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Steady-state kinetic studies of arginase with an improved direct spectrophotometric assay
ANAl.YTICAL
BIOCHEMISTRY
Steady-State
C. NICK
109,
Kinetic
261-265
(1980)
Studies of Arginase with an Improved Spectrophotometric Assay’
PACE, ANDRES
BUONANNO,
Received A direct spectrophotometric t 1967. At~crl. Biochem. 18, 102) substrate concentrations, for kinetic studies of the enzyme. reported and compared with number of problems encountered
AND
was supported by Grant Institutes of Health.
SIMMONS-HANSEN
July 7. 1980
assay for arginase developed by R. L. Ward and P. A. Srere is extended so that it can be used at pH 7.5 or 9.5. at higher assaying crude sources of arginase. and for steady-state Steady-state kinetic studies of beef and rat liver arginase are Gmilar results obtained using other assay procedures. A in steady-state kinetic studies of argina\e are discussed.
Arginase (t-arginine amidinohydrolase, EC 3.5.3. I) catalyzes the cleavage of arginine to ornithine and urea. A surprisingly large number of techniques have been developed for assaying arginase (see ( 1) and references therein). The most popular of these measures urea formation and requires a 60-min boiling step (2). In addition, the product is light labile and requires special precautions during boiling and cooling (3). This inconvenience coupled with the fact that arginase generally requires prior activation by heating (4) result in a tedious assay of limited precision. The wide range of Michaelis constants reported for rat and beef liver arginase (0.3 to over 20 mM). and the diversity of conclusions about the role of Mn’+ in the activation of the enzyme are, in part, reflections of the assays available. In 1967, Ward and Srere (5) described a direct, spectrophotometric assay for arginase based on the difference in absorption between the products and reactants at 205.7 nm. This approach is much simpler and faster than the indirect assays and has since been used by several investigators (6-10). We have extended this method so that it can be used at higher ’ This research from the National
JEANNIE
Direct
AM 191 12-02
substrate concentrations, for assaying crude samples of arginase, and at either pH 7.5 of 9.5. We show that the method can be used for steady-state kinetic studies of the enzyme and report Michaelis constants determined for the rat and beef liver enzymes under various conditions. MATERIALS
AND METHODS
Partially purified beef liver arginase (33.3 unitsimg) was purchased from Worthington Biochemical Company (Lot AR36C869). Schimke’s procedure ( 11) was used for purifying rat liver arginase. The specific activity of the final product ranged from 650 to 1050 unitsimg in different purifications. Enzymes and chemicals for the coupled-enzyme assay were purchased from Boehringer-Mannheim. For the data in Fig. I. the A grade hydrochlorides of arginine and ornithine from Calbiochem. and “ultrapure” urea from SchwarziMann were used. All other chemicals were the best grade available from Sigma Chemical Company. The absorption spectra and some of the initial velocities were measured with a Cary Model 15 spectrophotometer. Other initial velocities were recorded with a Gilford Model 250 spectrophotometer. The
262
PACE.
BUONANNO.
Wavelength FIG. and and
I.
Absorption
spectra
for
urea at pH 9.5. E = Molar k = tArC ~ Ed,,,, - t,,,-, .,,.
arginine.
absorption
ornithine. coefficient.
