ANALYTICAL
BIOCHEMISTRY
A Rapid
MAKOTO
84,
361-369 (1978)
and Simple Spectrophotometric Assay of Angiotensin-Converting Enzyme
HAYAKARI,*
* Pharmaceutical tDepartment
YOSHIKAZU
KONDO,*
AND HIROSHI
IZUMI?
Institute, Tohoku University, Aobayama, Sendai, Japan of Physiology, Tohoku University School of Dentistry, Seityo-machi, Sendai, Japan
and
Received March 14, 1977; accepted August 26, 1977 A rapid, simple, and accurate method for the chemical assay of angiotensin-converting enzyme has been developed. The method relies on previously published method for spectrophotometric assay of angiotensinconverting enzyme activity and on the use of 2,4,6-trichloro-s-triazine (TT) as a calorimetric reagent of hippuric acid (N-benzoylglycine). When 3% TT in dioxane was added to the incubation medium of the angiotensin-converting enzyme after stopping the incubation by the immersion of the test tubes in a boiling-water bath, the absorbance at 382 nm increased linearly as a function of both enzyme concentration and incubation time. Neither hippuryl-L-histidyl-tleucine (HHL, substrate for this assay system) nor histidyl-leucine was positive in color reaction with TT. Accordingly, this method does not require any procedures for separation of hippuric acid from HHL. The enzyme activity was found to be highest at pH 8.3, at chloride ion concentration of 600 mM, and at HHL concentration of 3 mM. when the SOOOg supematant fluid of the rat lung was used.
Although the angiotensin-converting enzyme is widespread in animal organs (l), it plays an especially important biological role in pulmonary circulation. At present, a suitable method for determining the level of serum angiotensin-converting enzyme is required for clinical diagnosis of sarcoidosis and Gaucher’s disease. Several methods for determination of this enzyme activity have been reported. Piquilloud ef al. (2) reported a method in which L-histidyl-L-leucine released from the synthetic substrates hippuryk-histidyk-leucine (HHL) or hippuryl-glycyl-glycine by the enzymic hydrolysis was assayed fluorometically using o-phthalaldehyde. Cushman and Cheung (3) have demonstrated the other method in which hippuric acid released from HHL was extracted with ethylacetate and determined spectrophotometically. However, these methods are tedious for clinical purposes. In this paper, we report a direct spectrophotometric assay of the angiotensin-converting enzyme, the method of which is based upon the specific calorimetric reaction of liberating hippuric acid with 2,4,6-trichloro-s361
0003-2697/78/0842-0361$02.00/O Copyright 0 1978 by Academic Ress, Inc. All rights of reproduction in any form reserved.
362
HAYAKARI,
KONDO
AND
IZUMI
triazine (TT) (4). This method has these advantages: direct and accurate determination without separation of hippuric acid from HHL and a singlebottle procedure. This method provides for sensitive, accurate, and simplified estimation of the angiotensin-converting enzyme. EXPERIMENTAL
Materials. HHL was purchased from Protein Research Foundation, Mino, Osaka, Japan. TT and dioxane were obtained from Nakarai Chemicals, Ltd. (Japan). Dioxane was purified by fractional distillation after removal of the peroxides on addition of lithium aluminum hydride. Hippuric acid was purchased from Wako Pure Chemical Industry, Ltd. (Japan). All other chemicals were analytical grade or best commercially available. Preparation of angiotensin-converting enzyme. Angiotensin-converting enzyme was prepared by using a modification of method described by Cushman and