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A fluorimetric kinetic method for the determination of tonin activity in rat submaxillary glands
MICROCHEMICAL
32,
JOURNAL
A Fluorimetric
3 17-323
Kinetic Method for the Determination Activity in Rat Submaxillary Glands
C.BENTABOL, Department
(1985)
A. REYES, J.A.
of Biochemistry,
Faculty
NARVAEZ,AND
of Medicine,
University
of Tonin
M. MORELL
of Mrilaga,
Mdlaga,
Spain
AND
J. J. LASERNAAND Department
of Analytical
Chemistry,
Faculty
F. GARCIA SANCHEZ* of Sciences,
University
of Mdlaga,
Mrilaga,
Spain
Received October 29, 1983 A method for the kinetic determination of tonin activity in rat submaxillary gland extracts is described. The method is based on the fluorogenic reaction of o-phthalaldehyde with the dipeptide histidyl-leucine generated in the enzymatic hydrolysis of angiotensin I. The reaction is monitored by fluorescence at 440 nm. At pH 7.5, the method allows the determination of 2-15 nmol/ml histidyl-leucine with relative standard deviation of 6%. o 1985 Academic Press, Inc.
INTRODUCTION Tonin, an enzyme found in submaxillary glands (Z), has promoted a remarkable interest by its relation with the reninangiotensin system (6) and its connection with the control of the blood irrigation at a local level (4, 5). Tonin converts angiotensin I to angiotensin II by releasing the C-terminal dipeptide histidylleucine (2, 12), the amount of dipeptide released being a measure of the enzyme activity. In order to have a quantitative estimation of this activity, the determination of histidyl-leucine has been performed by the fluorescence developed in its reaction with o-phthalaldehyde, the measurement of the fluorescence being carried out by an automated method (I), or after the equilibrium or at least in steady state during the reaction with the fluorogenic reagent (II). This paper deals with the kinetic monitoring of the histidyl-leucinelo-phthalaldehyde reaction as a means of determining the tonin activity in rat submaxillary glands. Kinetic methods of analysis, frequently cited for speed and versatility, can be used for the analysis of mixtures of closely related substances without prior separation, and, as a result of the relative character of the measurement cuvette imperfections, turbid samples, or accidental modifications in instrumental parameters have little or no effect on the quantitative methods. These and other characteristics have gained a wide acceptance for the reliable analysis of samples of mineral and biological origin (8, 9). The slow character of most of labeling reactions of peptide molecules with fluorogenic reagents (IO) provides a wide potential for the application of kinetic* To whom
reprint
requests
should
be addressed. 317 0026-265X/85
$1.50
Copyright Q 1985 by Academic Press, Inc. All rights of reproduction in any form reserved
318
BENTABOL
ET AL.
based methods to enzyme activity determinations. Although the generalized application of this methodology is basically restricted by kinetic considerations, the method reported here can be applied in a routine basis for the determination of tonin activity in a number of experimental samples of rat submaxillary glands (II), and it is expected to enlarge the possibilities for study of these complex analytical systems. MATERIALS Reagents
AND METHODS
and Solutions
Angiotensin
Z (isoleucine-5-angiotensin
Ltd.). A solution mixture was prepared.
I, Sigma Chemicals,
containing 500 pg/ml of angiotensin I in 1:3 methanol-water The solution was stored at - 18°C.
Dipeptide histidyl-leucine (Sigma Chemicals, Ltd.). Prepared weekly by dissolving 5 mg of the compound in 250 ml of ethanol and diluting to 1000 ml with deionized distilled water. The solution was stored in glass bottles at 4°C. o-Phthalaldehyde. One hundred milligrams of the reagent were dissolved in 25 ml of ethanol, diluting to 100 ml with water. Solutions of lower concentrations were made by dilution with water. Buffer solution. This incorporates the components needed to block nonspecific enzyme activities. The following solutions were mixed in proportion 15O:l: 1:2 v/v/v/v: phosphate buffer (pH 7.5), prepared by mixing the appropriate volumes of 0.1 M Na,HPO, and 0.1 it4 KH,PO,; diisopropylfluorophosphate (2% in isopropanol); o,a’-dipyridyl (20% in methanol); EDTA (1.3 x 10e3 M aqueous solution). In the pH study, phosphate buffer solutions were employed for pH values between 5 and 9; acetate buffer solutions for pH < 5; glycine solutions for pH > 9. The animals used were Albino-Wistar male rats. Submaxillary glands were removed and tonin was purified by homogenization and acetone extraction (3). Apparatus
A Perkin-Elmer fluorescence spectrophotometer, Model MPF-43A, with facilities for maintaining the cell holders at constant temperature was used. All the analytical information was obtained from initial rate and fixed time measurements. The fluorescence data are given without spectral correction. Procedure
In a 25-ml standard flask place 0.1 ml of angiotensin I solution and an aliquot of sample containing 50-375 nmol of histidyl-leucine or the equivalent amount of tonin. Add 5 ml of 1 m&f o-phthalaldehyde solution, 5 ml of buffer solution, and dilute to volume with deionized distilled water. Every 30 set measure the fluorescence intensity at 440 nm, excited at 355 nm. Plot the response curve and calculate the reaction rate (A&‘/At). Determine the content of histidyl-leucine by comparison with a calibration curve constructed with dipeptide standards. All solutions should be at 25°C.