Archibald method (2) and the coupledenzyme method ( 12) were used for measuring some of the initial velocities in Fig. 2. For the experiments in Fig. 2 and in Table 2. the arginase was dialyzed against 0.05 or 5 IIIM MnCl,, 10 mM Tris-HCl, pH 7. for 4 to 6 h at 4°C and then activated by heating for 1 hat 37°C. Identical results are obtained when the heating step is omitted. The reaction is initiated by adding 0.1 ml of the arginase to 2.5 ml of a substrate solution at either pH 7.5 or 9.5: consequently, the MP concentrations will be only 0.0019 or 0.19 mM when the velocity is measured. RESULTS The absorption spectra of arginine, ornithine, and urea at pH 9.5 are shown in Fig. 1. The difference absorption coefficient, Ae = E.~,, - Ed,,, ~ t,,,,;,, is also shown and can be seen to increase sharply below 215 nm. Consequently, the absorbance change accompanying the conversion of arginine to
AND
HANSEN
ornithine plus urea will be maximized by using the lowest possible wavelength. At pH 7.5 similar results are obtained but AC and the molar absorption coefficients for arginine and ornithine are reduced. The presence of I mM Mn” does not affect the he values at pH 7.5. Molar absorption coefficients for arginine and the k values at pH 7.5 and 9.5 are listed in Table I. In a typical assay of arginase. we use 2.5 ml of an I -arginine solution with a concentration in the range from 0. I to I5 KIM. Arginase (0.5 to 3.5 units) is added to this solution to initiate the reaction and the absorbance is recorded. The rate of absorbance change can be determined by drawing a tangent to the progress curve at the beginning of the reaction. Initial velocities in terms of concentrations can then be calculated from these rates using the ALE values in Table 1. With care, velocities can be determined with a precision of better than +5% over a wide range of substrate concentrations. Excellent agreement is obtained when different wavelengths are used for measuring the velocities. A wavelength ‘I‘ABLk MOLAR ANI)
ABSoRPrlON DII I t RFN(‘~ ‘,I
I
CO1.l I 1c1kh ABSORPTION pH pH
9.5
‘.NI)
15 I OR AR(~INI~I Corl~t IC II VI\ 7.5
7.5
pH
Y.5
Wavelength (nm) 215 214 213 217 211 110 7OY
42 hl
I34 177
85 123 I74 136 344 477
74 I Y71 I x4
205 104
I’ At
7’) 103
2 35 314 423 564
108 107 106
” hf-’
Art”
t”
643 X63 I I41 1478
IhI5 cm t\rc
‘. t
t,,n ‘8
t’l
At”
340
YX
27x 334
I I7 155
396 47’) SY3 734
IYY Xl 352 46X 6’0
YI II40 1471
X16 I Oh2
I754 7193
1361 17%
ARGINASE
263
ASSAY
c : I E
0.5
0
0.2
0.1
v/S
0.3
Imln-‘I
FIG. 2. Eadie-Hofstee plots for the hydrolysis of L-arginine by beef liver arginase. Initial velocities determined by Archibald assay at pH 7.0. 37°C (0): by the direct assay at pH 9.5. 25T (13): or by the coupled-enzyme assay at pH 9.5. 37°C (a).
must be selected which gives an absorbance in the usable range of the spectrophotometer (generally less than 3.0) when the substrate and enzyme are mixed. The data in Table 1 can be used to calculate the absorbance of the substrate solution; the absorbance due to the enzyme solution depends on the source of the enzyme and must be determined independently. As pointed out by Ward and Srere (5). this assay can be used with crude samples of arginase and gives results in excellent agreement with the Archibald procedure (2) when used to monitor the purification of arginase. It is common practice to maintain a high Mn’+ concentration (50 mM) during the purification of arginase 14). This creates a problem with the direct assay not previously noted. Since the pH optimum for arginase is near pH 9.5, the enzyme is generally assayed by adding a small aliquot of the enzyme solution at pH 7-7.5 to a solution of the substrate at pH 9.5. If 50 mM Mn”+ is present in the enzyme solution, there will be a sizable increase in the absorbance resulting from the exposure of Mn2+ to pH 9.5. This absorbance change will diminish the decrease in absorbance due to the hydrolysis
of arginine and lead to erroneous results. This problem can be avoided by assaying the arginase at pH 7.5. or minimized by maintaining the Mn”+ concentration of the arginase solution at 5 mM or less. The direct assay is particularly convenient for obtaining initial velocities for steadystate kinetic studies of arginase. Some results obtained using the direct assay are compared with results obtained using the Archibald method (2) and the coupledenzyme assay ( 12) in Eadie-Hofstee plots in Fig. 2. The data from the direct assay yield a Michaelis constant of 1.3 mM, while values of 1.6 and 1.4 mM are obtained from the Archibald and coupled-enzyme assays. respectively. We have also used the direct assay to determine the Michaelis constants for L-arginine with purified rat liver arginase. Initial velocities were measured at substrate concentrations ranging from 0.15 to 5.0 mM and the Michaelis constant and standard errors were estimated as described by Wilkinson (13). Corrections were made for substrate depletion (14) and corrections for product inhibition were shown to be insignificant. The results are presented in
264
PACE, TABLE
THE
MICHAELIS L-ARGININE
BUONANNO.