Cheung (3). Male Sprague-Dawley rats (CLEA, Japan, Inc.), weighing 250-300 g, were used in these experiments. After the animals were decapitated, the lungs were isolated and washed with icecold 10 mM potassium phosphate buffer, pH 8.3, and were kept frozen until used. Tissue sample was diced and homogenized in 10 volumes of the same ice-cold buffer. The homogenate was centrifuged at SOOOgfor 10 min, and the resulting supernatant fluid was dialyzed for 24 hr against 20 volumes of the same buffer at 4°C. The buffer was exchanged four times. The dialyzed 5OOOgsupernatant fluid was used as the enzyme source for angiotensin-converting enzyme. Assay of angiotensin-converting enzyme. The assay for the angiotensinconverting enzyme activity was carried out in a 0.5-ml incubation mixture containing 40 pmol of potassium phosphate buffer, pH 8.3, 300 pmol of sodium chloride, 1.5 pmol of HHL, and the enzyme. The reaction was initiated by the addition of the substrate, and the reaction mixture was incubated at 37°C for 30 min. Reactions were terminated by immersing the test tube in a boiling-water bath for 10 min. After the addition of 3 ml of 0.2 M phosphate buffer, pH 8.3, 1.5 ml of 3% TT solution (in dioxane) was added to the reaction mixture with vigorous stirring until the turbid solution became transparent, and the tubes were centrifuged at 1OOOg for 10 min. The enzyme activity in the resulting supernatant fluid was determined from the absorbance at 382 nm by the differential spectrophotometric method. The control run was identical to the above procedure minus the incubation. Definition of enzyme unit. One unit of enzyme activity was defined as the amount of enzyme that hydrolyzed 1 pmol of HHL to hippuric acid plus L-histidyl-L-leucine in 1 min at 37°C under the condi-
ASSAY
OF ANGIOTENSIN-CONVERTING
363
ENZYME
tions described above. The specific activity was the number of units per milligram of protein. Protein determination. The protein concentrations were determined according to the procedure described by Lowry et al. (5) in which crystalline bovine serum albumine was used at the standard. RESULTS
Calibration curve of hippuric acid. In preliminary experiments, the absorbance at 382 nm of known samples (up to 50 pg) containing hippuric acid-TT chromogen in either 0.2 M phosphate buffer, pH 8.3, or the incubation mixture of angiotensin-converting enzyme containing HHL was determined. A similar linearity between absorbance and hippuric acid concentration in each medium was observed (Fig. 1). These results suggest that the presence of substrate (HHL) does not interfere with the reaction of hippuric acid and TT. The critical factors are determined as follows. Influence of 2,4,6-trichloro-s-triazine concentration. The effects of various concentrations of TT on the intensity of the chromogen were investigated with a specific concentration of hippuric acid (25 pg) in the present assay system. When the final concentrations of TT were less than 0.8%, TT caused a dose-dependent intensity. But over that concentration, the intensity reached a plateau. In the present experiments, a final TT concentration of 0.9% was used (Fig. 2). 2.0
FIG.
1. Calibration
curve
Concentration
of Hippuric
for hippuric
acid quantity
Acid
versus
( pg/tube
relative
)
absorbance.
364
HAYAKARI,
KONDO
0
AND
IZUMI
1.5 Final
Concentration
3 of T.T.
Reagent
( % )
FIG. 2. Effect of TT reagent concentrations. Each concentration of TT reagent was added to the incubation mixture containing 40 pg of hippuric acid per tube used as a standard.
