- Email: [email protected]
A rapid assay for dipeptidyl aminopeptidase III in human erythrocytes
ANALYTICAL
BIOCHEMISTRY
119, 418-423
(1982)
A Rapid Assay for Dipeptidyl THEODORE Department
of Chemistry,
Aminopeptidase
III in Human Erythrocytes’
H. D. JONES AND ALEXIA University
of San Francisco,
Received August
KAPRALOU
San Francisco,
California
941 I7
11, 198 1
A simple, rapid procedure for the assay of dipeptidyl aminopeptidase III in human erythrocytes has been developed. The cells are lysed with Triton X-100, centrifuged, and the supernatant fractionated on small columns of carboxymethyl Bio-Gel A at pH 6.5. The enzyme is eluted in the void volume and can be obtained completely separated from hemoglobin; it is then assayed by hydrolysis of Arg-Arg-fl-naphthylamide. The procedure is suitable for the routine screening of large numbers of human blood samples for their DAP III activity.
Mammalian tissues contain a variety of proteinases and peptidases distinguishable by their catalytic mechanisms and molecular characteristics ( 1). The peptidases cleave peptide bonds at the ends of peptides and polypeptides and can be subdivided according to whether they act at the N- or C-terminus and whether they remove single amino acids or dipeptides from the substrate; many such enzymes are also specific for a particular amino acid or amino acid sequence. In recent years the detection and study of different peptidases has been made possible by the availability of artificial substrates containing particular amino acid sequences. The use of a wide range of dipeptidyl-fi-naphthylamide substrates led to the recognition of a new class of arylamidases: (a) dipeptidyl arylamidase I, catalyzing hydrolytic removal of Ser-Tyr from SerTyr-/3-NA2 (2); (b) dipeptidyl arylamidase II, removing Lys-Ala
from
Lys-Ala-P-NA
(3);
(c) dipeptidyl arylamidase III, hydrolyzing the arylamide bond of Arg-Arg-P-Na (4) and ’ This work was supported by a grant from the Lily Drake Research Fund of the University of San Francisco. 2 Abbreviations used: /3-NA, P-naphthylamide; RBC, red blood cell; Hb, hemoglobin. 0003-2697/82/020418-06$02.00/O Copyright 0 1982 by Academic Press. Inc. All rsghfs of reproduction in any form reserved.
418
(d) dipeptidyl arylamidase IV, removing Gly-Pro from Gly-Pro-P-NA (5). These enzymes also remove dipeptides sequentially from the unsubstituted N-termini of polypeptides and so were renamed dipeptidy1 aminopeptidases (DAP I, DAP II, DAP III, and DAP IV) and classified as dipeptidylpeptide hydrolases (EC 3.4.14). DAP III was first isolated from bovine pituitary gland and has been partially characterized (4). Arg-Arg-/3-NA is the only dipeptidyl+naphthylamide tested which it hydrolyzes. Among the naturally occurring polypeptides tested, only the octapeptide hormone, angiotensin II, is a substrate. The enzyme removes the first two N-terminal dipeptides, thus inactivating the hormone (6). Angiotensin II causes elevation in blood pressure and is removed from the circulation by enzymatic hydrolysis (7,8). Rabbit red blood cells (RBCs) contain significant amounts of an angiotensinase which cleaves the Arg2-Va13 and Tyr4-Ile’ bonds, as does bovine pituitary DAP III (6,9). Comparative studies on rabbit erythrocyte angiotensinase and bovine pituitary DAP III have shown that they behave similarly with respect to substrate specificity, pH optima, sensitivity to sulfhydryl reagents and to EDTA, have similar K,,, values for Arg-Arg-@-NA and
DIPEPTIDYL
AMINOPEPTIDASE
show substrate inhibition (6). It has been concluded that the two activities represent homologous enzymes. The assay of this enzyme in RBCs has required the use of angiotensin as substrate with measurement of the loss of its hormonal activity, and is thus dependent upon bioassays for the hormone which are both tedious and relatively inaccurate (7,9). We report here that direct measurement of human RBC enzyme by hydrolysis of Arg-Arg$NA is prevented by the interference of hemoglobin (Hb) and have developed a procedure by which the enzyme can be quickly separated from Hb and assayed. MATERIALS
AND METHODS
Citrate phosphate dextrose whole blood, containing 450 ml human blood and 63 ml citrate phosphate dextrose solution, was obtained from the Irwin Memorial Blood Bank of San Francisco. Arg-Arg-fl-NA, lyophilized human Hb, Drabkin’s reagent, and Sephadex G-25-80 were provided by Sigma Chemical Company (St. Louis, MO.). CM-Bio-Gel A was from Bio-Rad Laboratories (Oakland, Calif.) and Ultrogel AcA 44 from LKB Instruments (Rockville, Md.). Preparation of RBC lysates. Cells were collected from 1 ml of whole blood by centrifugation at 2000g for 10 min at 2°C washed with cold, isotonic NaCl (9 g/liter) and recentrifuged. The supernatant, containing plasma, leukocytes, and thrombocytes, was collected following centrifugation and used in subsequent experiments. Assay of DAP III. DAP III activity was measured by its hydrolysis of the nonfluorescent Arg-Arg-P-NA to form the fluorescent-P-NA according to the procedure used in the study of DAP III in bovine pituitary gland (4). Lysate (0.1 ml) was incubated with 0.1 ml 0.1 M 2-mercaptoethanol and 2.8 ml 62.5 mM Tris-HCl buffer, pH 9.0, for 15 min at 23°C. Two milliliters of 90 gM Arg-Arg-/?-NA was added and the
419
IN RBCs
rate of increase of intensity of fluorescence at 410 nm was measured in a Turner lluorometer, Model III. A standard curve, with O-25 nmol @-NA in 5.0 ml Tris-HCl buffer was used to convert fluorometer units to amount of /3-NA produced. One unit of enzyme activity was defined as the amount of enzyme which catalyzes the production of 1 nmol /3-NA/min under the conditions given. Effect
of other compounds
on DAP
III.
