- Email: [email protected]
Stability Properties of Calf Intestinal Alkaline Phosphatase
Stability and Stabilization of Biocatalysts A. Ballesteros, F.J. Plou, J.L. Iborra and P.J. Hailing (Editors) 9 1998 Elsevier Science B.V. All rights reserved.
47
Stability Properties o f C a l f Intestinal Alkaline P h o s p h a t a s e Ultan McKEON, Brendan O'CONNOR & Ciarhn 0'F/kG.~qq" School of Biological Sciences, Dublin City University, Dublin 9, Ireland.
Inactivation of alkaline phosphatase (APase) in the range 48-65~ fits a first order decay; an Arrhenius plot gives estimates of low-temperature half-lives. APase tolerates urea but not GdC1. Micromolar amounts EDTA lead to inactivation, as do millimolar amounts 1,10phenanthroline. APase is sensitive to SDS but retains significant activity in presence of the solvents DMSO, DMF or THF (up to 60% (v/v) in buffer).
1. INTRODUCTION Alkaline phosphatase (Orthophosphate monoester phosphohydrolase; EC 3.1.3.1; APase) is used to prevent self-annealing of DNA by cleavage of 3'-terminal phosphate groups (1). Its ability to utilize luminescent substrates has led to its use as a reporter enzyme in nonradioactive DNA probes (2) and in sensitive enzyme immunoassays (3). Its levels in serum can be diagnostic in clinical chemistry (4) and it is included in commercial control sera. Long-term persistence of enzyme activity can be a critical parameter in these various types of diagnostic or molecular biology products. It would be useful to have some indication of the baseline stability characteristics of APase and of its ability to withstand various kinds of inactivating influences. The tetrameric, zinc-containing calf intestinal APase (5) is widely used. Here we explore the intrinsic stability properties and limitations of this enzyme and we report on its tolerances of heat, denaturants, chelating agents, detergent and water-miscible organic solvents.
2. EXPERIMENTAL Boehringer Mannheim calf intestinal APase (product # 108 138) was diluted 1/1500 in 2amino-2-methyl-1-propanol/metal ion buffer (6) for use and assayed by the method of IFCC [intemational Federation of Clinical Chemistry] (6). 4-nitrophenyl phosphate and other assay reagents were from Sigma, as were urea, guanidine chloride [GdC1], EDTA, 1,10phenanthroline and SDS. Aldrich supplied 1,7- and 4,7-phenanthrolines.
"Author for correspondence.Tel +35317045288; Fax +35317045412; Email [email protected]
48 Dimethylsulphoxide [DMSO] was from BDH while acetone, dimethylformamide [DMF] and tetrahydrofuran [THF] were from Labsean (Dublin, Ireland). For the temperature profile, aliquots of APase were placed (after preheating to 35-40~ for 10 min at temperatures in the range 35-70~ then removed onto ice to prevent further denaturation. Residual activity was later determined under optimal conditions. APase was heated continuously at 48~ 50~ 55~ 60~ and 650C to obtain thermoinactivation curves. Residual activities of samples, removed at intervals and stored on ice, were assayed. An exponential decay curve of remaining activity against time was plotted to estimate the first order rate constant, k, at each temperature. Urea and GdC1 stock solutions (5M) were prepared according to (7), but using Tris, pH 8.3, instead of MOPS buffer. Incubation of the enzyme in the denaturant took place at 30~ for 2 h, after which time enzyme activity was determined. The effects of EDTA (pH 8.3), 1,7-phenanthroline, 1,10-phenanthroline and 4,7phenanthroline on activity were determined. After incubation at 370C for 4h, samples were extensively dialysed (40C) against 10mM Tris-HC1, pH 8.3. Activities were then measured. Enzyme was treated with various v/v concentrations of THF, DMF and DMSO and assayed after 90 min at room temperature. APase was treated with various w/v concentrations of SDS. After 2h at 30~ catalytic activity was measured.
3. RESULTS & DISCUSSION
Thermostability Studies. The temperature profile experiment gave 57~ as halfinactivation temperature, Tso. All thermoinactivation curves in the range 48~176 fitted a first order decay curve and gave linear semi-log replots. Values for the first-order rate constant, k, and half lives (t~t2= 0.693/k) are shown in Table 1. Table 1 Values of first-order rate constant, k, for APase thermoinactivation Temperature
48~
50~
55~
60~
65~
k, min"
0.021+ 0.001
0.028+ 0.001
0.063+ 0.002
0.143+ 0.008
0.30+ 0.04
An Arrhenius plot of these data yielded a value of 142 kJ.mol-! for the inactivating event under the conditions used. Extrapolation of the Arrhenius plot permits estimation of k (and of likely half lives) at temperatures of interest - see Table 2. Note that the value for 0~ takes no account of possible freezing effects. The values obtained under our particular experimental conditions may not be generally applicable. Franks (8) has discussed strengths and weaknesses of accelerated stability testing.
