Arginase from human blood serum

BIOCHEMICAL MEDICINE 26, 174-184 (1981)

Arginase from Human Blood Serum

ANNA BARA~CZYK-K&MA, IWONA SKRZYPEK-OSIECKA, AND ZOFIA POREMBSKA’

Department of Biochemistry, Institute of Biopharmacy, Medical School, ul. Banacha 1 I 02-097 Warsaw. Poland

Received March 3, 1981

Arginase is present in many human tissues (1). Data from the literature concerning arginase occurrence in blood of healthy humans are incon- sistent. Some authors (2-4) claim that the enzyme is absent in serum and others (5-7) that it appears there in only small amounts.

The absence of arginase in blood serum and the differences in its activity are no doubt due to differences in methods and experimental conditions.

In our earlier studies a simple a:rd sensitive method for arginase activity determination was developed (8). This activity in blood serum from healthy subjects averaged 1.5 units, expressed as micromoles ornithine per minute per 1000 ml serum. In about 30% of the subjects the enzyme was not observed.

On the other hand, considerable arginase activity has been observed in serum of patients with myocardial infarction (9). It has also been found that arginase determination in serum may be a useful test for early detection of infarction and for its differentiation from other cardiac dis- eases. The suitability of this test for the diagnosis of myocardial infarction has also been confirmed by other authors (IO).

The fact that arginase occurs in human tissues in various molecular forms (I), as well as the usefulness of the arginase test, led us to attempt arginase isolation from the blood of healthy subjects and to compare its properties with those of arginases from other human tissues.

Reagents EXPERIMENTAL

Reagents were purchased as follows: L-arginine, o-arginine, L-homoarginine hydrochloride, P-guanidinepropionic acid, L-a-amino-p-guani-

I To whom requests for reprints should be addressed.

174 0006-2944/81/050174-11$02.00/O Copyright 6 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.

ARGINASE FROM HUMAN BLOOD SERUM 175

dinepropionic acid hydrochloride, y-guanidinebutyrate, a-amino-y-guanidinebutyrate (Calbiochem, Los Angeles, Calif.), Acrylamide and methylenebisacrylamide (Fluka, Buchs, Switzerland), N,N,N’,N’-tetra- methylethylenediamide (Eastman Organic Chemicals, Rochester, N.Y .), Sephadex G-150 and Dextran 2000 (Pharmacia, Uppsala, Sweden), and CM-cellulose (CM-52) and DEAE-cellulose (DE-l 1) (Whatman Biochem- icals, Maidstone, Kent, England).

Marker proteins. Bovine albumin, chicken ovalbumin, bovine globulin, and horse myoglobin (Sigma Chemical Co., St. Louis, MO.) were used. All other chemicals were of the purest available grades from standard commercial sources.

Materials

Blood for the investigations was obtained from a blood bank. Fresh blood samples were kept at 4°C for 2-3 hr, and then centrifuged at 4°C for 5 min at 2000 rpm. The resulting serum was again centrifuged for 20 min at 5000 rpm. Frozen serum from the blood bank was also used in the studies. Sera of groups 0, A, B, and AB were examined. Inde- pendently of the blood group, 70% of sera showed arginase activity. In 30% of sera the enzyme was not observed.

On account of the relatively high arginase level in the erythrocytes, it was essential for the determination of serum enzyme activity to exclude samples with hemolysis.

The degree of erythrocyte hemolysis was determined by the benzidine test (11) and by microscopic inspection. Sera showing traces of hemolysis were discarded.

Arginase Assay

Arginase activity was determined from the increase in the reaction products, urea and ornithine. Urea was estimated according to Ratner (12) and ornithine by the method of Chinard (13) as modified by Po- rembska and Baranczyk-Kuima (8). At a final volume of I ml the incubation mixture for urea determination contained: 250 mM glycine-NaOH buffer, pH 9.5, 5 mM MnCl,, 20 mM L-arginine, and the enzyme preparation. For ornithine determination 100 mM sodium Verona1 buffer, pH 9.5, was used instead of the glycine-NaOH buffer mainly because glycine interferes with the orithine determination. Samples of the incubation mixtures were kept for lo-20 min at 37°C. Extinction was measured in a Bausch & Lomb Spectronic 20 photocolorimeter at 540 nm for urea and 515 nm for ornithine. One unit of enzymic activity was defined as 1 kmole of product formed per minute at 37°C.