2
CoNsrAN r COR THY HYDROLYSIS BY RAI LIVER ARGINASI ”
ok
K,,, (mhil M”‘/ (mM)
pH 7.5
pH 9.5
0.05 .s
I.55 ? 0.16 1.33 t 0.13
1.03 + 0.19 1.13 -t 0.13
” Km values Wilkinson (13).
? SE
estimated
as
described
by
Table 2. For reasons discussed below, estimates of K,,, obtained using the direct assay are generally lower than estimates obtained using other assays. DISCUSSION Michaelis constants ranging from 0.3 ( 10) to 20 mM (15) have been reported for the hydrolysis of arginine by beef and rat liver arginase. Several factors which may contribute to this poor agreement are considered below. The evidence is good that both beef and rat liver arginase bind 1 mol of Mn” per subunit (7.16) and that the enzyme can be activated by heating in the presence of a high concentration of Mn” + ( IZ- 17). typically 5 min at 55°C in the presence of 50 mM Mn’)+ (4). It is less certain, however, that the resulting enzyme resembles arginase as it exists in \lit*o. The total concentration of Mn’+ in rat liver is approximately 0.024 mM ( I .3 pgig liver) (18) and the free Mn’+ concentration, considerably less (19). Thus, distinctly nonphysiological conditions are used to activate arginase and there are substantial discrepancies among published results. (Compare, for example, the results in ( 16) and (20) for rat liver arginase.) Furthermore. high Mn” concentrations interfere with both the direct (see Results) and Archibald (21) assays. Finally. Mn”+ binds to zwitterionic arginine with an association constant of 335 Mm’ (22) and can thereby
AND
HANSEN
reduce the free substrate concentration. For these reasons, we recommend the use of lower Mn’+ concentrations and lower temperatures for the activation of arginase. The results in Table 2 show that very similar results are obtained when arginase is activated at 37°C in the presence of 0.05 or 5 mM Mn’+. The pH optima for beef and rat liver arginase are between pH 9.0 and 10.5 ( 15). and the enzymes have been assayed at several different pH values in this range. This complicates kinetic studies of arginase. The pk’ of the cu-amino group of arginine is 9.04 (23) or 9.36 (22). Consequently, the relative concentration of monocationic and zwitterionic arginine will vary depending on the pH used for the assay and it is not established with certainty which is the better substrate (24). In addition. the degree of complex formation between Mn?’ and arginine will increase with increasing pH. and, finally. Mn’+ solutions turn brown when exposed to high pH. Thus, even though the activity of arginase is lower, much more consistent results can be obtained when kinetic studies are carried out at physiological pH. Failure to correct for substrate depletion can substantially increase the K,,, value determined from a steady-state kinetic study (14). For example, in a typical kinetic study of arginase using the Archibald assay and a 5-min incubation of substrate and enzyme, calculations show that the K,,, would be over 25% greater if no correction were made for substrate depletion. Since 5and IO-min incubations are commonly used in kinetic studies of arginase, this factor probably contributes to the wide range ofk’,,, values observed. A related problem is the substrate concentration range employed in a steady-state kinetic study. Cleland recommends a range of substrate concentrations from 0.2 to 5 K,,, for determining K,,, (25). Rosenfeld cf trl. ( 10) report K,,, = 0.3-0.4 mM for beef liver arginase but use only a 0.5 to 1.O mM range
ARCINASE
of arginine
concentrations.