The effects of human Hb and Triton X- 100 were tested by adding appropriate volumes of each compound dissolved in Tris buffer to the lysate-mercaptoethanol-Tris solution to give a volume of 3.0 ml and incubating for 15 min. Substrate was then added and the reaction rates determined as before. Ion-exchange chromatography of cell lysate. CM-Bio-Gel A, purchased as a swollen
gel, was poured to form a small column ( 1.3cm diameter, 7.5-cm height) and equilibrated by washing with 80 ml of 10 mM potassium phosphate buffer, pH 6.5. A mixture containing 0.28 ml RBC lysate and 0.10 20% sucrose was prepared and 0.2 ml was applied to the column. The column was eluted with the phosphate buffer at a flow rate of 0.6 ml/min and 15 0.7-ml fractions were collected; analysis showed that all of the DAP III was eluted in the first 8 fractions and none of the Hb was eluted. The column was then washed with 62.5 mM TrisHCl, pH 9.0, to remove the Hb and reequilibrated with phosphate buffer, pH 6.5. The same column could be used repeatedly to separate the DAP III from the Hb in the RBC lysate. Gel-Jiltration chromatography. In other experiments designed to separate DAP III and Hb, 2.0 ml of a sample containing 1.4 ml RBC lysate, 0.4 ml 20% sucrose, and 0.2 ml Dextran Blue was applied to an Ultrogel AcA 44 column (2.5-cm diameter, 58-cm height, equilibrated with 62.5 mrvr Tris-HCl, pH 9.0, at 4°C) and eluted at how rates between 1.4 and 7.0 ml/h). Fractions were collected and analyzed for DAP III and Hb.
420
JONES
AND
The column was calibrated with proteins of known molecular weight as standards under the same conditions as used for elution of the DAP III. In experiments where Hb was to be separated from ,&NA, columns of Sephadex G25-80 (1.3~cm diameter, 7.5-cm height) were equilibrated with 62.5 mM Tris-HCl buffer, pH 9.0. Reaction mixtures to be analyzed were mixed with 0.2 vol 20% sucrose, applied to the column, and eluted with the Tris buffer at a flow rate of 0.6 ml/min. Fractions of 5 ml were collected; P-NA was eluted only in fractions 5-7, partially separated from the Hb which eluted at the void volume, and measured with the fluorometer. Measurement of Hb and protein. Hb concentration was measured by Drabkin’s procedure ( 11) and protein by the Bradford assay (12). RESULTS
When cell lysate and Arg-Arg-/3-NA were incubated together, immediate and continuous hydrolysis of the substrate occurred with the release of /3-NA, demonstrating the presence in red blood cells of substantial amounts of DAP activity. The rate of hydrolysis, however, was not proportional to the amount of cell lysate (Fig. 1). This result might be due to the presence in the cell lysate of an inhibitor of the enzyme or of a compound which quenched the fluorescence of the P-NA product. The latter was considered likely since the fluorescence maximum for @-NA occurs at 410 nm while the absorption spectrum of human Hb shows a peak around 410 to 420 nm. Addition of Hb to reaction mixtures containing unfractionated cell lysate decreased the observed rate of substrate hydrolysis. To develop an assay which is unaffected by Hb and which would be rapid and easy to perform routinely, two approaches were explored: separation of DAP III from Hb before enzyme assay and separation of /3-NA product from Hb after enzyme reaction.
KAPRALOU VOLUME
OF ELUATE (ml)
0.6 -
0.04 VOLUME
FIG. 1. Amount
of enzyme
0.08
0.12
0.16
0.20
OF RSC LYSATE (ml)
activity
observed as a funcunfractionated from CM-Bio-Gel
tion of sample volume. Samples were RBC lysate, (O), A column, (0).
Separation
or Hb-free
eluate
of DAP II1 jkom hemoglobin.
