (2,3).
Of these, p-dimethylaminocinnamaldehyde has been
proven to be effective (4,s). It has also been applied as a detecting re-
Vol. 13,rjo.l
CHROMOGEXIC
STjBSTRATE
11
ASSAYS
Reaction Velocity
I.U.
per
ml Digest
FIGURE 5 Reaction velocity of urokinase on substrate S-2227 as measured using the micromethod described in the text.
N”2 &NH (JH
$02”
0
‘F “2’2 ‘F”24 H,N-C-C+-C-C-V-C-C-NA6HHGHk6A
0
-NO2
S-2227 Urokinase
+
0
H2N- 0
+ Peptide -NO2
PNA
+ (CH3)2N-
0 0
-::;.c”,O
(CH3j2N-
DACA
0 0
Schiff
-?=!-!=N-O-N,
Base
FIGURE 6 Reaction scheme for the amplification of the PNA liberated from a chromogenic substrate by urokinase.
CHROMOGENIC
12
agent in chromatography (6,7).
SL'BSTHATE
ASSAYS
Vol. 13,No.l
The present study utilizes this aldehyde re-
agent for amplifying the color developed during the enzyme catalyzed hydrolysis of synthetic substrates.
Most of the commercially available synthetic
peptide substrates for coagulation and fibrinolytic enzymes use PNA as the chromogen.
The color yield from this chromogen can be unsatisfactory when
the enzyme activity is low.
This is especially apparent in the case of uro-
kinase, where the color yield may be in the area of 0.005 O.D. units per min. or less.
We have successfully amplified the color (1) by diazotizing the
liberated PNA and then coupling it with N-(1-naphthyl)-ethylenediamine (NED). This procedure, however, requires several intermediary steps.
The aldehyde
reagnet 4-dimethylaminocinnamaldehyde (D&CA), on the other hand , permits a one-step process for amplifying the color or PNA. advantage in that the color yield of the MCA-PNA than that of the NED reagent.
It provides an additional complex is even greater
The reaction scheme is depicted in Figure 6.
The color obtained is stable and the method is applicable under a variety of enzyme digest conditions.
The method may be used in any enzyme as-
say in which PNA is the liberated chromogen, as is the case with our earlier method (1).
When HCl is used as the acid, the procedure is simplified fur-
ther since a centrifugation step is eliminated.
This suggests that the DACA
method using HCl should be particularly attractive for adaptation to automated systems of analysis.
DACA has been used for the assay of para-amino-
hippuric acid in kidney function tests in automated systems (8.9).
This
should greatly facilitate the automation of these hematological assays for which synthetic substrates are available. ACKNOWLEDGEMENTS We gratefully thank Dr. Lars Mellstam of AB Kabi for the S-2227 and Dr. Kurt Stocker of Pentapharm for the Chromozym UK-analog. REFERENCES 1.
FRIEMN, R.B., KWAAN, H.C., and SZCZECINSKI, M. Improved sensitivity of chromogenic substrate assays for urokinase and plasmin. Thromb. Res. (in press) 1978.
2.
BRUN, C. A rapid method for the determination of para-aminohippuric acid in kidney function tests. 3. Lab. Clin. Med. 37, 995, 1951.
3.
SAWIC'KI, E., STANLEY, T.W., and JOHNSON, H. Comparison of spectrophotometric and spectrophluorometric methods for the determination of malonaldehyde. Anal. Chem. 35, 199, 1963.
4.
PESEZ, M., and BARTOS, J. Sur la colorimetrie des arylamines primairer et des derives indoliques a laide du p-dimethylaminocinnamaldehyde.
Vol.'3.~0.'
CHRWlOGE?;IC
Bul. Sot. Chem. France.
ST,XiSTRXTE
ASS_AYS
13
3082, 1966.
5.
STRELL, M., and JOHNSON, S. Neue Polymethinfarbstoffe durch Umsetzung von p-Dimethylamino-zimtaldehyd mit primaren aromatischen Aminen, verschiedenen Heterocyclen und Sulfonamiden. Arch. Pharm. 293, 984, 1960.
6.
HARLN-MASON, J., and ARCHER, A.A.P.G. Use of p-dimethylaminocinnamaldehyde as a spray reagent for indole derivatives on paper chromatograms. Biochem. J. 69, 60, 1958.
7.
BAUMANN,P. Quantitative determination of indolic compounds in the rat brain using p-dimethylaminocinnamaldehyde as reagent. J. Chromatog. 109, 313, 1975.
8.
PAREKH, C.K., KIRPAN, J., PETERSON, A., RASSERT, G.L., and MURPHY, B.F. Automated simultaneous determination of p-aminohippurate and creatinine in plasma or urine. Clin. Chem. 20, 348, 1974.
9.
YATZIDIS, H. Simplified determination of plasma p-aminohippurate in kidney function tests. Nephron 15, 78, 1975.