Hirsch-Kolb
rt (11. (15). on the other hand, report K,,,
values of 7 and 20 mM for beef and rat liver arginase, respectively, and use a substrate concentration range from 5 to 100 mM arginine. The use of an inappropriate range of substrate concentration is likely to contribute to errors in the K,,, values. The values of K,,, we report are in best agreement with values of 1.0 mM for beef (7). 2.4 mM for rat (26), and 1.4 tItM for rabbit (27) liver arginase. Most of the k’,,, values reported for liver arginases are considerably higher than these values, probably, in part, for reasons discussed above. In summary, the information presented here will allow arginase to be assayed under a wider range of conditions and should prove useful for steady-state kinetic studies of the enzyme. REFERENCES I. Ruegg. U. T.. and Russell. A. S. (1980) And/. Eiochem. 102, 206-212. 2. Archibald. R. M. (1945) J. Biol. Ch~nr. 157, 507517. 3. Van Slyke. D. D.. and Archibald, R. M. (1946) J. Bid. C‘hem. 165, 293-309. 4. Schimke. R. T. (1970) in Methods Enzymology (Tabor. H., and Tabor. C. W.. eds.). Vol. l7A, pp. 313-317. Academic Press, New York. 5. Ward, R. L.. and Srere. P. A. (1967) ,4no/. Riothem. 18, IOZ- 106. 6. Hirsch-Kolb. H.. and Greenberg. D. M. (1968) J. Bic~f. C-hem. 243, 6123-6129. 7. Harell, D.. and Sokolovsky. M. (1972) Eur. J. Bioc~hrm. 25, 102-108.
ASSAY
365
8. Kaysen. G. A., and Strecker, H. J. (1973) Bioc~hertl. J. 133, 779-788. 9. Kuchel. P. W.. Nichol, L. W.. and Jeffrey. P. D. (1975) J. Bird. Chenz. 2.50, 8222-8227. 10. Rosenfeld. .I. L.. Dutta. S. P., Chheda. G. B., and Tritsch, G. L. (1975) Bioc,him. Bioph.vt. Ac,rc~ 410, 164- 166. I I. Schimke, R. T. (1964) .!. Rio/. Ckcm. 239, 380% 3817. I?. Bergmeyer, H. U. (1974) in Methods of Enzymatic Analysis, Vol. 4. pp. 1794-1798. Academic Press, New York. 13. Wilkinson, G. N. (1961) Biochern. J. 80, 324-332. 14. Pace. C. N. (1980) Trends Eiochrnr. Sci. 5.9910. IS. Hirsch-Kolb, H., Heine. J. P., Kolb, H. J.. and Greenberg, D. M. t 1970) Con~p. Bioc,hem. Phy.tio/. 37, 345-359. 16. Hirsch-Kolb. H.. Kolb, H. J.. and Greenberg. D. M. (197l)J. Viol. Chcnr. 246, 395-401. 17. Campbell, J. W. (1966) Camp. Biochcm. Physird. 18, l79- 199. 18. Thiers. R. E., and Vallee. B. L. (1957) J. Bid. Chern. 226, 91 l-920. 19. Scrutton, M. C.. Utter. M. F.. and Mildvan. A. S. (1966) J. Bird. C‘henr. 241, 3480-3487. 20. Musznska. G., and Ber. E. (1979). frrr. .I. Hi+ chen1. 9, 757-759. 21. Munakata. M.. Niina. S.. and Ueda, I. (1976) Rioinorgnn. C‘hern. 6. l33- 143. 22. Clarke, E. R.. and Mat-tell, A. E. (1970) .I. Inrq. Nut,/. Chrm. 32, 91 l-926. 23. Cohn. E. J.. and Edsall. J. T. (1943) Proteins, Amino Acids, and Peptides, p. 85, Hafner, New York. 34. Greenberg. D. M. (1960) in The Enzymes (Bayer, P. D., Lardy, H.. and Myrback. K.. eds.). 2nd rd.. Vol. 4. pp. 257-367. Academic Press. New York. 25. Cleland, W. W. (1967)Adr,un. Enynrol. 29, l-33. 26. Schimke. R. T. ( 196?).1. Viol. Clren). 237,459-468. 27. Vielle-Breitburd. F., and Orth. G. (1972) J. Biol. C‘hrrn. 247, 1327- 1’3.5.