Activity as a function of time and enzyme concentration. On the basis of the above results, we have determined the amounts of hippuric acid released from HHL by the action of angiotensin-converting enzyme prepared from lung at various times of incubation and with various enzyme concentrations. As can be seen in Fig. 3, the formation of hippuric acid is linear for at least 40 min with enzyme concentrations of up to 1.3 mg of protein/assay. On the high-concentration, 60-min incubation, however, the apparent absorbance deviated from the reliable linearity, presumably because of inactivation of the enzyme. Znjluence of pH. The rate of hydrolysis of HHL by angiotensinconverting enzyme of rat lung was determined at pH values from 6.0 to 9.0 in the presence of 600 mM NaCl (Fig. 4). The maximum rate was found to occur at pH 8.3. Influence of chloride ion. The effect of chloride ion concentration on the angiotensin-converting enzyme activity in rat lung was examined (Fig. 5). The enzymic activity was very low in the absence of chloride ion, and an increase in chloride ion concentration caused a progressive increase in the activity of angiotensin-converting enzyme, reaching a plateau at above a 600 mM chloride ion concentration. Activity as a function of substrate concentration. Assays of angiotensin-converting enzyme in the rat lung were conducted in various HHL concentrations (Fig. 6). The optimal concentration of HHL was 3
ASSAY OF ANGIOTENSIN-CONVERTING
365
ENZYME
600
400
200
0 0
10
20
30 Time
( minutes
60 )
FIG. 3. Effect of incubation time on the activity of angiotensin-converting enzyme of rat lung. The enzyme activities were assayed as described in Experimental. Three different protein concentrations of rat lung enzyme were employed: 139 pg of protein/assay (A); 0.69 mg of protein/assay (0); and 1.39 mg of protein/assay (0).
mM; higher concentrations of substrate reduced enzymic activity. The apparent K, value for HHL was calculated to be approximately 1.11 mM. In the present experiments, a 3 mM concentration of HHL was used. Znjluence of chelating reagents. Two chelating compounds were tested for their inhibitory activity on the angiotensin-converting enzyme. As shown in Table 1, both EDTA and 8-hydroxyquinoline were potent inhibitors. In particular, EDTA produced a marked inhibition even at concentrations as low as 0.1 mh4. DISCUSSION
Angiotensin-converting enzyme splits a histidyl-leucine dipeptide from the decapeptide structure of angiotensin I, converting it to the active octapeptide angiotensin II; its properties and distribution in the body have been studied by others (6-8). Furthermore, recent reports have revealed that patients with clinical sarcoidosis and Gaucher’s disease exhibit an elevated level of angiotensin-converting enzyme activity in the serum (9-12). Previously, the system was assayed by biological measurement of the formation of angiotensin II from an angiotensin I substrate (13). Huggins and Thampi (14) described a radiometric assay using labeled angiotensin
HAYAKARI,
366
KONDO AND IZLJMI
I
0
7
6
I 9
8 PH
4. Effect of pH on the activity of angiotensin-converting enzyme of rat lung. Assay conditions were as described in Experimental. The buffer used was 0.2 M phosphate buffer. The enzyme (1.39 mg of protein/assay) was incubated at 37°C for 30 min in the presence of 0.3 M NaCl. FIG.
0 0
200
400 Chloride
600 Ion
Concentration
800
1,000 ( mM )
FIG. 5. Effect of chloride ion concentration on the activity of angiotensin-converting enzyme of rat lung. Enzyme (1.39 mg of protein/assay) was incubated at 37°C for 30 min under conditions described in Experimental without 0.3 M NaCl.
ASSAY
OF
ANGIOTENSIN-CONVERTING
367
ENZYME
20
0 10
5
0 Substrate
( mM ]
FIG. 6. Effect of varying concentrations of substrate on the activity of angiotensinconverting enzyme of rat lung. Assay conditions were as described in Experimental. The reaction was started by the addition of varying concentrations of HHL, and the incubation was carried out at 37°C for 30 min. V, reaction velocity in nanomoles per minute; S, substrate concentration (millimolar).
I as the substrate, in which the release of radioactive histidyl-leucine served as an index of enzymic activity. On the other hand, histidylleucine was also determined by fluorometrically (2,15). Recently, Cushman and Cheung (3) developed an assay system employing ‘hippurylTABLE INHIBITION
1
OF ANGIOTENSIN-CONVERTING ENZYME BY THE CHELATING REAGENTS”
Compound EDTA
8-Hydroxyquinoline
Concentration (M) 1 x 10-S 1 x 10-4 1 x 10-S 1 x IO-3 1 x 10-4 1 x 10-S
OF RAT LUNG
Percentage of control activity 4 8
18 44 98
loo
a The enzyme (1.39 mg of protein/assay) was incubated with each compound at the indicated final concentration. Incubations were carried out at 37°C for 30 min under condition described in Experimental.