DETERMINATION
4
OF TONIN
6
a
319
ACTIVITY
IO
‘* pH
FIG. 1. Effect of pH on the rate of the reaction between o-phthalaldehyde leucine (DP): [OPT] = 1 x 10m5M; [DP] = 2 X 10e6M.
(OPT) and histidyl-
RESULTS AND DISCUSSION
Tonin converts angiotensin I to angiotensin II by releasing the C-terminal dipeptide histidyl-leucine, which reacts with o-phthalaldehyde to give a product of intense blue fluorescence (hex 355, h,, 440 nm). Owing to the slow nature of this reaction, needing 60 min for full fluorescence development, the fluorescence monitoring of the reaction rate can give the basis for a kinetic method for the determination of histidyl-leucine. The application of this method for the determination of tonin activity in biological tissues can be performed if purified extracts of the enzyme are made to act on a prefixed amount of angiotensin I, the concentration of histidyl-leucine released being measured kinetically. However, it must be borne in mind that, as in most of the enzyme-substrate reactions, the action of tonin on angiotensin I is not an instantaneous process. Consequently, as the experimental optimization
FIG. 2. Influence of OPT concentration
10PTl.104 M on the reaction rate: [DP] = 1.5 x 1O-s M.
320
BENTABOL
I
IO
20
ET AL.
30
40 50 T.mpamcun, le
FIG. 3. Effect of temperature on the reaction rate: [OPT] = 2 x 10e4 M; [DP] = 1 x 10e5 M.
and the analytical calibration graph are established for the reaction his-leulophthalaldehyde, it is required for the analysis of biological samples that the kinetics of the fluorogenic reaction (instead the kinetics of the enzymatic hydrolysis) govern the rate of the overall reaction. Otherwise, the rate-determining character of the hydrolysis reaction would require a different calibration system based on tonin standards. The o-phthalaldehyde fluorogenic reaction of his-leu needs 60 min to reach completion at pH 7.4-7.6 (II), while, for the tonin activity occurring in rat submaxillary glands an angiotensin concentration of 2 x 10P3 g/ liter is adequate, depletion of the substrate being reached in 10 min. Both facts account for the slower character of the fluorogenic reaction, allowing the determination of tonin in biological tissues by the kinetic method for his-leu. Effect of Experimental
Variables on the His-Leulo-Phthalaldehyde
Reaction
A study on the effect of acidity showed that the rate of reaction reaches a maximum in the pH range 7.2-7.8 (Fig. I), this fact being relevant as it includes the pH values for the enzymatic action (physiological pH 7.4-7.6). A phosphate buffer, 0.1 M, pH 7.5, provides adequate buffering capacity in the optimum range. Figure 2 shows the effect of reagent concentration on the reaction rate. ReproTABLE 1 Summary of Kinetic Parameters of the Histidyl-Leucinelo-Phthalaldehyde Dependent variable
W+l
Observed orders
1.04 0.01
[His-Leu]
0.97
’ pH values.