BIOCHEMISTRY
Steady-State
C. NICK
109,
Kinetic
261-265
(1980)
Studies of Arginase with an Improved Spectrophotometric Assay’
PACE, ANDRES
BUONANNO,
Received A direct spectrophotometric t 1967. At~crl. Biochem. 18, 102) substrate concentrations, for kinetic studies of the enzyme. reported and compared with number of problems encountered
AND
was supported by Grant Institutes of Health.
SIMMONS-HANSEN
July 7. 1980
assay for arginase developed by R. L. Ward and P. A. Srere is extended so that it can be used at pH 7.5 or 9.5. at higher assaying crude sources of arginase. and for steady-state Steady-state kinetic studies of beef and rat liver arginase are Gmilar results obtained using other assay procedures. A in steady-state kinetic studies of argina\e are discussed.
Arginase (t-arginine amidinohydrolase, EC 3.5.3. I) catalyzes the cleavage of arginine to ornithine and urea. A surprisingly large number of techniques have been developed for assaying arginase (see ( 1) and references therein). The most popular of these measures urea formation and requires a 60-min boiling step (2). In addition, the product is light labile and requires special precautions during boiling and cooling (3). This inconvenience coupled with the fact that arginase generally requires prior activation by heating (4) result in a tedious assay of limited precision. The wide range of Michaelis constants reported for rat and beef liver arginase (0.3 to over 20 mM). and the diversity of conclusions about the role of Mn’+ in the activation of the enzyme are, in part, reflections of the assays available. In 1967, Ward and Srere (5) described a direct, spectrophotometric assay for arginase based on the difference in absorption between the products and reactants at 205.7 nm. This approach is much simpler and faster than the indirect assays and has since been used by several investigators (6-10). We have extended this method so that it can be used at higher ’ This research from the National
JEANNIE
Direct
AM 191 12-02
substrate concentrations, for assaying crude samples of arginase, and at either pH 7.5 of 9.5. We show that the method can be used for steady-state kinetic studies of the enzyme and report Michaelis constants determined for the rat and beef liver enzymes under various conditions. MATERIALS
AND METHODS
Partially purified beef liver arginase (33.3 unitsimg) was purchased from Worthington Biochemical Company (Lot AR36C869). Schimke’s procedure ( 11) was used for purifying rat liver arginase. The specific activity of the final product ranged from 650 to 1050 unitsimg in different purifications. Enzymes and chemicals for the coupled-enzyme assay were purchased from Boehringer-Mannheim. For the data in Fig. I. the A grade hydrochlorides of arginine and ornithine from Calbiochem. and “ultrapure” urea from SchwarziMann were used. All other chemicals were the best grade available from Sigma Chemical Company. The absorption spectra and some of the initial velocities were measured with a Cary Model 15 spectrophotometer. Other initial velocities were recorded with a Gilford Model 250 spectrophotometer. The
262
PACE.
BUONANNO.
Wavelength FIG. and and
I.
Absorption
spectra
for
urea at pH 9.5. E = Molar k = tArC ~ Ed,,,, - t,,,-, .,,.
arginine.
absorption
ornithine. coefficient.