Three techniques were tested for their ability to separate DAP III and Hb in RBC lysates. Cell lysate was fractionated by gel-filtration chromatography on Ultrogel AcA 44. DAP III activity eluted as a single peak slightly before Hb but always overlapping the Hb peak, which prevented accurate measurement of the total amount of DAP III activity recovered from the column, Pituitary DAP III can be partially purified by precipitation with ammonium sulfate at a final concentration of 40 to 60% (4). When RBC lysate was fractionated by precipitation with ammonium sulfate, most of the DAP III precipitated in the 45-63s range of saturation, but Hb was present in this precipitate in significant amounts, preventing accurate assay of total DAP III activity. When RBC lysate was applied to CMBio-Gel A columns at pH 6.5, DAP III activity was not bound to the resin and eluted within the first 6 ml of eluate. Hb was fully
DIPEPTIDYL
AMINOPEPTIDASE
retained on the column and could be removed by elution at pH 9.0. The column, following equilibration at pH 6.5, could then be reused for the fractionation of further samples of cell lysate. Hb-free enzyme obtained from this ionexchange procedure gave a linear response in the enzyme assay procedure up to 1.5 enzyme units (Fig. 1). Hb added to reaction mixtures containing such Hb-free enzyme decreased the amount of activity observed: 2.0 mg Hb in the 5.0-ml reaction mixture resulted in 91% decrease (Fig. 2). The concentration of Hb in the unfractionated cell lysate was determined to be 38 mg/ml; thus assay of the DAP III activity in 0.1 ml of unfractionated cell lysate is under conditions producing more than 9 1% apparent decrease in enzyme activity. Separation of /3-NA and Hb. To determine whether Hb inhibits DAP III or interferes with the measurement of /3-NA through quenching, reaction mixtures containing Hbfree DAP III were mixed with 0 and 2.0 mg human Hb. Substrate was added and reaction was allowed to proceed for 30 min. The total production of /3-NA in the two samples was measured. In the reaction containing Hb, 8-9% of the &NA present in the control (reaction without Hb) could be observed (Table 1). The P-NA produced was thenseparated from Hb by two procedures. Forty
421
IN RBCs TABLE PRODUCTION
OF &NA IN REACTION CONTAINING Hb
@NA produced/30 min/ml original RBC lysatc (nmol) Expt. I 2 3 *Reaction components;
1
-Hb”
+Hb
594 614 560
46 56 51
MIXTURES
&NA recovered from Heating pP3WdU~C (nmol)
Gel filtration (nmol) -Hb 238 224 190
+Hb
-Hb
+Hb
107 119 120
356 390 369
151 182 239
mixtures contained 0.0 or 2.0 m g Hb in addition Hb-free DAP III was used as enzyme.
to the usual
percent of each reaction mixture was fractionated by gel-filtration chromatography on Sephadex G-25 and the P-NA measured. The remaining 60% was heated in a boilingwater bath for 10 min, the precipitate of protein and Hb was removed by centrifugation, and the P-NA in each supernatant was measured. The results showed that partial resolution of reaction mixtures into /3NA and Hb resulted in the detection of much higher amounts of fl-NA than were observable in the original reaction mixture (Table 1). Further experiments in which reaction mixtures were completely resolved into separate Hb and /3-NA peaks on large Sephadex columns showed that recovery of /3-NA was then equal to that obtained in the control reaction mixture without Hb. Thus the Hb does not inhibit DAP III but does quench the fluorescence of the ,&NA. Effect of Triton X-100 on DAP Ifl. The effect of Triton-X-100 on DAP III activity was studied to determine whether this detergent could be used in the preparation of cell lysates. Triton X-100, in concentrations up to 1.0% v/v, had no significant effect upon the enzyme. Recommended procedure for DAP III assay. The final assay developed for mea-
HEMOGLOBIN
(mg)
FIG. 2. Effect of Hb, added to reaction observed activity of Hb-free DAP III.