176 BARArjCZYK-KUiMA,SKRZYPEK-OSIECKA,ANDPOREMBSKA

Protein Determination

Protein was determined according to Lowry et al. (14) or spectro- photometrically by the method of Warburg and Christian (15), with crys- talline bovine serum albumin as standard.

Polyacrylamide Gel Electrophoresis

All gels (7.5% w/v polyacrylamide) were prepared according to the methods of Omstein (16), Davis (17), or Sakai and Gross (18). Electro- phoresis was performed in Tris-glycine buffer, pH 8.9, and in acetate buffer, pH 5.5, using a current of 3 mA per tube. Protein was stained with 0.5% amido black in 7% v/v acetic acid. Arginase activity was determined in 2-mm gel slices eluted with Verona1 buffer, pH 9.5.

Molecular Weight Determination

The molecular weight of human serum arginase was determined by Sephadex G-100 and Sephadex G-150 chromatography, as described by Andrews (19). The column (2 x 40 cm) was previously equilibrated with 100 mM KC1 in 50 mM Tris-HCL buffer, pH 7.5.

Horse myoglobin (17,000 MW), ovalbumin (46,000 MW), bovine serum albumin (69,000 MW), bovine y-globulin (150,000 MW) were used as standards (5 mg each). Fractions of 2 ml were checked for enzyme activity and protein content.

EDTA Treatment of Human Serum Arginase

The enzyme obtained after the sixth purification step (Table 1) was concentrated in the presence of Ficoll and then dialyzed against 50 mM Tris-HCl buffer, pH 7.5. This preparation (2 mg of protein) was incubated with 50 mM EDTA at pH 7.5 at 37°C for 30 min. Arginase activity was determined in the presence and absence of Mn*+. The molecular weight of EDTA-treated arginase was determined by Sephadex G-150 chromatography; the column was previously equilibrated with 10 mM EDTA.

RESULTS

Purification Procedure for Human Serum Arginase

For purification, some of the steps recommended by other authors were followed. For enzyme stabilization, 5 mM MnC1,(20) was used; 1 mM 2-mercaptoethanol was applied to prevent possible arginase aggre- gation (21). Unless otherwise stated, all procedures were carried out at 4°C. The results of purification are summarized in Table 1.

Step I: (NH,) #04 fractionation. Serum samples showing no trace of hemolysis were fractionated using solid ammonium sulfate at pH 7.5.


TABLE 1 PURIFICATION OF HUMAN SERUM ARGINASE (VALUES NORMALIZED TO A PREPARATION FROM

100 ml OF SERUM)

Procedure

Serum 30-75%

(NH,), SO,- satur.

Heated at WC, 20 min

Ethanol precipitation

Heated at 6O”C, 20 min

4s75% (NH,) $0, satur.

CM-cellulose chromatography

Sephadex G-150 chromatography

Activity Total ----

protein Total specific Purification Yield b-d (wmole/min) ( pmolelminimg) factor (%o)

6750

3769 0.49 1.3 x 1om4 1.6 90.7

1044 0.47 4.5 x lo-’ 5.6 87

190 0.44 2.3 x 10-j 28 81.5

65 0.30 4.3 x 10-j 56 55.6

12.5 0.25 2.0 x lo-’ 250 46.3

2.9 0.23 8.0 x IO-’ 1000 42.6

1.1 0.21 1.9 x lo-’ 2375 39.0

0.54 8.0 x lo-’ - 100

The material sedimenting at 0.3-0.75% (NH,) SO4 saturation was refrigerated for 60 min, then collected by centrifugation and dissolved in a mixture of 5 mM MnCl,, 1 mM 2-mercaptoethanol, and 10 mM Tris-HCl buffer, pH 8.3, whereupon it was dialyzed for 20 hr against the same solution.

Step 2: Heat treatment. Advantage was taken of the stability of the enzyme up to 60°C. The dialysate (2-ml portions) was placed for 20 min in a water bath (6O”C), with mild stirring. Then all portions were pooled rapidly, cooled with ice, and centrifuged at 13,000g for 15 min.