Archibald method (2) and the coupledenzyme method ( 12) were used for measuring some of the initial velocities in Fig. 2. For the experiments in Fig. 2 and in Table 2. the arginase was dialyzed against 0.05 or 5 IIIM MnCl,, 10 mM Tris-HCl, pH 7. for 4 to 6 h at 4°C and then activated by heating for 1 hat 37°C. Identical results are obtained when the heating step is omitted. The reaction is initiated by adding 0.1 ml of the arginase to 2.5 ml of a substrate solution at either pH 7.5 or 9.5: consequently, the MP concentrations will be only 0.0019 or 0.19 mM when the velocity is measured. RESULTS The absorption spectra of arginine, ornithine, and urea at pH 9.5 are shown in Fig. 1. The difference absorption coefficient, Ae = E.~,, - Ed,,, ~ t,,,,;,, is also shown and can be seen to increase sharply below 215 nm. Consequently, the absorbance change accompanying the conversion of arginine to
AND
HANSEN
ornithine plus urea will be maximized by using the lowest possible wavelength. At pH 7.5 similar results are obtained but AC and the molar absorption coefficients for arginine and ornithine are reduced. The presence of I mM Mn” does not affect the he values at pH 7.5. Molar absorption coefficients for arginine and the k values at pH 7.5 and 9.5 are listed in Table I. In a typical assay of arginase. we use 2.5 ml of an I -arginine solution with a concentration in the range from 0. I to I5 KIM. Arginase (0.5 to 3.5 units) is added to this solution to initiate the reaction and the absorbance is recorded. The rate of absorbance change can be determined by drawing a tangent to the progress curve at the beginning of the reaction. Initial velocities in terms of concentrations can then be calculated from these rates using the ALE values in Table 1. With care, velocities can be determined with a precision of better than +5% over a wide range of substrate concentrations. Excellent agreement is obtained when different wavelengths are used for measuring the velocities. A wavelength ‘I‘ABLk MOLAR ANI)
ABSoRPrlON DII I t RFN(‘~ ‘,I
I
CO1.l I 1c1kh ABSORPTION pH pH
9.5
‘.NI)
15 I OR AR(~INI~I Corl~t IC II VI\ 7.5
7.5
pH
Y.5
Wavelength (nm) 215 214 213 217 211 110 7OY
42 hl
I34 177
85 123 I74 136 344 477
74 I Y71 I x4
205 104
I’ At
7’) 103
2 35 314 423 564
108 107 106
” hf-’
Art”
t”
643 X63 I I41 1478
IhI5 cm t\rc
‘. t
t,,n ‘8
t’l
At”
340
YX
27x 334
I I7 155
396 47’) SY3 734
IYY Xl 352 46X 6’0
YI II40 1471
X16 I Oh2
I754 7193
1361 17%
ARGINASE
263
ASSAY
c : I E
0.5
0
0.2
0.1
v/S
0.3
Imln-‘I
FIG. 2. Eadie-Hofstee plots for the hydrolysis of L-arginine by beef liver arginase. Initial velocities determined by Archibald assay at pH 7.0. 37°C (0): by the direct assay at pH 9.5. 25T (13): or by the coupled-enzyme assay at pH 9.5. 37°C (a).
must be selected which gives an absorbance in the usable range of the spectrophotometer (generally less than 3.0) when the substrate and enzyme are mixed. The data in Table 1 can be used to calculate the absorbance of the substrate solution; the absorbance due to the enzyme solution depends on the source of the enzyme and must be determined independently. As pointed out by Ward and Srere (5). this assay can be used with crude samples of arginase and gives results in excellent agreement with the Archibald procedure (2) when used to monitor the purification of arginase. It is common practice to maintain a high Mn’+ concentration (50 mM) during the purification of arginase 14). This creates a problem with the direct assay not previously noted. Since the pH optimum for arginase is near pH 9.5, the enzyme is generally assayed by adding a small aliquot of the enzyme solution at pH 7-7.5 to a solution of the substrate at pH 9.5. If 50 mM Mn”+ is present in the enzyme solution, there will be a sizable increase in the absorbance resulting from the exposure of Mn2+ to pH 9.5. This absorbance change will diminish the decrease in absorbance due to the hydrolysis
of arginine and lead to erroneous results. This problem can be avoided by assaying the arginase at pH 7.5. or minimized by maintaining the Mn”+ concentration of the arginase solution at 5 mM or less. The direct assay is particularly convenient for obtaining initial velocities for steadystate kinetic studies of arginase. Some results obtained using the direct assay are compared with results obtained using the Archibald method (2) and the coupledenzyme assay ( 12) in Eadie-Hofstee plots in Fig. 2. The data from the direct assay yield a Michaelis constant of 1.3 mM, while values of 1.6 and 1.4 mM are obtained from the Archibald and coupled-enzyme assays. respectively. We have also used the direct assay to determine the Michaelis constants for L-arginine with purified rat liver arginase. Initial velocities were measured at substrate concentrations ranging from 0.15 to 5.0 mM and the Michaelis constant and standard errors were estimated as described by Wilkinson (13). Corrections were made for substrate depletion (14) and corrections for product inhibition were shown to be insignificant. The results are presented in
264
PACE, TABLE
THE
MICHAELIS L-ARGININE
BUONANNO.