Concentration range WI
-0.30 0.04 0.61
[o-Phthalaldehyde]
Reaction
5.0-7.1” 7.2-7.8” 7.8-8.6a
x 10-5-l x 10-4 1 x 10-4-3 x 10-4 2 x 10-G-15 x 10-b 4
DETERMINATION
OF TONIN
/ / / / / /’ / / / / // / /’ t/ 15.0 5.5
321
ACTIVITY
3
6
2.7
FIG. 4. Determination of tonin activity in extracts of rat submaxillary glands by the standard addition method: [OPT] = 2 x 10m4M; (W) 0.5 ml extract; (0) 1.O ml extract; (A) 3.0 ml extract.
ducibility considerations advise the use of an o-phthalaldehyde concentration of 2 x lop4 M, since small fluctuations in concentration will not affect the reaction rate. The graph on the influence of temperature (Fig. 3) reveals the existence of two opposite effects. The reaction rate increases with temperature for values up to 20°C. However, the quenching effect of temperature on the fluorescence becomes apparent as a decrease in rate when the temperature rises above 30°C. Both effects counterbalance each other for temperature values between 20 and 3O”C, thermostatic control being at 25°C of sample, when the solutions and measurement cell are appropriate. A survey of the kinetic parameters of the his-leulo-phthalaldehyde reaction is presented in Table 1. Under the selected reaction conditions, 25”C, pH 7.5, 0.1 M phosphate buffer, and 2 x 10W4M o-phthalaldehyde, the reaction is pseudo first order in histidyl-leucine. Calibration and Analytical
Parameters
Calibration graphs for his-leu were established by tangent and fixed time (4.0 min from the reaction start) methods. The reaction rate was linear with the hisleu concentration in the range 2-15 x lop6 M, this linear dependence ceasing for higher dipeptide concentrations. The fixed time method permits the same concentration range to be covered. Table 2 summarizes the analytical parameters TABLE 2 Analytical Parameters of the Kinetic Methods for the Determination of Histidyl-Leucine Method
Taken M
n
Found CM)
RSD” (‘?c)
G” (5%)
Initial rate Fixed time
9 x 10-h 9 x 10-h
IO II
9.8 x 10-h 9.8 x 10-h
6.0 1.1
4.2 5.5
n Relative standard deviation. ’ Relative error (100rs/n1’2 X, confidence level 9S%).
BENTABOL
322
ET AL.
TABLE 3 Tonin Activity Levels in Extracts of Rat Submaxillary Glands Tonin activity Volume of extract b-4
Measured solution (nmol his-leu/ml)
Tissue (pmol his-leu/g tissue)
0.5 1.0 3.0
2.7 5.5 15.0
3.63 3.69 3.36
of both methods, where the better specifications offered by the tangent method can be observed. The precision of rate measurements decreases notably for hisleu concentrations lower than 2 x lop6 M, considered the minimum determinable concentration. The establishment of the limit of detection on the basis of blank reproducibility considerations is not allowed for this method by the lack of background kinetic reaction. Analysis of Biological Samples
As discussed above, a substrate concentration of 2 x 10e3 g/liter is adequate for the determination of tonin activity in rat submaxillary glands. The action of potential interferent enzymes, which can also produce the hydrolysis of angiotensin I, has been blocked by addition of suitable chemical agents (7). The conversion enzyme has been inhibited by EDTA and ol,a’-dipyridyl, while trypsin and chymotrypsin are blocked by diisopropylfluorophosphate. These agents are incorporated into the reaction mixture by means of the buffer solution. No interferences of these agents with the kinetic determination of his-leu have been observed. The results for the analysis of preparations of rat submaxillary glands by the standard addition method are presented in Fig. 4. As shown, the graphs are linear for the use of 0.5, 1.0, and 3.0 ml of extract, indicating the persistence of the first-order dependence of reaction rate on the concentration of dipeptide. On the other hand, the change in slope as the sample volume increases reveals the existence of a matrix effect for high extract contents, which can be overcome by application of the standard addition method. Table 3 summarizes the results obtained by the tangent method. The precision of this method when applied to biological samples was determined by repetitively monitoring the reaction of 2 x lop6 M his-leu added to OS-ml extract samples with 2 x lop4 M o-phthalaldehyde. The relative standard deviation was found to be 9.5%. Although the application of the present methodology to other enzyme-substrate reactions is basically restricted by kinetic considerations, verification of the ratedetermining character of the fluorogenic reaction can lead to useful information on enzyme activities. The method has the advantage of freedom of blank, since no background kinetic reaction is possible in the absence of fluorogenic reagent. Another advantage is simplicity; special instrumentation is not required.