368
HAYAKARI,
KONDO
AND
IZUMI
L-histidyl-L-leucine (HHL) as substrate which measures the formation of free hippuric acid by the action of angiotensin-converting enzyme. In their report, however, hippuric acid was determined after its separation from unreacted HHL by extraction into ethylacetate. Accordingly, it is necessary to calculate the conversion factor. It has been reported by Suzuki et al. (4) that N-monosubstituted derivatives of glycine produce a colored material (chromogen) on reaction with 2,4,6-trichloro-s-triazine (TT) (4). In the present experiments, we have investigated whether or not TT is available for the determination of hippuric acid in the incubation medium of the angiotensin-converting enzyme assay without the extraction of hippuric acid. The hippuric acid-TT chromogen was found to be quite stable in the incubation medium. There were no observable changes in the absorbances of the solution in the course of the experiment and even after standing for 3-6 hr at room temperature. These results suggested a practically complete recovery of hippuric acid without separation from HHL under the optimal conditions. Several critical factors in this enzymatic reaction are similar to results obtained by Cushman and Cheung (3). The enzymic reaction was interfered with by reagents such as EDTA and 8-oxyquinoline. HHL, a tripeptide substrate first synthesized by Cushman and Cheung, was preferred as substrate for the assay of angiotensin-converting enzyme because HHL can be employed over a wide range of substrate and enzyme concentrations (3). It has also been reported that chymotrypsin and aminopeptidase show no activity on HHL (3). On the other hand, many other tripeptide substrates besides HHL for angiotensin-converting enzyme have previously been reported, such as Bz-Gly-Phe-Phe, Bz-GlyGly-Pro, Bz-Gly-Gly-Gly, Bz-Phe(NOz)-His-Leu, Bz-Phe(NO,)-Gly-Gly, and Z-Gly-Lys-Gly (2,7,16).’ In the experiment, we measured hippuric acid liberated from HHL. Accordingly, tripeptide substrates such as Bz-Gly-Phe-Phe and Bz-Gly-Gly-Pro could be also available as substrates of the angiotensin-converting enzyme in the present method. However, since N-substituted derivatives of serine, threonine, and tryptophan and glycyl-glycine and N-acetyl-glycine also gave positive color reactions with TT (4), it is necessary to use caution in selecting the substrate. REFERENCES 1. 2. 3. 4. 5.
Skeggs, L. T., Kahn, J. R., and Shumway, N. P. (1956) J. Exp. Med. 103, 295-299. Piquilloud, Y., Reinharz, A., and Roth, M. (1970)Biochim. Biophys. Acta 206; 136-142. Cushman, D. W., and Cheung, H. S. (1971) Biochem. Pharmacol. 20, 1637-1648. Suzuki, S., Hachimori, Y., and Yaoeda, U. (1970)Anal. Chem. 42, 101-103. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951)3. Biol. Chem. 193, 265-275.
1 Abbreviations
used:
Bz, benzoyl;
Z-, benzoylcarbonyl-.
ASSAY OF ANGIOTENSIN-CONVERTING
ENZYME
369
6. Cushman, D. W., and Cheung, H. S. (1971) Biochem. Biophys. Acta 250, 261-265. 7. Yang, H. Y. T., Erdos, E. G., and Levin, Y. (1971) J. Pharmacol. Exp. Med. 177, 291-300. 8. Ryan, J. W., Ryan, U. S., Schultz, D. R., Whitaker, C., and Chung, A. (1975)Biochem. J. 146, 497-499. 9. Lieberman, J. (1975)Amer. J. Med. 59, 365-372. 10. Lieberman, J. (1976) N. Engl. J. Med. 294, 1442-1444. 11. Silberstein, E., Friedland, J., Lyons, H. A., and Gourin, A. (1975) C/in. Res. 23, 352A. 12. Silberstein, E., Friedland, J., Lyons, H. A., and Gourin, A. (1976) Proc. Nat. Acad. Sci. USA 73, 2137-2141. 13. Boucher, R., Kurihara, H., Grise, G., and Genest, J. (1970) Circ. Res. 26, 27, Suppl. 1, 83-91. 14. Huggins, C. G., and Thampi, N. P. (1969) Life Sci. 7, 633-639. 15. Friedland, J., and Silberstein, E. (1976) Amer. J. Clin. Puthol. 66, 416-424.