2
CoNsrAN r COR THY HYDROLYSIS BY RAI LIVER ARGINASI ”
ok
K,,, (mhil M”‘/ (mM)
pH 7.5
pH 9.5
0.05 .s
I.55 ? 0.16 1.33 t 0.13
1.03 + 0.19 1.13 -t 0.13
” Km values Wilkinson (13).
? SE
estimated
as
described
by
Table 2. For reasons discussed below, estimates of K,,, obtained using the direct assay are generally lower than estimates obtained using other assays. DISCUSSION Michaelis constants ranging from 0.3 ( 10) to 20 mM (15) have been reported for the hydrolysis of arginine by beef and rat liver arginase. Several factors which may contribute to this poor agreement are considered below. The evidence is good that both beef and rat liver arginase bind 1 mol of Mn” per subunit (7.16) and that the enzyme can be activated by heating in the presence of a high concentration of Mn” + ( IZ- 17). typically 5 min at 55°C in the presence of 50 mM Mn’)+ (4). It is less certain, however, that the resulting enzyme resembles arginase as it exists in \lit*o. The total concentration of Mn’+ in rat liver is approximately 0.024 mM ( I .3 pgig liver) (18) and the free Mn’+ concentration, considerably less (19). Thus, distinctly nonphysiological conditions are used to activate arginase and there are substantial discrepancies among published results. (Compare, for example, the results in ( 16) and (20) for rat liver arginase.) Furthermore. high Mn” concentrations interfere with both the direct (see Results) and Archibald (21) assays. Finally. Mn”+ binds to zwitterionic arginine with an association constant of 335 Mm’ (22) and can thereby
AND
HANSEN
reduce the free substrate concentration. For these reasons, we recommend the use of lower Mn’+ concentrations and lower temperatures for the activation of arginase. The results in Table 2 show that very similar results are obtained when arginase is activated at 37°C in the presence of 0.05 or 5 mM Mn’+. The pH optima for beef and rat liver arginase are between pH 9.0 and 10.5 ( 15). and the enzymes have been assayed at several different pH values in this range. This complicates kinetic studies of arginase. The pk’ of the cu-amino group of arginine is 9.04 (23) or 9.36 (22). Consequently, the relative concentration of monocationic and zwitterionic arginine will vary depending on the pH used for the assay and it is not established with certainty which is the better substrate (24). In addition. the degree of complex formation between Mn?’ and arginine will increase with increasing pH. and, finally. Mn’+ solutions turn brown when exposed to high pH. Thus, even though the activity of arginase is lower, much more consistent results can be obtained when kinetic studies are carried out at physiological pH. Failure to correct for substrate depletion can substantially increase the K,,, value determined from a steady-state kinetic study (14). For example, in a typical kinetic study of arginase using the Archibald assay and a 5-min incubation of substrate and enzyme, calculations show that the K,,, would be over 25% greater if no correction were made for substrate depletion. Since 5and IO-min incubations are commonly used in kinetic studies of arginase, this factor probably contributes to the wide range ofk’,,, values observed. A related problem is the substrate concentration range employed in a steady-state kinetic study. Cleland recommends a range of substrate concentrations from 0.2 to 5 K,,, for determining K,,, (25). Rosenfeld cf trl. ( 10) report K,,, = 0.3-0.4 mM for beef liver arginase but use only a 0.5 to 1.O mM range
ARCINASE
of arginine
concentrations.
