A rapid and sensitive fluorescence assay for angiotensin-converting enzyme

ANALYTICAL

BIOCHEMISTRY

87, 556-561 (1978)

A Rapid and Sensitive Fluorescence Assay for Angiotensin-Converting Enzyme J. M. CONROY*

AND

C. Y. LAI?’

*Department of Biochemistry. Cornell University Medical College, New York, New 10021 and tDepartment of Physiological Chemistry and Pharmacology, Roche Institute of Molecular Biology, Nutley, New Jersey 07110

York

Received October 3, 1977; accepted February 8, 1978 A rapid and sensitive assay for angiotensin-converting enzyme with a variety of substrates has been developed. With acetylated angiotensin I or hippurylhistidylleucine as substrate, the liberated histidylleucine was quantitated by reaction with fluorescamine. The hydrolytic activity of the converting enzyme, ranging from 0.2 to 10 mu, may be assayed within 20 min by this procedure. The assay was found applicable in the measurement of enzyme activity in a crude extract of rabbit lung as well as in the determination of anticatalytic activity of antisera specific for the enzyme.

Angiotensin-converting enzyme (EC 3.4.15.1) is a peptidyldipeptide hydrolase which participates in the renin-angiotensin system of bloodpressure regulation. The enzyme hydrolyzes angiotensin I to give the potent vasopressor angiotensin II and the COOH-terminal dipeptide histidylleucine. It also degrades the vasodepressor peptide bradykinin (for review, see Ref. 1). For assays of the converting enzyme, both the physiological substrate, angiotensin I, and model substrates, usually tripeptides with blocked amino terminals, have been used. With radiolabeled angiotensin I, hydrolysis of the substrate has been monitored by separation and quantitation of the radioactive dipeptide liberated (2,3). Generation of histidylleucine from angiotensin I or from model substrates has also been measured fluorimetrically after reaction with o-phthalaldehyde (4-6). This fluorimetric assay is sensitive and specific for substrates containing histidylleucine at the COOH-terminus. With hippurylhistidylleucine as the substrate, enzyme activity has also been determined by extraction of hippuric acid from the reaction mixture followed by spectrophotometric estimation of the hippuric acid generated (7). Fluorescamine, a fluorimetric reagent for primary amines (8), has been successfully used for the general detection of peptides (9). A ready access ’ Author to whom all correspondence 0003-2697/78/0872-0556$02.00/O Copyright All rights

0 1978 by Academic Press, Inc. of reproduction in any form reserved.

should be addressed. 556

ANGIOTENSIN-CONVERTING

ENZYME

ASSAY

557

to the fluorescamine procedure in the laboratory has prompted us to test its use in the assay of the angiotensin-converting enzyme. The procedure described here is useful with a variety of substrates and can be performed in a single tube within 20 min. MATERIALS

AND METHODS

Materials. The crude extract of rabbit lung, purified angiotensinconverting enzyme, and goat antisera specific for this enzyme were prepared as previously described (10,ll). Angiotensin I (I-Asp, 5-Ile), glycylglycine and serylglycine were from Schwarz-Mann, histidylleucine was from Cycle Chemicals, and hippurylhistidylleucine was from Research Plus. Fluorescamine (Fluram) ando-phthalaldehyde (Fluoropa) were products of Hoffmann-La Roche and Durrum, respectively. N-acetyl angiotensin I was prepared by acetylation (12) of angiotensin I as described below. Acetylation ofangiotensin I. Angiotensin I(5 mg) was dissolved in 200 ~1 of 50% saturated sodium acetate, and 5 ~1 of acetic anhydride was added in l-p1 aliquots over a period of 1 hr at 0°C. A slight precipitate which formed in the reaction mixture was solubilized by the addition of 50 ~1 of 10 M urea. Acetylated angiotensin I was then separated from the reaction mixture by Sephadex G-10 chromatography (1 x 39-cm column). Fractions from gel filtration were monitored with absorbance at 230 nm. Fractions containing N-acetyl angiotensin I were pooled, and the concentration of angiotensin I in the pooled fractions was determined by amino acid analysis. The optical absorbance of this material in aqueous solution (1 mg/ml) was 0.325 at 280 nm (E, = 4.35 x 105) and 7.05 at 230 nm (E, = 9.43 x 106). The reactivity of fluorescamine with angiotensin I was essentially eliminated by acetylation. Assays. Assays (50 ~1 reaction mixture) were performed in siliconized glass tubes (13 x 100 mm). Reaction mixtures contained 0.4 mM N-acetyl angiotensin I, and 30 mM NaCl in 100 mM potassium phosphate buffer, pH 7.5. When the model substrate was used, the reaction mixture contained 5 mM hippurylhistidylleucine and 300 mM NaCl in 100 mM potassium phosphate buffer, pH 8.3. Reactions were started by the addition of enzyme. At indicated times after incubation at 37°C the reaction was stopped by the addition of 50 ~1 of 1 N HCl. To determine the amount of histidylleucine released, the reaction mixture was neutralized with 50 ~1 of 1 N NaOH, brought to a final volume of 1.5 ml with 0.5 M sodium borate buffer, pH 8.5, and 0.3 ml of fluorescamine (20 mg/lOO ml solution in acetone) added with mixing (9). The relative fluorescence was determined against a blank which contained all reagents but the enzyme. Fluorescence determinations were made with a Aminco-Bowman spectrofluorimeter (American Instruments Co.) with excitation and emission wavelengths of