mixtures,
upon
suring DAP III activity in red blood cells was as follows: 1. 1.O ml whole blood was centrifuged at 2000g for 10 min at 2°C; the cells were
422
JONES
AND
gently washed with cold isotonic NaCl solution and recentrifuged. The cells were then lysed with 5.0 ml cold 1% Triton X- 100, centrifuged, and the supernatant retained. 2. This cell lysate was mixed with 0.25 vol 20% sucrose and 0.1 ml was applied to a CM-Bio-Gel A column ( 1.3 X 7.5 cm) and eluted with 10 mM potassium phosphate buffer, pH 6.5. A single 7.4-ml fraction was collected; 1.0 ml of this was added to the standard reaction mixture and the rate of production of &NA was determined over a period of 15 min. Under these conditions a linear assay was obtained with 0.0 to 1.5 units of DAP III per reaction mixture. Reproducibility of the assay. To determine the reproducibility of this assay, three types of experiments were performed. (a) A single RBC lysate was prepared, using 1% Triton X- 100 to lyse the cells, and four 0.1 -ml samples were applied to ion-exchange columns. Each sample was eluted with phosphate buffer, collected as a single 7.4-ml fraction, and 1.O ml was assayed. The amounts of enzyme found were 42.2, 39.2, 41.6, and 39.5 units/ml lysate. (b) Four separate cell lysates were prepared from a single sample of whole blood and were fractionated and assayed as in (a). The amounts of enzyme were found to be 42.6, 42.6, 49.9, and 42.7 units/ml lysate. (c) A single cell lysate was prepared and 6 different volumes covering the range O.OO0.10 ml were applied to columns and the eluates assayed as before. Enzyme activity per milliliter of eluate was directly proportional to the amount of enzyme applied to the cation-exchange column in four separate experiments. Samples of eluates were rechromatographed on the ion-exchange columns and enzyme recovery was consistently in the range 95-100%. DlScusslm The biosynthesis of angiotensin II has been the subject of continued study, but little attention has been focused upon its degra-
KAPRALOU
dation. The discovery of angiotensinase activity in circulating RBCs (9) suggests the need for detailed examination of the characteristics of this enzyme and its possible role in the catabolism of circulating angiotensin II. Such examination has been hindered by the lack of a simple accurate assay for the enzyme; its activity has been assayed by the rate of inactivation of angiotensin, the amount of angiotensin being determined by measuring the ability of samples to raise blood pressure or to cause the contraction of isolated strips of smooth muscle (7,9), an assay procedure which is tedious, relatively inaccurate, and nonspecific, being significantly affected by nonangiotensin compounds and ions. The discovery that RBC angiotensinase and pituitary DAP III are homologous proteins (6) suggested that an assay could be developed for RBC angiotensinase based upon the rate of hydrolysis of Arg-Arg-P-NA. In this work we report such an assay which is simple, rapid, specific, and reproducible. RBCs may contain additional angiotensinases which do not hydrolyze this artificial substrate and these would not be detected by this assay. Hb interfered significantly with measurement of the enzyme reaction product, /3-NA. When the B-NA was partially separated from the Hb by gel filtration or by heat precipitation of the Hb, the amount of product detected increased from 8 to 9% of that present in the control lacking Hb to about 50 to 60% of the control value. An amount of ,f3NA equal to that in the control was not observed because of incomplete separation of Hb from the ,&NA. The Hb was not fully precipitated from solution by the heating procedure nor was it totally separated from the @-NA when small Sephadex columns were used. Larger gel-filtration columns were used to separate the Hb and P-NA completely, and full recovery of &NA equal to that in control reaction mixtures was then obtained. Thus Hb inhibits the measurement of the P-NA product and not the DAP III enzyme. The use of the large gel-filtration
DIPEPTIDYL
AMINOPEPTIDASE
columns resulted in a usable assay procedure but one which was too lengthy for convenient routine use. Separation of enzyme from Hb proved a better approach. DAP III was completely separated from Hb in a single ion-exchange step which could be performed rapidly and reproducibly and the enzyme activity could then be measured by fluorimetry. The availability of the simple procedure described here should be of value in further studies on the nature and function of this enzyme in circulating red blood cells.
4. 5. 6.
7. 8.
9.
REFERENCES 1. Barrett, A. J. (1977) in Proteinases in Mammalian Cells and Tissues (Barrett, A. J., ed.), pp. l-56, North-Holland, New York. 2. McDonald, J. K., Ellis, S., and Reilly, T. J. (1966) J. Biol. Chem. 241, 1494-1501. 3. McDonald, J. K., Leibach, F. H., Grindeland, R.,
10.
11. 12.
IN RBCs
423
and Ellis, S. (1968) J. Biol. Chem. 243, 41434150. Ellis, S., and Nuenke, J. M. (1967) J. Biol. Chem. 242,4623-4629. Hopsu-Havu, V. K., and Glenner, G. G. (1966) Histochemie 7, 197-201. McDonald, J. K., and Schwabe, C. (1977) in Proteinases in Mammalian Cells and Tissues (Barrett, A. J., ed.), pp. 31 I-391, North-Holland, New York. Page, I. H., and Bumpus, F. M. ( 1974) Angiotensin, Springer-Verlag, New York. Ryan, J. W. (1974) in Angiotensin (Page, I. H., and Bumpus, F. M., eds.), pp. 81-110, SpringerVerlag, New York. Kokubu, T., Akutsu, H., Fujimoto, S., Vieda, F., Hiwada, K., and Yamamura, V. (1969) Biochim. Biophys. Acta 191, 668-676. Schroter, W. (1974) in Clinical Biochemistry, Principles and Methods (Curtius, H. C., and Roth, M. eds.), pp. 1178- 1207, de Gruyter, New York. Sigma Technical Bull., No. 525 (1980) Sigma Chem. Co., St. Louis, MO. Bradford, M. (1976) Anal. Biochem. 72, 248-254.