Hirsch-Kolb
rt (11. (15). on the other hand, report K,,,
values of 7 and 20 mM for beef and rat liver arginase, respectively, and use a substrate concentration range from 5 to 100 mM arginine. The use of an inappropriate range of substrate concentration is likely to contribute to errors in the K,,, values. The values of K,,, we report are in best agreement with values of 1.0 mM for beef (7). 2.4 mM for rat (26), and 1.4 tItM for rabbit (27) liver arginase. Most of the k’,,, values reported for liver arginases are considerably higher than these values, probably, in part, for reasons discussed above. In summary, the information presented here will allow arginase to be assayed under a wider range of conditions and should prove useful for steady-state kinetic studies of the enzyme. REFERENCES I. Ruegg. U. T.. and Russell. A. S. (1980) And/. Eiochem. 102, 206-212. 2. Archibald. R. M. (1945) J. Biol. Ch~nr. 157, 507517. 3. Van Slyke. D. D.. and Archibald, R. M. (1946) J. Bid. C‘hem. 165, 293-309. 4. Schimke. R. T. (1970) in Methods Enzymology (Tabor. H., and Tabor. C. W.. eds.). Vol. l7A, pp. 313-317. Academic Press, New York. 5. Ward, R. L.. and Srere. P. A. (1967) ,4no/. Riothem. 18, IOZ- 106. 6. Hirsch-Kolb. H.. and Greenberg. D. M. (1968) J. Bic~f. C-hem. 243, 6123-6129. 7. Harell, D.. and Sokolovsky. M. (1972) Eur. J. Bioc~hrm. 25, 102-108.
ASSAY
365
8. Kaysen. G. A., and Strecker, H. J. (1973) Bioc~hertl. J. 133, 779-788. 9. Kuchel. P. W.. Nichol, L. W.. and Jeffrey. P. D. (1975) J. Bird. Chenz. 2.50, 8222-8227. 10. Rosenfeld. .I. L.. Dutta. S. P., Chheda. G. B., and Tritsch, G. L. (1975) Bioc,him. Bioph.vt. Ac,rc~ 410, 164- 166. I I. Schimke, R. T. (1964) .!. Rio/. Ckcm. 239, 380% 3817. I?. Bergmeyer, H. U. (1974) in Methods of Enzymatic Analysis, Vol. 4. pp. 1794-1798. Academic Press, New York. 13. Wilkinson, G. N. (1961) Biochern. J. 80, 324-332. 14. Pace. C. N. (1980) Trends Eiochrnr. Sci. 5.9910. IS. Hirsch-Kolb, H., Heine. J. P., Kolb, H. J.. and Greenberg, D. M. t 1970) Con~p. Bioc,hem. Phy.tio/. 37, 345-359. 16. Hirsch-Kolb. H.. Kolb, H. J.. and Greenberg. D. M. (197l)J. Viol. Chcnr. 246, 395-401. 17. Campbell, J. W. (1966) Camp. Biochcm. Physird. 18, l79- 199. 18. Thiers. R. E., and Vallee. B. L. (1957) J. Bid. Chern. 226, 91 l-920. 19. Scrutton, M. C.. Utter. M. F.. and Mildvan. A. S. (1966) J. Bird. C‘henr. 241, 3480-3487. 20. Musznska. G., and Ber. E. (1979). frrr. .I. Hi+ chen1. 9, 757-759. 21. Munakata. M.. Niina. S.. and Ueda, I. (1976) Rioinorgnn. C‘hern. 6. l33- 143. 22. Clarke, E. R.. and Mat-tell, A. E. (1970) .I. Inrq. Nut,/. Chrm. 32, 91 l-926. 23. Cohn. E. J.. and Edsall. J. T. (1943) Proteins, Amino Acids, and Peptides, p. 85, Hafner, New York. 34. Greenberg. D. M. (1960) in The Enzymes (Bayer, P. D., Lardy, H.. and Myrback. K.. eds.). 2nd rd.. Vol. 4. pp. 257-367. Academic Press. New York. 25. Cleland, W. W. (1967)Adr,un. Enynrol. 29, l-33. 26. Schimke. R. T. ( 196?).1. Viol. Clren). 237,459-468. 27. Vielle-Breitburd. F., and Orth. G. (1972) J. Biol. C‘hrrn. 247, 1327- 1’3.5.