558

CONROY AND LA1

390 and 475 nm, respectively. The amount of either substrate hydrolyzed was calculated from a reference curve of histidylleucine, which gave linear fluorescence over the range from 1 to 20 nmol. Fluorimetry of dipeptides using o-phthalaldehyde (OPA) andjuorescamine. To each tube containing 2, 5, and 10 nmol of the dipeptides

His-Leu, Gly-Gly and Ser-Gly in 0.25 ml 0.1 M potassium phosphate buffer, pH 7.5, 0.03 M NaCl, 1.45 ml of 0.3 M NaOH, and 0.1 ml of 0.2% OPA in methanol were added. After 4 min, 0.2 ml of 3 M HCl was added, and fluorescence was measured at 500 nm with excitation at 365 nm, as described by Cheung and Cushman (4). For the OPA reagents including mercaptoethanol (13), 1.5 ml of 0.5 M sodium borate buffer, pH 10, containing 0.05% 2-mercaptoethanol and 0.3 ml of 0.02% OPA in water were added to the peptide samples, and the fluorescence was measured as above. The same samples were treated with fluorescamine as described in the previous section. Anticatalytic activity of anti-converting enzyme antibody. Antibody and enzyme were incubated for 30 min in 100 mrvr potassium phosphate and 0.15 M NaCl. Residual enzyme activity was then determined by the assay procedure described above and the spectrophotometric method of Cushman and Cheung (7). RESULTS

Under the conditions used, the yield of fluorescence in the reaction of o-phthalaldehyde-mercaptoethanol with dipeptides was found to be about one-fifth of that obtained with fluorescamine, and the background fluorescence was nearly 10 times greater (Table 1). In the absence of mercaptoethanol (4), o-phthalaldehyde yielded higher fluorescence with histidylleucine than did fluorescamine, but no fluorescence was obtained with the other peptides tested. TABLE

1

FLUORESCENCEOF DIPEPTIDES AFTER REACTION WITH FLUOROGENIC REAGENTS Relative fluorescence” Method Fluorescamine o-Phtbalaldehyde o-phthalaldehydemercaptoethanol

Reagent blank

His-Leu

Gly-Gly

Ser-Gly

5 1

470 705

365 2

210 2

32

43

64

46

U Experiments were performed as described in the Materials and Methods Section. Relative fluorescence was measured against distilled water. The values are those obtained with 10 nmol of each dipeptide.

ANGIOTENSIN-CONVERTING

ENZYME

559

ASSAY

6

B 4

0

16 14

. IO 26 2 IO

20 30 TIME (mid

40

50

iii3 0

50 ENZYME ADDED

100

FIG. I. Hydrolysis of N-acetyl angiotensin I (0.4 mM) and hippurylhistidylleucine (5 mM) by angiotensin-converting enzyme (90 U/m@. The generation of histidylleucine was measured as a function of (A) incubation time and (B) amount of enzyme added (for a 15-min incubation time). Amounts of enzyme added were 50 ng with N-acetyl angiotensin I and 10 ng with Hip-His-Leu.

Hydrolysis of N-acetyl angiotensin I or hippurylhistidylleucine by angiotensin-converting enzyme proceeded linearly with time up to 30 min (Fig. IA). Using the 15-min incubation time, the extent of hydrolysis of these substrates was proportional to the amount of enzyme added, up to 25 ng (2.25 muJ2 with hippurylhistidylleucine and 100 ng with N-acetyl angiotensin I (Fig. 1B). The reciprocal plot of Lineweaver-Burk (14) for hydrolysis of hippurylhistidylleucine gave a K, value of 2.4 mM and a turnover number of 1.55 x lo4 mol/min/mol of enzyme (Fig. 2). Similarly, the K, value and turnover number for N-acetyl angiotensin I were determined as 0.11 mM and 650 mol/min/mol of enzyme, respectively (data not shown). Based on these observations, the assay procedures as described in the Materials and Methods section have been formulated for the enzyme in the range from 2 to 100 ng (0.18-9 mu). The ability to measure low levels of the enzyme activity would suggest that the method was useful for assaying crude tissue extracts. It was indeed possible to assay the hippurylhistidylleucinecleavage activity of the converting enzyme in an extract of rabbit lung having a specific activity of 0.33 u/mg. One example of the usefulness of the fluorimetric assay is illustrated by determination of anticatalytic activity of a number of antisera specific for angiotensin-converting enzyme. Estimates of anticatalytic activity obtained from the fluorescent assay were in close agreement with determinations made spectrophotometrically (Fig. 3). In antisera with low anticatalytic activity, problems were encountered with high background fluorescence. Amino acids and other primary amines contained in the large volume of serum required for the reaction contributed to the high back’ One unit of the angiotensin-converting enzyme is defined as the amount which hydrolyzes 1 Fmol of hippurylhistidylleucine/min at 37°C (1).