BIOCHEMISTRY
119, 418-423
(1982)
A Rapid Assay for Dipeptidyl THEODORE Department
of Chemistry,
Aminopeptidase
III in Human Erythrocytes’
H. D. JONES AND ALEXIA University
of San Francisco,
Received August
KAPRALOU
San Francisco,
California
941 I7
11, 198 1
A simple, rapid procedure for the assay of dipeptidyl aminopeptidase III in human erythrocytes has been developed. The cells are lysed with Triton X-100, centrifuged, and the supernatant fractionated on small columns of carboxymethyl Bio-Gel A at pH 6.5. The enzyme is eluted in the void volume and can be obtained completely separated from hemoglobin; it is then assayed by hydrolysis of Arg-Arg-fl-naphthylamide. The procedure is suitable for the routine screening of large numbers of human blood samples for their DAP III activity.
Mammalian tissues contain a variety of proteinases and peptidases distinguishable by their catalytic mechanisms and molecular characteristics ( 1). The peptidases cleave peptide bonds at the ends of peptides and polypeptides and can be subdivided according to whether they act at the N- or C-terminus and whether they remove single amino acids or dipeptides from the substrate; many such enzymes are also specific for a particular amino acid or amino acid sequence. In recent years the detection and study of different peptidases has been made possible by the availability of artificial substrates containing particular amino acid sequences. The use of a wide range of dipeptidyl-fi-naphthylamide substrates led to the recognition of a new class of arylamidases: (a) dipeptidyl arylamidase I, catalyzing hydrolytic removal of Ser-Tyr from SerTyr-/3-NA2 (2); (b) dipeptidyl arylamidase II, removing Lys-Ala
from
Lys-Ala-P-NA
(3);
(c) dipeptidyl arylamidase III, hydrolyzing the arylamide bond of Arg-Arg-P-Na (4) and ’ This work was supported by a grant from the Lily Drake Research Fund of the University of San Francisco. 2 Abbreviations used: /3-NA, P-naphthylamide; RBC, red blood cell; Hb, hemoglobin. 0003-2697/82/020418-06$02.00/O Copyright 0 1982 by Academic Press. Inc. All rsghfs of reproduction in any form reserved.
418
(d) dipeptidyl arylamidase IV, removing Gly-Pro from Gly-Pro-P-NA (5). These enzymes also remove dipeptides sequentially from the unsubstituted N-termini of polypeptides and so were renamed dipeptidy1 aminopeptidases (DAP I, DAP II, DAP III, and DAP IV) and classified as dipeptidylpeptide hydrolases (EC 3.4.14). DAP III was first isolated from bovine pituitary gland and has been partially characterized (4). Arg-Arg-/3-NA is the only dipeptidyl+naphthylamide tested which it hydrolyzes. Among the naturally occurring polypeptides tested, only the octapeptide hormone, angiotensin II, is a substrate. The enzyme removes the first two N-terminal dipeptides, thus inactivating the hormone (6). Angiotensin II causes elevation in blood pressure and is removed from the circulation by enzymatic hydrolysis (7,8). Rabbit red blood cells (RBCs) contain significant amounts of an angiotensinase which cleaves the Arg2-Va13 and Tyr4-Ile’ bonds, as does bovine pituitary DAP III (6,9). Comparative studies on rabbit erythrocyte angiotensinase and bovine pituitary DAP III have shown that they behave similarly with respect to substrate specificity, pH optima, sensitivity to sulfhydryl reagents and to EDTA, have similar K,,, values for Arg-Arg-@-NA and
DIPEPTIDYL
AMINOPEPTIDASE
show substrate inhibition (6). It has been concluded that the two activities represent homologous enzymes. The assay of this enzyme in RBCs has required the use of angiotensin as substrate with measurement of the loss of its hormonal activity, and is thus dependent upon bioassays for the hormone which are both tedious and relatively inaccurate (7,9). We report here that direct measurement of human RBC enzyme by hydrolysis of Arg-Arg$NA is prevented by the interference of hemoglobin (Hb) and have developed a procedure by which the enzyme can be quickly separated from Hb and assayed. MATERIALS
AND METHODS
Citrate phosphate dextrose whole blood, containing 450 ml human blood and 63 ml citrate phosphate dextrose solution, was obtained from the Irwin Memorial Blood Bank of San Francisco. Arg-Arg-fl-NA, lyophilized human Hb, Drabkin’s reagent, and Sephadex G-25-80 were provided by Sigma Chemical Company (St. Louis, MO.). CM-Bio-Gel A was from Bio-Rad Laboratories (Oakland, Calif.) and Ultrogel AcA 44 from LKB Instruments (Rockville, Md.). Preparation of RBC lysates. Cells were collected from 1 ml of whole blood by centrifugation at 2000g for 10 min at 2°C washed with cold, isotonic NaCl (9 g/liter) and recentrifuged. The supernatant, containing plasma, leukocytes, and thrombocytes, was collected following centrifugation and used in subsequent experiments. Assay of DAP III. DAP III activity was measured by its hydrolysis of the nonfluorescent Arg-Arg-P-NA to form the fluorescent-P-NA according to the procedure used in the study of DAP III in bovine pituitary gland (4). Lysate (0.1 ml) was incubated with 0.1 ml 0.1 M 2-mercaptoethanol and 2.8 ml 62.5 mM Tris-HCl buffer, pH 9.0, for 15 min at 23°C. Two milliliters of 90 gM Arg-Arg-/?-NA was added and the
419
IN RBCs
rate of increase of intensity of fluorescence at 410 nm was measured in a Turner lluorometer, Model III. A standard curve, with O-25 nmol @-NA in 5.0 ml Tris-HCl buffer was used to convert fluorometer units to amount of /3-NA produced. One unit of enzyme activity was defined as the amount of enzyme which catalyzes the production of 1 nmol /3-NA/min under the conditions given. Effect
of other compounds
on DAP
III.