BIOCHEMISTRY
A Rapid
MAKOTO
84,
361-369 (1978)
and Simple Spectrophotometric Assay of Angiotensin-Converting Enzyme
HAYAKARI,*
* Pharmaceutical tDepartment
YOSHIKAZU
KONDO,*
AND HIROSHI
IZUMI?
Institute, Tohoku University, Aobayama, Sendai, Japan of Physiology, Tohoku University School of Dentistry, Seityo-machi, Sendai, Japan
and
Received March 14, 1977; accepted August 26, 1977 A rapid, simple, and accurate method for the chemical assay of angiotensin-converting enzyme has been developed. The method relies on previously published method for spectrophotometric assay of angiotensinconverting enzyme activity and on the use of 2,4,6-trichloro-s-triazine (TT) as a calorimetric reagent of hippuric acid (N-benzoylglycine). When 3% TT in dioxane was added to the incubation medium of the angiotensin-converting enzyme after stopping the incubation by the immersion of the test tubes in a boiling-water bath, the absorbance at 382 nm increased linearly as a function of both enzyme concentration and incubation time. Neither hippuryl-L-histidyl-tleucine (HHL, substrate for this assay system) nor histidyl-leucine was positive in color reaction with TT. Accordingly, this method does not require any procedures for separation of hippuric acid from HHL. The enzyme activity was found to be highest at pH 8.3, at chloride ion concentration of 600 mM, and at HHL concentration of 3 mM. when the SOOOg supematant fluid of the rat lung was used.
Although the angiotensin-converting enzyme is widespread in animal organs (l), it plays an especially important biological role in pulmonary circulation. At present, a suitable method for determining the level of serum angiotensin-converting enzyme is required for clinical diagnosis of sarcoidosis and Gaucher’s disease. Several methods for determination of this enzyme activity have been reported. Piquilloud ef al. (2) reported a method in which L-histidyl-L-leucine released from the synthetic substrates hippuryk-histidyk-leucine (HHL) or hippuryl-glycyl-glycine by the enzymic hydrolysis was assayed fluorometically using o-phthalaldehyde. Cushman and Cheung (3) have demonstrated the other method in which hippuric acid released from HHL was extracted with ethylacetate and determined spectrophotometically. However, these methods are tedious for clinical purposes. In this paper, we report a direct spectrophotometric assay of the angiotensin-converting enzyme, the method of which is based upon the specific calorimetric reaction of liberating hippuric acid with 2,4,6-trichloro-s361
0003-2697/78/0842-0361$02.00/O Copyright 0 1978 by Academic Ress, Inc. All rights of reproduction in any form reserved.