DETERMINATION
OF TONIN
ACTIVITY
323
REFERENCES Boucher, R., Saidi, M., and Genest, J., A new “angiotensin I converting enzyme” system. In “Hypertension 72” (J. Genest and E. Koiw, Eds.), pp. 512-523. Springer-Verlag, Heidelberg, 1972. 2. Boucher, R., Asselin, J., and Genest, J., A new enzyme leading to the direct formation of angiotensin II. Circ. Res. 34-35, suppl. 1, 203-212 (1974). 3. Boucher, R., Kurihara, H., Grise, C., and Genest, J., Conversion of angiotensin I: measurement of plasma angiotensin I converting enzyme activity. Circ. Res. 26-27, suppl. 1, 83-91 (1970). 4. Garcia, R., Kondo, K., Scholkens, B., Boucher, R., and Genest, J., Effects in vivo of several drugs upon tonin concentration in rat saliva, and submaxillary gland. Canad. J. Physiol. Phar1.
macol.
55, 983 (1976).
5. Garcia, R., Boucher, R., and Genest, J., Tonin activity in rat saliva: effect of sympathomimetic and parasympathomimetic drugs. Canad. J. Physiol. Pharmacol. 54, 443-445 (1976). 6. Gutman, Y., Levy, M., and Shorr, J., Renin-like activity of the rat submaxillary gland: characterization and the effect of several drugs and stimuli. Brir. J. Pharmacol. 47, 59 (1970). 7. Lis, M., Boucher, R., Chretien, M., and Genest, J., Dependence of tonin activity in rat submaxillary gland on growth hormone and testosterone. Am. J. Physiol. 232, 522 (1977). 8. Mottola, H. A., and Mark, H. B., Kinetic determinations and some kinetic aspects of Analytical Chemistry. Anal. Chem. 52, 3lR-40R (1980). 9. Mottola, H. A., and Mark, H. B., Kinetic determination and some kinetic aspects of Analytical Chemistry. Anal. Chem. 54, 62R-70R (1982). 10. Nairn, R. C. (Ed.), “Fluorescent Protein Tracing,” p. 628, Longman, Edinburg, 1976. 11. Reyes, A., Narvaez, J. A., Fernandez Pastor, J. M., Morell, M., Laserna, J. J., and Garcia Sanchez, F., to be published. 12. Schiller, P. W., Demassieux, S., and Boucher, R., Substrate specificity of tonin from rat submaxillary gland. Circ. Res. 39, 629-632 (1976).
32,
JOURNAL
A Fluorimetric
3 17-323
Kinetic Method for the Determination Activity in Rat Submaxillary Glands
C.BENTABOL, Department
(1985)
A. REYES, J.A.
of Biochemistry,
Faculty
NARVAEZ,AND
of Medicine,
University
of Tonin
M. MORELL
of Mrilaga,
Mdlaga,
Spain
AND
J. J. LASERNAAND Department
of Analytical
Chemistry,
Faculty
F. GARCIA SANCHEZ* of Sciences,
University
of Mdlaga,
Mrilaga,
Spain
Received October 29, 1983 A method for the kinetic determination of tonin activity in rat submaxillary gland extracts is described. The method is based on the fluorogenic reaction of o-phthalaldehyde with the dipeptide histidyl-leucine generated in the enzymatic hydrolysis of angiotensin I. The reaction is monitored by fluorescence at 440 nm. At pH 7.5, the method allows the determination of 2-15 nmol/ml histidyl-leucine with relative standard deviation of 6%. o 1985 Academic Press, Inc.
INTRODUCTION Tonin, an enzyme found in submaxillary glands (Z), has promoted a remarkable interest by its relation with the reninangiotensin system (6) and its connection with the control of the blood irrigation at a local level (4, 5). Tonin converts angiotensin I to angiotensin II by releasing the C-terminal dipeptide histidylleucine (2, 12), the amount of dipeptide released being a measure of the enzyme activity. In order to have a quantitative estimation of this activity, the determination of histidyl-leucine has been performed by the fluorescence developed in its reaction with o-phthalaldehyde, the measurement of the fluorescence being carried out by an automated method (I), or after the equilibrium or at least in steady state during the reaction with the fluorogenic reagent (II). This paper deals with the kinetic monitoring of the histidyl-leucinelo-phthalaldehyde reaction as a means of determining the tonin activity in rat submaxillary glands. Kinetic methods of analysis, frequently cited for speed and versatility, can be used for the analysis of mixtures of closely related substances without prior separation, and, as a result of the relative character of the measurement cuvette imperfections, turbid samples, or accidental modifications in instrumental parameters have little or no effect on the quantitative methods. These and other characteristics have gained a wide acceptance for the reliable analysis of samples of mineral and biological origin (8, 9). The slow character of most of labeling reactions of peptide molecules with fluorogenic reagents (IO) provides a wide potential for the application of kinetic* To whom
reprint
requests
should
be addressed. 317 0026-265X/85
$1.50
Copyright Q 1985 by Academic Press, Inc. All rights of reproduction in any form reserved
318
BENTABOL
ET AL.