Step 3: Precipitation with ethanol. Three volumes of ethanol cooled to - 10°C and containing 5 mM MnCl*, was slowly added to the supernatant. The mixture was centrifuged at 13,OOOg at - 10°C for 15 min. The pellet was dried, extracted with 10 vol of solution containing 5 mM MnCL 1 mM 2-mercaptoethanol, 10 mM Tris-HCl buffer, pH 7.5, and then dialyzed overnight against the same solution.

Step 4: Second heat treatment (60°C for 20 min). The dialysate was centrifuged at 13,000g for 15 min and the supernatant was heated as in step 2.

Step 5: Second (NH.,) zS04fiactionation. Solid ammonium sulfate was used. The precipitate obtained for 0.45-0.75% saturation was refrigerated

178 BARANCZYK-K&MA, SKRZYPEK-OSIECKA, AND POREMBSKA

for 60 min, collected by centrifugation, dissolved in a solution containing 5 mM MnCl,, 1 mM 2-mercaptoethanol, 10 mM Tris-HCl buffer, pH 7.5, and then diaiyzed for 25 hr against the same solution.

Step 6: CM-cellulose chromatography. The dialysate was centrifuged and placed on a column packed with CM-cellulose (18 x 1 cm), stabilized with 500 ml of 10 mM Tris-HCl buffer, pH 7.5. The enzyme adsorbed on the column was eluted with a KC1 concentration gradient (0.0-0.3 M) (Fig. 1). The active fractions were concentrated using Ficoll, in the presence of MnCl, (at a final concentration of about 5 mM), and dialyzed for 24 hr against a solution of 5 mM MnC&, 1 mM 2-mercaptoethanol, and 10 mM Tris-HCI buffer, pH 7.5).

Step 7: Sephadex G-150 gel Jiltration. The dialysate was applied to a Sephadex G-150 column (2 x 40 cm) equilibrated with 100 mM KC1 in 50 mM Tris-HCI buffer, pH 7.5, and eluted with the same solution. A single peak of enzyme activity was observed (Fig. 2). The fractions showing arginase activity were pooled, concentrated, and dialyzed as described for step 1. The specific activity of the arginase preparation so obtained was about 2400 times greater than that of serum arginase; it

Elution volume ( ml) FIG. 1. CM-cellulose chromatography of human serum arginase. The enzyme obtained

after step 5 was applied (about 25 mg) to a CM-cellulose column (1 x 18 cm) equilibrated with 10 mM Tris-HCI, pH 7.5, eluted with a KC1 concentration gradient. Fractions (5 ml) were collected at a flow rate of 1-l .5 ml/mitt. Protein and arginase activity were determined by standard assays. (0) Arginase activity: (0) protein content; (-) KC1 gradient.


0.50 .E al

Q25 z

Eiution volume ( ml) FIG. 2. Sephadex G-150 filtration. The enzyme obtained after step 6 (about 6 mg) was

applied to a Sephadex G-150 column (2 x 40 cm) and eluted with 100 mM KCI, 50 mM Tris-HCI buffer, pH 7.5. Fractions (2 ml) were collected at a flow rate of 25 ml/hr. (0) Arginase activity; (0) protein content.

contained 1.1 mg protein/ml, with a specific activity of 0.19 pmole ornithine/mg protein/min.

The preparation was not homogeneous. Disc polyacrylamide gel electrophoresis at pH 5.5 and 8.9 failed to reveal the presence of contami- nating materials (Fig. 3).

Attempts at applying affinity chromatography on Sepharose bound with lysine did not increase the degree of purity. The very low concentration of enzyme in serum and its low stability in the final steps of preparation rendered further purification very difficult.

PROPERTIES OF THE ENZYME

Electrophoretic Properties

Figure 3 illustrates polyacrylamide gel electrophoresis of human serum arginase after the seventh step of purification. At pH 5.5 the enzyme migrated toward the cathode as three protein bands. The whole arginase activity was found in the faster-moving fraction. During electrophoresis at pH 8.9 the enzyme exhibited very low electrophoretic mobility and was detected near the start.