The effects of human Hb and Triton X- 100 were tested by adding appropriate volumes of each compound dissolved in Tris buffer to the lysate-mercaptoethanol-Tris solution to give a volume of 3.0 ml and incubating for 15 min. Substrate was then added and the reaction rates determined as before. Ion-exchange chromatography of cell lysate. CM-Bio-Gel A, purchased as a swollen
gel, was poured to form a small column ( 1.3cm diameter, 7.5-cm height) and equilibrated by washing with 80 ml of 10 mM potassium phosphate buffer, pH 6.5. A mixture containing 0.28 ml RBC lysate and 0.10 20% sucrose was prepared and 0.2 ml was applied to the column. The column was eluted with the phosphate buffer at a flow rate of 0.6 ml/min and 15 0.7-ml fractions were collected; analysis showed that all of the DAP III was eluted in the first 8 fractions and none of the Hb was eluted. The column was then washed with 62.5 mM TrisHCl, pH 9.0, to remove the Hb and reequilibrated with phosphate buffer, pH 6.5. The same column could be used repeatedly to separate the DAP III from the Hb in the RBC lysate. Gel-Jiltration chromatography. In other experiments designed to separate DAP III and Hb, 2.0 ml of a sample containing 1.4 ml RBC lysate, 0.4 ml 20% sucrose, and 0.2 ml Dextran Blue was applied to an Ultrogel AcA 44 column (2.5-cm diameter, 58-cm height, equilibrated with 62.5 mrvr Tris-HCl, pH 9.0, at 4°C) and eluted at how rates between 1.4 and 7.0 ml/h). Fractions were collected and analyzed for DAP III and Hb.
420
JONES
AND
The column was calibrated with proteins of known molecular weight as standards under the same conditions as used for elution of the DAP III. In experiments where Hb was to be separated from ,&NA, columns of Sephadex G25-80 (1.3~cm diameter, 7.5-cm height) were equilibrated with 62.5 mM Tris-HCl buffer, pH 9.0. Reaction mixtures to be analyzed were mixed with 0.2 vol 20% sucrose, applied to the column, and eluted with the Tris buffer at a flow rate of 0.6 ml/min. Fractions of 5 ml were collected; P-NA was eluted only in fractions 5-7, partially separated from the Hb which eluted at the void volume, and measured with the fluorometer. Measurement of Hb and protein. Hb concentration was measured by Drabkin’s procedure ( 11) and protein by the Bradford assay (12). RESULTS
When cell lysate and Arg-Arg-/3-NA were incubated together, immediate and continuous hydrolysis of the substrate occurred with the release of /3-NA, demonstrating the presence in red blood cells of substantial amounts of DAP activity. The rate of hydrolysis, however, was not proportional to the amount of cell lysate (Fig. 1). This result might be due to the presence in the cell lysate of an inhibitor of the enzyme or of a compound which quenched the fluorescence of the P-NA product. The latter was considered likely since the fluorescence maximum for @-NA occurs at 410 nm while the absorption spectrum of human Hb shows a peak around 410 to 420 nm. Addition of Hb to reaction mixtures containing unfractionated cell lysate decreased the observed rate of substrate hydrolysis. To develop an assay which is unaffected by Hb and which would be rapid and easy to perform routinely, two approaches were explored: separation of DAP III from Hb before enzyme assay and separation of /3-NA product from Hb after enzyme reaction.
KAPRALOU VOLUME
OF ELUATE (ml)
0.6 -
0.04 VOLUME
FIG. 1. Amount
of enzyme
0.08
0.12
0.16
0.20
OF RSC LYSATE (ml)
activity
observed as a funcunfractionated from CM-Bio-Gel
tion of sample volume. Samples were RBC lysate, (O), A column, (0).
Separation
or Hb-free
eluate
of DAP II1 jkom hemoglobin.