362
HAYAKARI,
KONDO
AND
IZUMI
triazine (TT) (4). This method has these advantages: direct and accurate determination without separation of hippuric acid from HHL and a singlebottle procedure. This method provides for sensitive, accurate, and simplified estimation of the angiotensin-converting enzyme. EXPERIMENTAL
Materials. HHL was purchased from Protein Research Foundation, Mino, Osaka, Japan. TT and dioxane were obtained from Nakarai Chemicals, Ltd. (Japan). Dioxane was purified by fractional distillation after removal of the peroxides on addition of lithium aluminum hydride. Hippuric acid was purchased from Wako Pure Chemical Industry, Ltd. (Japan). All other chemicals were analytical grade or best commercially available. Preparation of angiotensin-converting enzyme. Angiotensin-converting enzyme was prepared by using a modification of method described by Cushman and Cheung (3). Male Sprague-Dawley rats (CLEA, Japan, Inc.), weighing 250-300 g, were used in these experiments. After the animals were decapitated, the lungs were isolated and washed with icecold 10 mM potassium phosphate buffer, pH 8.3, and were kept frozen until used. Tissue sample was diced and homogenized in 10 volumes of the same ice-cold buffer. The homogenate was centrifuged at SOOOgfor 10 min, and the resulting supernatant fluid was dialyzed for 24 hr against 20 volumes of the same buffer at 4°C. The buffer was exchanged four times. The dialyzed 5OOOgsupernatant fluid was used as the enzyme source for angiotensin-converting enzyme. Assay of angiotensin-converting enzyme. The assay for the angiotensinconverting enzyme activity was carried out in a 0.5-ml incubation mixture containing 40 pmol of potassium phosphate buffer, pH 8.3, 300 pmol of sodium chloride, 1.5 pmol of HHL, and the enzyme. The reaction was initiated by the addition of the substrate, and the reaction mixture was incubated at 37°C for 30 min. Reactions were terminated by immersing the test tube in a boiling-water bath for 10 min. After the addition of 3 ml of 0.2 M phosphate buffer, pH 8.3, 1.5 ml of 3% TT solution (in dioxane) was added to the reaction mixture with vigorous stirring until the turbid solution became transparent, and the tubes were centrifuged at 1OOOg for 10 min. The enzyme activity in the resulting supernatant fluid was determined from the absorbance at 382 nm by the differential spectrophotometric method. The control run was identical to the above procedure minus the incubation. Definition of enzyme unit. One unit of enzyme activity was defined as the amount of enzyme that hydrolyzed 1 pmol of HHL to hippuric acid plus L-histidyl-L-leucine in 1 min at 37°C under the condi-
ASSAY
OF ANGIOTENSIN-CONVERTING
363
ENZYME
tions described above. The specific activity was the number of units per milligram of protein. Protein determination. The protein concentrations were determined according to the procedure described by Lowry et al. (5) in which crystalline bovine serum albumine was used at the standard. RESULTS
Calibration curve of hippuric acid. In preliminary experiments, the absorbance at 382 nm of known samples (up to 50 pg) containing hippuric acid-TT chromogen in either 0.2 M phosphate buffer, pH 8.3, or the incubation mixture of angiotensin-converting enzyme containing HHL was determined. A similar linearity between absorbance and hippuric acid concentration in each medium was observed (Fig. 1). These results suggest that the presence of substrate (HHL) does not interfere with the reaction of hippuric acid and TT. The critical factors are determined as follows. Influence of 2,4,6-trichloro-s-triazine concentration. The effects of various concentrations of TT on the intensity of the chromogen were investigated with a specific concentration of hippuric acid (25 pg) in the present assay system. When the final concentrations of TT were less than 0.8%, TT caused a dose-dependent intensity. But over that concentration, the intensity reached a plateau. In the present experiments, a final TT concentration of 0.9% was used (Fig. 2). 2.0
FIG.
1. Calibration
curve
Concentration
of Hippuric
for hippuric
acid quantity
Acid
versus
( pg/tube
relative
)
absorbance.
364
HAYAKARI,
KONDO
0
AND
IZUMI
1.5 Final
Concentration
3 of T.T.
Reagent
( % )
FIG. 2. Effect of TT reagent concentrations. Each concentration of TT reagent was added to the incubation mixture containing 40 pg of hippuric acid per tube used as a standard.