180 BARAfiCZYK-K&MA, SKRZYPEK-OSIECKA, AND POREMBSKA

pH 5,5 pH 89 / * \ / .A 7

a b a FIG. 3. Diagram of electrophoretic patterns of human serum arginase. Polyacrylamide

gel electrophoresis was performed as described under Experimental. The enzyme obtained (40 pg) after step 7 was used. (a) Protein; (b) activity.

Human serum arginase is a cationic protein, and its mobility at both pH was similar to the mobility of the A, form from human liver and kidney (1) as well as to that from human heart (A. Baranczyk-Kuima, I. Skrzypek-Osiecka, and Z. Porembska, unpublished observations).

In a manner similar to that of the other above-mentioned arginases, human serum arginase was adsorbed on CM-cellulose but not retained by DEAE-cellulose (Fig. 1).

Molecular Weight

The molecular weight of human serum arginase was found to be 120,000 + 5000 (Fig. 4).

Optimal pH

The effect of pH on the enzymic activity was studied in 100 mM Tris-HCl buffer, 100 mM Verona1 buffer, and 250 mM glycine-NaOH buffer, from pH 6 to Il. For human serum arginase the optimal pH was 9.2-9.6.

Substrate Specificity

Human serum arginase was highly specific for L-arginine; it did not hydrolyze n-arginine, L-homoarginine, and the tested guanidine deriva- tives: p-guanidinepropionate, L-a-amino-p-guanidinepropionate, y-guanidinebutyrate, and cx-amino-y-guanidinebutyrate.


Ve Imll ,

70

60

50

40

30

20

10

1

J

\

-

\

molecular weight ( x l@) FIG. 4. Molecular weight determination of human serum arginase by Sephadex G-150

filtration. The enzyme obtained after step 6 (2 mg) or marker proteins (5 mg) were applied. (1) Horse myoglobin (17,000 MW); (2) chicken egg albumin (45,000 MW); (3) bovine serum albumin (69,000 MW). (4) bovine serum globulin (lSO,OOO MW). (0) Native human serum arginase; (A) human serum arginase inactivated by EDTA.

Effect of Substrate Concentration

The influence of substrate concentrations, varying from 0.2 to 8.0 mM, on enzymic activity was studied in 250 mM glycine-NaOH buffer, pH 9.4, in the presence of 5 mM MnCl,. From this data a Lineweaver-Burk plot with a Km value of 3.3 mM was calculated (Fig. 5).

Effect of EDTA

After the sixth step of purification human serum arginase was incubated with EDTA (100 umole of EDTA per 1 mg protein per 1 ml), at 37”C, for 30 min at pH 7.5. Under these conditions the enzyme was completely

182 BARAfiCZYK-KUiMA, SKRZYPEK-OSIECKA. AND POREMBSKA

I I I J

03 02 O,l 0 OJ QJ 03

l/iArgl(mM-‘1 FIG. 5. Lineweaver-Burk plot of human serum arginase (6 pg of enzyme obtained after

step 7 was used).

inactivated. EDTA-inactivated arginase was dialyzed at 4°C against 25 mM Tris-HCl buffer, pH 7.5, for 20 hr to remove the excess of EDTA, and it was subjected to gel filtration on a Sephadex G-150 column equilibrated and eluted with 100 mM KCl, 50 mM Tris-HCI buffer, pH 7.5, containing 1 mM EDTA. The enzyme was eluted as one fraction showing activity only after addition of Mn”. The molecular weight of inactivated arginase from human serum was about 30,000, indicating that the removal of Mn2+ ions by EDTA leads to dissociation into subunits. Addition of Mr?’ ions to the inactive subunits resulted in the reappearance of the enzymic activity. The molecular weight of the reassociated enzyme was found to approach that of the native form.

DISCUSSION

The presence of different molecular forms of arginase in mammalian tissues has been postulated by many authors (22).

In rat tissues we have found four forms of arginase, designated A,-A, according to the order in which they emerged from the DEAE-cellulose column (23). All forms had a similar molecular weight of about 120,000; they differed, however, in their behavior on DEAE-cellulose, and in their electrophoretic pattern, Michaelis constant, and stability.


In humans form A, has been found in all tissues examined, A, being present as the second form in liver and erythrocytes, and A, in the kidney and brain (I).