based methods to enzyme activity determinations. Although the generalized application of this methodology is basically restricted by kinetic considerations, the method reported here can be applied in a routine basis for the determination of tonin activity in a number of experimental samples of rat submaxillary glands (II), and it is expected to enlarge the possibilities for study of these complex analytical systems. MATERIALS Reagents
AND METHODS
and Solutions
Angiotensin
Z (isoleucine-5-angiotensin
Ltd.). A solution mixture was prepared.
I, Sigma Chemicals,
containing 500 pg/ml of angiotensin I in 1:3 methanol-water The solution was stored at - 18°C.
Dipeptide histidyl-leucine (Sigma Chemicals, Ltd.). Prepared weekly by dissolving 5 mg of the compound in 250 ml of ethanol and diluting to 1000 ml with deionized distilled water. The solution was stored in glass bottles at 4°C. o-Phthalaldehyde. One hundred milligrams of the reagent were dissolved in 25 ml of ethanol, diluting to 100 ml with water. Solutions of lower concentrations were made by dilution with water. Buffer solution. This incorporates the components needed to block nonspecific enzyme activities. The following solutions were mixed in proportion 15O:l: 1:2 v/v/v/v: phosphate buffer (pH 7.5), prepared by mixing the appropriate volumes of 0.1 M Na,HPO, and 0.1 it4 KH,PO,; diisopropylfluorophosphate (2% in isopropanol); o,a’-dipyridyl (20% in methanol); EDTA (1.3 x 10e3 M aqueous solution). In the pH study, phosphate buffer solutions were employed for pH values between 5 and 9; acetate buffer solutions for pH < 5; glycine solutions for pH > 9. The animals used were Albino-Wistar male rats. Submaxillary glands were removed and tonin was purified by homogenization and acetone extraction (3). Apparatus
A Perkin-Elmer fluorescence spectrophotometer, Model MPF-43A, with facilities for maintaining the cell holders at constant temperature was used. All the analytical information was obtained from initial rate and fixed time measurements. The fluorescence data are given without spectral correction. Procedure
In a 25-ml standard flask place 0.1 ml of angiotensin I solution and an aliquot of sample containing 50-375 nmol of histidyl-leucine or the equivalent amount of tonin. Add 5 ml of 1 m&f o-phthalaldehyde solution, 5 ml of buffer solution, and dilute to volume with deionized distilled water. Every 30 set measure the fluorescence intensity at 440 nm, excited at 355 nm. Plot the response curve and calculate the reaction rate (A&‘/At). Determine the content of histidyl-leucine by comparison with a calibration curve constructed with dipeptide standards. All solutions should be at 25°C.
DETERMINATION
4
OF TONIN
6
a
319
ACTIVITY
IO
‘* pH
FIG. 1. Effect of pH on the rate of the reaction between o-phthalaldehyde leucine (DP): [OPT] = 1 x 10m5M; [DP] = 2 X 10e6M.
(OPT) and histidyl-
RESULTS AND DISCUSSION
Tonin converts angiotensin I to angiotensin II by releasing the C-terminal dipeptide histidyl-leucine, which reacts with o-phthalaldehyde to give a product of intense blue fluorescence (hex 355, h,, 440 nm). Owing to the slow nature of this reaction, needing 60 min for full fluorescence development, the fluorescence monitoring of the reaction rate can give the basis for a kinetic method for the determination of histidyl-leucine. The application of this method for the determination of tonin activity in biological tissues can be performed if purified extracts of the enzyme are made to act on a prefixed amount of angiotensin I, the concentration of histidyl-leucine released being measured kinetically. However, it must be borne in mind that, as in most of the enzyme-substrate reactions, the action of tonin on angiotensin I is not an instantaneous process. Consequently, as the experimental optimization
FIG. 2. Influence of OPT concentration
10PTl.104 M on the reaction rate: [DP] = 1.5 x 1O-s M.
320
BENTABOL
I
IO
20
ET AL.