Activity as a function of time and enzyme concentration. On the basis of the above results, we have determined the amounts of hippuric acid released from HHL by the action of angiotensin-converting enzyme prepared from lung at various times of incubation and with various enzyme concentrations. As can be seen in Fig. 3, the formation of hippuric acid is linear for at least 40 min with enzyme concentrations of up to 1.3 mg of protein/assay. On the high-concentration, 60-min incubation, however, the apparent absorbance deviated from the reliable linearity, presumably because of inactivation of the enzyme. Znjluence of pH. The rate of hydrolysis of HHL by angiotensinconverting enzyme of rat lung was determined at pH values from 6.0 to 9.0 in the presence of 600 mM NaCl (Fig. 4). The maximum rate was found to occur at pH 8.3. Influence of chloride ion. The effect of chloride ion concentration on the angiotensin-converting enzyme activity in rat lung was examined (Fig. 5). The enzymic activity was very low in the absence of chloride ion, and an increase in chloride ion concentration caused a progressive increase in the activity of angiotensin-converting enzyme, reaching a plateau at above a 600 mM chloride ion concentration. Activity as a function of substrate concentration. Assays of angiotensin-converting enzyme in the rat lung were conducted in various HHL concentrations (Fig. 6). The optimal concentration of HHL was 3
ASSAY OF ANGIOTENSIN-CONVERTING
365
ENZYME
600
400
200
0 0
10
20
30 Time
( minutes
60 )
FIG. 3. Effect of incubation time on the activity of angiotensin-converting enzyme of rat lung. The enzyme activities were assayed as described in Experimental. Three different protein concentrations of rat lung enzyme were employed: 139 pg of protein/assay (A); 0.69 mg of protein/assay (0); and 1.39 mg of protein/assay (0).
mM; higher concentrations of substrate reduced enzymic activity. The apparent K, value for HHL was calculated to be approximately 1.11 mM. In the present experiments, a 3 mM concentration of HHL was used. Znjluence of chelating reagents. Two chelating compounds were tested for their inhibitory activity on the angiotensin-converting enzyme. As shown in Table 1, both EDTA and 8-hydroxyquinoline were potent inhibitors. In particular, EDTA produced a marked inhibition even at concentrations as low as 0.1 mh4. DISCUSSION
Angiotensin-converting enzyme splits a histidyl-leucine dipeptide from the decapeptide structure of angiotensin I, converting it to the active octapeptide angiotensin II; its properties and distribution in the body have been studied by others (6-8). Furthermore, recent reports have revealed that patients with clinical sarcoidosis and Gaucher’s disease exhibit an elevated level of angiotensin-converting enzyme activity in the serum (9-12). Previously, the system was assayed by biological measurement of the formation of angiotensin II from an angiotensin I substrate (13). Huggins and Thampi (14) described a radiometric assay using labeled angiotensin
HAYAKARI,
366
KONDO AND IZLJMI
I
0
7
6
I 9
8 PH
4. Effect of pH on the activity of angiotensin-converting enzyme of rat lung. Assay conditions were as described in Experimental. The buffer used was 0.2 M phosphate buffer. The enzyme (1.39 mg of protein/assay) was incubated at 37°C for 30 min in the presence of 0.3 M NaCl. FIG.
0 0
200
400 Chloride
600 Ion
Concentration
800
1,000 ( mM )
FIG. 5. Effect of chloride ion concentration on the activity of angiotensin-converting enzyme of rat lung. Enzyme (1.39 mg of protein/assay) was incubated at 37°C for 30 min under conditions described in Experimental without 0.3 M NaCl.
ASSAY
OF
ANGIOTENSIN-CONVERTING
367
ENZYME
20
0 10
5
0 Substrate
( mM ]
FIG. 6. Effect of varying concentrations of substrate on the activity of angiotensinconverting enzyme of rat lung. Assay conditions were as described in Experimental. The reaction was started by the addition of varying concentrations of HHL, and the incubation was carried out at 37°C for 30 min. V, reaction velocity in nanomoles per minute; S, substrate concentration (millimolar).