Three techniques were tested for their ability to separate DAP III and Hb in RBC lysates. Cell lysate was fractionated by gel-filtration chromatography on Ultrogel AcA 44. DAP III activity eluted as a single peak slightly before Hb but always overlapping the Hb peak, which prevented accurate measurement of the total amount of DAP III activity recovered from the column, Pituitary DAP III can be partially purified by precipitation with ammonium sulfate at a final concentration of 40 to 60% (4). When RBC lysate was fractionated by precipitation with ammonium sulfate, most of the DAP III precipitated in the 45-63s range of saturation, but Hb was present in this precipitate in significant amounts, preventing accurate assay of total DAP III activity. When RBC lysate was applied to CMBio-Gel A columns at pH 6.5, DAP III activity was not bound to the resin and eluted within the first 6 ml of eluate. Hb was fully
DIPEPTIDYL
AMINOPEPTIDASE
retained on the column and could be removed by elution at pH 9.0. The column, following equilibration at pH 6.5, could then be reused for the fractionation of further samples of cell lysate. Hb-free enzyme obtained from this ionexchange procedure gave a linear response in the enzyme assay procedure up to 1.5 enzyme units (Fig. 1). Hb added to reaction mixtures containing such Hb-free enzyme decreased the amount of activity observed: 2.0 mg Hb in the 5.0-ml reaction mixture resulted in 91% decrease (Fig. 2). The concentration of Hb in the unfractionated cell lysate was determined to be 38 mg/ml; thus assay of the DAP III activity in 0.1 ml of unfractionated cell lysate is under conditions producing more than 9 1% apparent decrease in enzyme activity. Separation of /3-NA and Hb. To determine whether Hb inhibits DAP III or interferes with the measurement of /3-NA through quenching, reaction mixtures containing Hbfree DAP III were mixed with 0 and 2.0 mg human Hb. Substrate was added and reaction was allowed to proceed for 30 min. The total production of /3-NA in the two samples was measured. In the reaction containing Hb, 8-9% of the &NA present in the control (reaction without Hb) could be observed (Table 1). The P-NA produced was thenseparated from Hb by two procedures. Forty
421
IN RBCs TABLE PRODUCTION
OF &NA IN REACTION CONTAINING Hb
@NA produced/30 min/ml original RBC lysatc (nmol) Expt. I 2 3 *Reaction components;
1
-Hb”
+Hb
594 614 560
46 56 51
MIXTURES
&NA recovered from Heating pP3WdU~C (nmol)
Gel filtration (nmol) -Hb 238 224 190
+Hb
-Hb
+Hb
107 119 120
356 390 369
151 182 239
mixtures contained 0.0 or 2.0 m g Hb in addition Hb-free DAP III was used as enzyme.
to the usual
percent of each reaction mixture was fractionated by gel-filtration chromatography on Sephadex G-25 and the P-NA measured. The remaining 60% was heated in a boilingwater bath for 10 min, the precipitate of protein and Hb was removed by centrifugation, and the P-NA in each supernatant was measured. The results showed that partial resolution of reaction mixtures into /3NA and Hb resulted in the detection of much higher amounts of fl-NA than were observable in the original reaction mixture (Table 1). Further experiments in which reaction mixtures were completely resolved into separate Hb and /3-NA peaks on large Sephadex columns showed that recovery of /3-NA was then equal to that obtained in the control reaction mixture without Hb. Thus the Hb does not inhibit DAP III but does quench the fluorescence of the ,&NA. Effect of Triton X-100 on DAP Ifl. The effect of Triton-X-100 on DAP III activity was studied to determine whether this detergent could be used in the preparation of cell lysates. Triton X-100, in concentrations up to 1.0% v/v, had no significant effect upon the enzyme. Recommended procedure for DAP III assay. The final assay developed for mea-
HEMOGLOBIN
(mg)
FIG. 2. Effect of Hb, added to reaction observed activity of Hb-free DAP III.