30
40 50 T.mpamcun, le
FIG. 3. Effect of temperature on the reaction rate: [OPT] = 2 x 10e4 M; [DP] = 1 x 10e5 M.
and the analytical calibration graph are established for the reaction his-leulophthalaldehyde, it is required for the analysis of biological samples that the kinetics of the fluorogenic reaction (instead the kinetics of the enzymatic hydrolysis) govern the rate of the overall reaction. Otherwise, the rate-determining character of the hydrolysis reaction would require a different calibration system based on tonin standards. The o-phthalaldehyde fluorogenic reaction of his-leu needs 60 min to reach completion at pH 7.4-7.6 (II), while, for the tonin activity occurring in rat submaxillary glands an angiotensin concentration of 2 x 10P3 g/ liter is adequate, depletion of the substrate being reached in 10 min. Both facts account for the slower character of the fluorogenic reaction, allowing the determination of tonin in biological tissues by the kinetic method for his-leu. Effect of Experimental
Variables on the His-Leulo-Phthalaldehyde
Reaction
A study on the effect of acidity showed that the rate of reaction reaches a maximum in the pH range 7.2-7.8 (Fig. I), this fact being relevant as it includes the pH values for the enzymatic action (physiological pH 7.4-7.6). A phosphate buffer, 0.1 M, pH 7.5, provides adequate buffering capacity in the optimum range. Figure 2 shows the effect of reagent concentration on the reaction rate. ReproTABLE 1 Summary of Kinetic Parameters of the Histidyl-Leucinelo-Phthalaldehyde Dependent variable
W+l
Observed orders
1.04 0.01
[His-Leu]
0.97
’ pH values.
Concentration range WI
-0.30 0.04 0.61
[o-Phthalaldehyde]
Reaction
5.0-7.1” 7.2-7.8” 7.8-8.6a
x 10-5-l x 10-4 1 x 10-4-3 x 10-4 2 x 10-G-15 x 10-b 4
DETERMINATION
OF TONIN
/ / / / / /’ / / / / // / /’ t/ 15.0 5.5
321
ACTIVITY
3
6
2.7
FIG. 4. Determination of tonin activity in extracts of rat submaxillary glands by the standard addition method: [OPT] = 2 x 10m4M; (W) 0.5 ml extract; (0) 1.O ml extract; (A) 3.0 ml extract.
ducibility considerations advise the use of an o-phthalaldehyde concentration of 2 x lop4 M, since small fluctuations in concentration will not affect the reaction rate. The graph on the influence of temperature (Fig. 3) reveals the existence of two opposite effects. The reaction rate increases with temperature for values up to 20°C. However, the quenching effect of temperature on the fluorescence becomes apparent as a decrease in rate when the temperature rises above 30°C. Both effects counterbalance each other for temperature values between 20 and 3O”C, thermostatic control being at 25°C of sample, when the solutions and measurement cell are appropriate. A survey of the kinetic parameters of the his-leulo-phthalaldehyde reaction is presented in Table 1. Under the selected reaction conditions, 25”C, pH 7.5, 0.1 M phosphate buffer, and 2 x 10W4M o-phthalaldehyde, the reaction is pseudo first order in histidyl-leucine. Calibration and Analytical
Parameters
Calibration graphs for his-leu were established by tangent and fixed time (4.0 min from the reaction start) methods. The reaction rate was linear with the hisleu concentration in the range 2-15 x lop6 M, this linear dependence ceasing for higher dipeptide concentrations. The fixed time method permits the same concentration range to be covered. Table 2 summarizes the analytical parameters TABLE 2 Analytical Parameters of the Kinetic Methods for the Determination of Histidyl-Leucine Method
Taken M
n
Found CM)
RSD” (‘?c)
G” (5%)
Initial rate Fixed time
9 x 10-h 9 x 10-h
IO II
9.8 x 10-h 9.8 x 10-h
6.0 1.1
4.2 5.5
n Relative standard deviation. ’ Relative error (100rs/n1’2 X, confidence level 9S%).
BENTABOL
322
ET AL.