I as the substrate, in which the release of radioactive histidyl-leucine served as an index of enzymic activity. On the other hand, histidylleucine was also determined by fluorometrically (2,15). Recently, Cushman and Cheung (3) developed an assay system employing ‘hippurylTABLE INHIBITION
1
OF ANGIOTENSIN-CONVERTING ENZYME BY THE CHELATING REAGENTS”
Compound EDTA
8-Hydroxyquinoline
Concentration (M) 1 x 10-S 1 x 10-4 1 x 10-S 1 x IO-3 1 x 10-4 1 x 10-S
OF RAT LUNG
Percentage of control activity 4 8
18 44 98
loo
a The enzyme (1.39 mg of protein/assay) was incubated with each compound at the indicated final concentration. Incubations were carried out at 37°C for 30 min under condition described in Experimental.
368
HAYAKARI,
KONDO
AND
IZUMI
L-histidyl-L-leucine (HHL) as substrate which measures the formation of free hippuric acid by the action of angiotensin-converting enzyme. In their report, however, hippuric acid was determined after its separation from unreacted HHL by extraction into ethylacetate. Accordingly, it is necessary to calculate the conversion factor. It has been reported by Suzuki et al. (4) that N-monosubstituted derivatives of glycine produce a colored material (chromogen) on reaction with 2,4,6-trichloro-s-triazine (TT) (4). In the present experiments, we have investigated whether or not TT is available for the determination of hippuric acid in the incubation medium of the angiotensin-converting enzyme assay without the extraction of hippuric acid. The hippuric acid-TT chromogen was found to be quite stable in the incubation medium. There were no observable changes in the absorbances of the solution in the course of the experiment and even after standing for 3-6 hr at room temperature. These results suggested a practically complete recovery of hippuric acid without separation from HHL under the optimal conditions. Several critical factors in this enzymatic reaction are similar to results obtained by Cushman and Cheung (3). The enzymic reaction was interfered with by reagents such as EDTA and 8-oxyquinoline. HHL, a tripeptide substrate first synthesized by Cushman and Cheung, was preferred as substrate for the assay of angiotensin-converting enzyme because HHL can be employed over a wide range of substrate and enzyme concentrations (3). It has also been reported that chymotrypsin and aminopeptidase show no activity on HHL (3). On the other hand, many other tripeptide substrates besides HHL for angiotensin-converting enzyme have previously been reported, such as Bz-Gly-Phe-Phe, Bz-GlyGly-Pro, Bz-Gly-Gly-Gly, Bz-Phe(NOz)-His-Leu, Bz-Phe(NO,)-Gly-Gly, and Z-Gly-Lys-Gly (2,7,16).’ In the experiment, we measured hippuric acid liberated from HHL. Accordingly, tripeptide substrates such as Bz-Gly-Phe-Phe and Bz-Gly-Gly-Pro could be also available as substrates of the angiotensin-converting enzyme in the present method. However, since N-substituted derivatives of serine, threonine, and tryptophan and glycyl-glycine and N-acetyl-glycine also gave positive color reactions with TT (4), it is necessary to use caution in selecting the substrate. REFERENCES 1. 2. 3. 4. 5.
Skeggs, L. T., Kahn, J. R., and Shumway, N. P. (1956) J. Exp. Med. 103, 295-299. Piquilloud, Y., Reinharz, A., and Roth, M. (1970)Biochim. Biophys. Acta 206; 136-142. Cushman, D. W., and Cheung, H. S. (1971) Biochem. Pharmacol. 20, 1637-1648. Suzuki, S., Hachimori, Y., and Yaoeda, U. (1970)Anal. Chem. 42, 101-103. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951)3. Biol. Chem. 193, 265-275.
1 Abbreviations
used:
Bz, benzoyl;
Z-, benzoylcarbonyl-.
ASSAY OF ANGIOTENSIN-CONVERTING
ENZYME
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