mixtures,
upon
suring DAP III activity in red blood cells was as follows: 1. 1.O ml whole blood was centrifuged at 2000g for 10 min at 2°C; the cells were
422
JONES
AND
gently washed with cold isotonic NaCl solution and recentrifuged. The cells were then lysed with 5.0 ml cold 1% Triton X- 100, centrifuged, and the supernatant retained. 2. This cell lysate was mixed with 0.25 vol 20% sucrose and 0.1 ml was applied to a CM-Bio-Gel A column ( 1.3 X 7.5 cm) and eluted with 10 mM potassium phosphate buffer, pH 6.5. A single 7.4-ml fraction was collected; 1.0 ml of this was added to the standard reaction mixture and the rate of production of &NA was determined over a period of 15 min. Under these conditions a linear assay was obtained with 0.0 to 1.5 units of DAP III per reaction mixture. Reproducibility of the assay. To determine the reproducibility of this assay, three types of experiments were performed. (a) A single RBC lysate was prepared, using 1% Triton X- 100 to lyse the cells, and four 0.1 -ml samples were applied to ion-exchange columns. Each sample was eluted with phosphate buffer, collected as a single 7.4-ml fraction, and 1.O ml was assayed. The amounts of enzyme found were 42.2, 39.2, 41.6, and 39.5 units/ml lysate. (b) Four separate cell lysates were prepared from a single sample of whole blood and were fractionated and assayed as in (a). The amounts of enzyme were found to be 42.6, 42.6, 49.9, and 42.7 units/ml lysate. (c) A single cell lysate was prepared and 6 different volumes covering the range O.OO0.10 ml were applied to columns and the eluates assayed as before. Enzyme activity per milliliter of eluate was directly proportional to the amount of enzyme applied to the cation-exchange column in four separate experiments. Samples of eluates were rechromatographed on the ion-exchange columns and enzyme recovery was consistently in the range 95-100%. DlScusslm The biosynthesis of angiotensin II has been the subject of continued study, but little attention has been focused upon its degra-
KAPRALOU
dation. The discovery of angiotensinase activity in circulating RBCs (9) suggests the need for detailed examination of the characteristics of this enzyme and its possible role in the catabolism of circulating angiotensin II. Such examination has been hindered by the lack of a simple accurate assay for the enzyme; its activity has been assayed by the rate of inactivation of angiotensin, the amount of angiotensin being determined by measuring the ability of samples to raise blood pressure or to cause the contraction of isolated strips of smooth muscle (7,9), an assay procedure which is tedious, relatively inaccurate, and nonspecific, being significantly affected by nonangiotensin compounds and ions. The discovery that RBC angiotensinase and pituitary DAP III are homologous proteins (6) suggested that an assay could be developed for RBC angiotensinase based upon the rate of hydrolysis of Arg-Arg-P-NA. In this work we report such an assay which is simple, rapid, specific, and reproducible. RBCs may contain additional angiotensinases which do not hydrolyze this artificial substrate and these would not be detected by this assay. Hb interfered significantly with measurement of the enzyme reaction product, /3-NA. When the B-NA was partially separated from the Hb by gel filtration or by heat precipitation of the Hb, the amount of product detected increased from 8 to 9% of that present in the control lacking Hb to about 50 to 60% of the control value. An amount of ,f3NA equal to that in the control was not observed because of incomplete separation of Hb from the ,&NA. The Hb was not fully precipitated from solution by the heating procedure nor was it totally separated from the @-NA when small Sephadex columns were used. Larger gel-filtration columns were used to separate the Hb and P-NA completely, and full recovery of &NA equal to that in control reaction mixtures was then obtained. Thus Hb inhibits the measurement of the P-NA product and not the DAP III enzyme. The use of the large gel-filtration
DIPEPTIDYL
AMINOPEPTIDASE
columns resulted in a usable assay procedure but one which was too lengthy for convenient routine use. Separation of enzyme from Hb proved a better approach. DAP III was completely separated from Hb in a single ion-exchange step which could be performed rapidly and reproducibly and the enzyme activity could then be measured by fluorimetry. The availability of the simple procedure described here should be of value in further studies on the nature and function of this enzyme in circulating red blood cells.
4. 5. 6.
7. 8.
9.
REFERENCES 1. Barrett, A. J. (1977) in Proteinases in Mammalian Cells and Tissues (Barrett, A. J., ed.), pp. l-56, North-Holland, New York. 2. McDonald, J. K., Ellis, S., and Reilly, T. J. (1966) J. Biol. Chem. 241, 1494-1501. 3. McDonald, J. K., Leibach, F. H., Grindeland, R.,
10.
11. 12.
IN RBCs
423
and Ellis, S. (1968) J. Biol. Chem. 243, 41434150. Ellis, S., and Nuenke, J. M. (1967) J. Biol. Chem. 242,4623-4629. Hopsu-Havu, V. K., and Glenner, G. G. (1966) Histochemie 7, 197-201. McDonald, J. K., and Schwabe, C. (1977) in Proteinases in Mammalian Cells and Tissues (Barrett, A. J., ed.), pp. 31 I-391, North-Holland, New York. Page, I. H., and Bumpus, F. M. ( 1974) Angiotensin, Springer-Verlag, New York. Ryan, J. W. (1974) in Angiotensin (Page, I. H., and Bumpus, F. M., eds.), pp. 81-110, SpringerVerlag, New York. Kokubu, T., Akutsu, H., Fujimoto, S., Vieda, F., Hiwada, K., and Yamamura, V. (1969) Biochim. Biophys. Acta 191, 668-676. Schroter, W. (1974) in Clinical Biochemistry, Principles and Methods (Curtius, H. C., and Roth, M. eds.), pp. 1178- 1207, de Gruyter, New York. Sigma Technical Bull., No. 525 (1980) Sigma Chem. Co., St. Louis, MO. Bradford, M. (1976) Anal. Biochem. 72, 248-254.