TABLE 3 Tonin Activity Levels in Extracts of Rat Submaxillary Glands Tonin activity Volume of extract b-4
Measured solution (nmol his-leu/ml)
Tissue (pmol his-leu/g tissue)
0.5 1.0 3.0
2.7 5.5 15.0
3.63 3.69 3.36
of both methods, where the better specifications offered by the tangent method can be observed. The precision of rate measurements decreases notably for hisleu concentrations lower than 2 x lop6 M, considered the minimum determinable concentration. The establishment of the limit of detection on the basis of blank reproducibility considerations is not allowed for this method by the lack of background kinetic reaction. Analysis of Biological Samples
As discussed above, a substrate concentration of 2 x 10e3 g/liter is adequate for the determination of tonin activity in rat submaxillary glands. The action of potential interferent enzymes, which can also produce the hydrolysis of angiotensin I, has been blocked by addition of suitable chemical agents (7). The conversion enzyme has been inhibited by EDTA and ol,a’-dipyridyl, while trypsin and chymotrypsin are blocked by diisopropylfluorophosphate. These agents are incorporated into the reaction mixture by means of the buffer solution. No interferences of these agents with the kinetic determination of his-leu have been observed. The results for the analysis of preparations of rat submaxillary glands by the standard addition method are presented in Fig. 4. As shown, the graphs are linear for the use of 0.5, 1.0, and 3.0 ml of extract, indicating the persistence of the first-order dependence of reaction rate on the concentration of dipeptide. On the other hand, the change in slope as the sample volume increases reveals the existence of a matrix effect for high extract contents, which can be overcome by application of the standard addition method. Table 3 summarizes the results obtained by the tangent method. The precision of this method when applied to biological samples was determined by repetitively monitoring the reaction of 2 x lop6 M his-leu added to OS-ml extract samples with 2 x lop4 M o-phthalaldehyde. The relative standard deviation was found to be 9.5%. Although the application of the present methodology to other enzyme-substrate reactions is basically restricted by kinetic considerations, verification of the ratedetermining character of the fluorogenic reaction can lead to useful information on enzyme activities. The method has the advantage of freedom of blank, since no background kinetic reaction is possible in the absence of fluorogenic reagent. Another advantage is simplicity; special instrumentation is not required.
DETERMINATION
OF TONIN
ACTIVITY
323
REFERENCES Boucher, R., Saidi, M., and Genest, J., A new “angiotensin I converting enzyme” system. In “Hypertension 72” (J. Genest and E. Koiw, Eds.), pp. 512-523. Springer-Verlag, Heidelberg, 1972. 2. Boucher, R., Asselin, J., and Genest, J., A new enzyme leading to the direct formation of angiotensin II. Circ. Res. 34-35, suppl. 1, 203-212 (1974). 3. Boucher, R., Kurihara, H., Grise, C., and Genest, J., Conversion of angiotensin I: measurement of plasma angiotensin I converting enzyme activity. Circ. Res. 26-27, suppl. 1, 83-91 (1970). 4. Garcia, R., Kondo, K., Scholkens, B., Boucher, R., and Genest, J., Effects in vivo of several drugs upon tonin concentration in rat saliva, and submaxillary gland. Canad. J. Physiol. Phar1.
macol.
55, 983 (1976).
5. Garcia, R., Boucher, R., and Genest, J., Tonin activity in rat saliva: effect of sympathomimetic and parasympathomimetic drugs. Canad. J. Physiol. Pharmacol. 54, 443-445 (1976). 6. Gutman, Y., Levy, M., and Shorr, J., Renin-like activity of the rat submaxillary gland: characterization and the effect of several drugs and stimuli. Brir. J. Pharmacol. 47, 59 (1970). 7. Lis, M., Boucher, R., Chretien, M., and Genest, J., Dependence of tonin activity in rat submaxillary gland on growth hormone and testosterone. Am. J. Physiol. 232, 522 (1977). 8. Mottola, H. A., and Mark, H. B., Kinetic determinations and some kinetic aspects of Analytical Chemistry. Anal. Chem. 52, 3lR-40R (1980). 9. Mottola, H. A., and Mark, H. B., Kinetic determination and some kinetic aspects of Analytical Chemistry. Anal. Chem. 54, 62R-70R (1982). 10. Nairn, R. C. (Ed.), “Fluorescent Protein Tracing,” p. 628, Longman, Edinburg, 1976. 11. Reyes, A., Narvaez, J. A., Fernandez Pastor, J. M., Morell, M., Laserna, J. J., and Garcia Sanchez, F., to be published. 12. Schiller, P. W., Demassieux, S., and Boucher, R., Substrate specificity of tonin from rat submaxillary gland. Circ. Res. 39, 629-632 (1976).