In this study human serum was found to contain only one form of arginase as has previously been established for the enzyme from lung (24), placenta (2% leukemic lymphocytes (26), and heart (A. Baradczyk- Kuima, I. Skrzypek-Osiecka, Z. Porembska, unpublished observations).

Human serum arginase resembled other arginases from different mammalian tissues in many properties such as molecular weight, Km, optimum pH, and heat stability. The electrophoretic mobility as well as the affinity to CM-cellulose of this enzyme resembled those of form A, but differed from those of forms A3 and A,.

SUMMARY

1. An isolation and purification procedure for human serum arginase is presented.

2. The specific activity of the purified enzyme was 2400 times greater than that of crude serum arginase.

3. Multiple molecular forms of the enzyme were not detected in serum. 4. The enzyme showed high specificity for L-arginine. K,,, of the enzyme

was 3.3 mM. The enzyme was found to be cationic protein; its molecular weight amounted to 120,000.

5. EDTA inhibited the enzyme. This inhibition could be completely reversed by Mn” ions. The enzyme had an oligomeric structure; upon treatment with EDTA it dissociated into subunits with a molecular weight of 30,000.

REFERENCES 1. Porembska, Z., and Kedra, M., BUN. Acad. Polon. Sci. Ser. Sci. Biol. 19, 633 (1971). 2. Forsell, 0. M., and Palva, I. P., Stand. J. Clin. Lab. invest. 12, 131 (1961). 3. Cabello, J., Prajoux, V., Basilio, C., and Plaza, M., Comp. Biochem. Physio/. 6, 11 I

(1%2). 4. Loeb, W. F., and Stuhlman, R. A., Clin. Chem. 15, 1962 (1969). 5. Manning, R. T., and Grisolia, S., Proc. Sot. Exp. Biol. Med. 95, 225 (1957). 6. Zapf, P. W., Clin. Chim. Acta 26, 547 (1969). 7. Jergovic, I., Zuzuc, I., Fiser-Herman, M., and Straus, B., C/in. Chim. Acra 30, 765

(1970). 8. Porembska, Z., and Baradczyk-Kuima, A., Diugn. Lab. 3, 239 (1974). 9. Porembska, Z., and Kedra, M., C/in. Chim. Acta 60, 355 (1975).

10. Dosiak, J., Pal. Tyg. Lek. 31, 153 (1976). 11. Bing, F., and Baker, R., J. Biol. Chem. 92, 589 (1931). 12. Ratner, S., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan. Eds.).

Vol. 2, p. 356. Academic Press, New York, 1955. 13. Chinard, F. P., J. Biol. Chem. 199, 91 (1952). 14. Lowry, 0. H., Rosebrough, N. J. Farr, A. L., and Randall, R. J. J. Biol. Chem. 193,

265 (1951).

184 BARAtiCZYK-KUiMA, SKRZYPEK-OSIECKA, AND POREMBSKA

15. Warburg, O., and Christian, W., Biochem. Z. 310, 384 (1941). 16. Omstein, L., Ann. N.Y. Acad. Sci. 121, 321 (1964). 17. Davis, B. J., Ann. N.Y. Acad. Sci. 121, 404 (1964). 18. Sakai, T., and Gross, J., Biochemistry 6, 518 (1%7). 19. Andrews, G., Biochem. J. 91, 222 (1964). 20. Greenberg, D. M., and Mahamed, M. S., Arch. B&hem. 8, 365 (1949). 21. Sakai, T., and Murachi, T., Physiol. Chem. Phys. 1, 317 (1969). 22. Porembska, Z., Enzyme IS, 198 (1973). 23. Ggsiorowska, I., Porembska, Z., Jackimowicz, J., and Mochnacka, I., Acra Biochim.

Polon. 17, 19 (1970). 24. Dahlig, E., Porembska, Z., and Mochnacka, I., Acta Biochim. Polon. 22, 77 (1975). 25. Porta, R., Esposito, C., Martin, A., and Della Pietra, G., B&hem. J. 159, 579 (1976). 26. Reyero, C., and Domer, F., Eur. J. Biochem. 56, 137 (1975).

Documents

Arginase from human blood serum