560

CONROY AND LA1

FIG. 2. Lineweaver-Burk plot of hydrolysis of hippurylhistidylleucine by angiotensinconverting enzyme. The initial velocity at each substrate concentration was determined after 2, 5, 10, and 15 min of incubation with 20 ng of angiotensin-converting enzyme (90 U/mg).

ground. Immunoglobulin prepared by ammonium sulfate fractionation these antisera reduced background fluorescence to negligible levels.

of

DISCUSSION

The fluorescamine assay allows rapid and sensitive quantitation of angiotensin-converting enzyme activity with hippurylhistidylleucine and angiotensin I. Michaelis constants and turnover numbers reported here for enzymatic hydrolysis of these substrates are in agreement with those previously determined (7,lO). Acetylation of the amino-terminal aspartic acid residue of angiotensin I therefore appears to have little effect on the enzyme’s utilization of this substrate. The use of fluorescamine represents a significant improvement in fluorescence assay of the enzyme because of its greater fluorescence yield with numerous dipeptides as compared to o-phthalaldehyde. In the method used by Cheung and Cushman, o-phthalaldehyde without mercaptoethanol (43) gives a higher fluorescence yield than does

0 SERUM

500 PROTEIN

IO00 tug)

f’lG. 3. Anticatalytic activity of antiserum on the hydrolysis of hippurylhistidylleucine by angiotensin-converting enzyme. Residual enzyme activity after incubation with antibody was determined by (0) fluorescent assay or (0) the spectrophotometric assay of Cushman and Cheung.

ANGIOTENSIN-CONVERTING

ENZYME

ASSAY

561

fluorescamine with histidylleucine (but not with other dipeptides). This greater reactivity allows the assay to be used with numerous physiological and model substrates which do not contain histidine as the penultimate carboxyl terminal residue, e.g., bradykinin, “fowl” angiotensin I (15), and hippurylglycylglycine. The enhanced reactivity allows shorter incubation times for the assay, thus minimizing inhibition by the products of the reaction, angiotensin II and the released dipeptide (10). In addition, the reaction of fluorescamine with primary amines is essentially instantaneous, allowing immediate measurement of fluorescence (8). All operations for the assay can be performed in 20 min in a single tube. With this procedure, direct assay of the enzyme even in crude tissue extracts was possible. Since only small aliquots of the extracts are required for the assay, the extraneous amines in the sample did not interfere with the fluorescence measurement. The assay is also useful for determining the anticatalytic activity of antisera specific for angiotensinconverting enzyme. We have recently obtained accurate measurement of the convertingenzyme activity without stopping the enzymatic reaction at the end of the incubation period. Since the excess fluorescamine added was rapidly hydrolyzed in the aqueous medium, further formation of histidylleucine by the enzyme would no longer by detected. ACKNOWLEDGMENT James M. Conroy is a postdoctoral fellow of the New York Heart Association under the auspices of Dr. Richard L. Soffer. The authors wish to thank Dr. Soffer for his encouragement and advice in this study.

REFERENCES I. Soffer, R. L. (1976) Ann. Rev. Biochem. 45, 73-94. 2. Huggins, C. G.. and Thampi, N. S. (1968) Life Sci. 7, 633-639. 3. Lee, H. J., Larue, J. N., and Wilson, I. B. (1971) Biochim. Biophys. Acra 235, 521-528. 4. Cheung, H. S., and Cushman, D. W. (1973) Biochim. Biophys. Acta 293, 451-463. 5. Piquilloud, Y., Reinharz, A., and Roth. M. (1970)Biochim. Biophys. Acta 206, 136-142. 6. Depierre, D., and Roth, M. (1975) Enzyme 19, 65-70. 7. Cushman, D. W., and Cheung, H. S. (1971) Biochem. Pharmacol. 20, 1637-1648. 8. Udenfriend, S., Stein, S., Bohlen. P.. Dairman, W., Leimgruber, W., and Weigele, M. (1972) Science 178, 871-872. 9. Nakai, N., Lai, C. Y., and Horecker, B. L. (1974) Anal. Biochem. 58, 563-570. 10. Das, M., and Soffer, R. L. (1975) J. Biol. Chem. 250, 6762-6768. 11. Conroy, J. M., Hoffman, H., Hirzel, H. O., Kirk, E. S., Sonnenblick, E. H., and Soffer, R. L. (1976) J. Bid. Chem. 251, 4828-4832. 12. Frankel-Conrat, H. (1957) in Methods in Enzymology (Colewick, S. P., and Kaplan, N. O., eds.), Vol. 4, p. 251. Academic Press, New York. 13. Mendez, E.. and Gavilanes, J. G. (1976) Anal. Biochem. 72, 473-479. 14. Lineweaver, J., and Burk, D. (1934) J. Amer. Chem. Sot. 56, 658-666. 15. Nakajima, T., Nakayama. T., and Sokabe, H. (1973)Chem. Pharm. Bull. 21,X%5-2087.