49 Table 2 APase half-lives estimated from Arrhenius plot. Temperature
0~
4~
20~
25~
37~
Half-life, h
6360
2568
88.8
33.6
3.6
Half-life, days
265
107
3.7
1.4
0.15
The DNA dephosphorylation protocol of Maniatis et al. (1) used calf intestinal APase for 1 h at 37~ followed by inactivation at 68~ for 15 min in SDS. The present results indicate that the enzyme remains active throughout the 37~ incubation and will quickly undergo inactivation at 68~ In the measurement of PCR products, Yang et al. (9) incubated APase at 37 ~ for 30 min in an enzyme-substrate reaction. Nakagami et al. (10) used APase in an enzyme-conjugated DNA probe system; they carried out hybridization of probe with target DNA at 42~ and found that a 30 min hybridization period gave the greatest sensitivity, with decreased sensitivities at longer times. These protocols appear to make good use of the intrinsic thermal stability characteristics of APase. de la Fourniere and colleagues (11) studied thermal and pH stabilities of bovine intestinal APase by Fourier transform infrared spectroscopy [FTIR]. At temperatures up to 70~ (or at pH values down to 5.4), activity loss was not accompanied by significant FTIR changes; i.e. there were no notable conformational alterations. At 80~ (or at pH 3.4), however, APase began to unfold after it had completely lost activity. FTIR results were confirmed by H/2H exchange and circular dichroism. Denaturant Stability. Fig. 1 shows the effects of increasing concentrations of urea and GdCI on APase activity. Urea up to 3M exerts a slight activation effect and approximately 80% activity is retained even at 5M urea. GdC1 has a similar activating effect up to 0.5M but APase activity declines steeply at GdC1 concentrations above 0.5M and is almost absent at 2M GdC1. GdC1 is a more potent denaturant than urea (7). At low concentrations, both denaturants slightly activate APase, perhaps by altering its conformation slightly to favour catalysis. 0.1M GdC1 caused tertiary structural changes in the monomeric horseradish peroxidase without affecting activity (12). Nakagami et al. (10) noted that APase retained 40% activity after 3 h in 6M urea. Effect of EDTA and Phenanthrolines. Micromolar EDTA concentrations have a potent adverse effect on APase (Fig. 2), as one would expect for a Zn-containing enzyme. This is most likely due to chelation of the catalytically-essential Zn 2§ ion and is confirmed by Fig. 3, which shows the effects of phenanthrolines on APase activity. The chelating 1,10phenanthroline adversely affects APase (but at millimolar concentrations) while the nonchelating 4,7- and 1,7-phenanthrolines (13) have little or no effect. Note that exhaustive dialysis of EDTA-containing samples took place prior to assay to prevent EDTA interference in the assay mix. Femley (5) has stated that the kinetics of EDTA inhibition are complicated and depend on both time and substrate. Satoh (14) has used the immobilized apoenzyme [Zn-free APase] for microanalysis of Zn(II) ions. This system could be used at least 120 times; treatment with a chelating agent regenerated the reactor following each measurement. Maniatis et al. (1) used EDTA together with high temperature to terminate the dephosphorylation reaction.
50 140 120 100 9.,..4
p. 80
0
<
60
.,.,.1
o
GdCI
o
Urea
l,.,,,d
40 20 I
I--
1
2
I
I
I
3
4
5
C oncentration(M
)
Fig. 1. Effects of denaturant on APase activity.
100
p.
80
0
< r .!.=,
60
40
:
"--....
20 0
10
20
30
EDTA(~tM)
Fig. 2. Effects ofEDTA on APasr activity.
40
50
60
70
51 120 100
80
~-
e A
0
<
t~
1,10 Phenanthroline 4,7 P h e n a n t h r o l i n e
60
".=. 40
0
5
10
15
C oncentration(m
20
M )
Fig. 3. Effects of Phenantrolines on APase activity.
120
9-~
80
<
60
A m
,ab
100
o.,,t
,..,
o
40
THF
m
DMF
A
20
DMSO
A
0
20
40
%
60
v/v
Fig. 4. Effects of Solvents on APase activity.
solvent
80
100
52
Effect of Solvents. APase showed great tolerance of the solvents DMF, DMSO and THF as their concentrations (% v/v) increased (Fig. 4). APase completely withstands up to 60% (v/v) DMSO (with a slight activation effect); above 63%, activity declines in a threshold manner and is absent at 66%. Similarly, APase is completely tolerant of DMF up to 66% (v/v). Here also, a steep threshold effect occurs and activity is virtually absent at 80% DMF. THF has no effect on activity up to 82% (v/v) and approximately 30% activity is retained in 100% THF. This last result is remarkable in view of THF's high denaturation capacity value for enzymes (15). Effect of SDS. Low SDS concentrations adversely affect APase; however, more than 50% of initial activity remains at 1% (w/v) SDS and approximately 30% activity is retained at 10% (w/v). Clearly, APase retains significant activity at the 0.5% level of SDS used by Nakagami et al. (10) in their prehybridization mixture. This study helps to define tolerable storage and reaction conditions for native APase; use beyond these limits will likely require the implementation of one or more stabilization strategies (reviewed in 16). Boivin et al. (17) have described the stabilization of calf intestinal APase by immobilization and chemical modification. Acknowledgement. We thank Mr Damien O'Brien for help in preparing this manuscript.
REFERENCES 1. Maniatis T, Fritsch EF, Sambrook J. Molecular Cloning: a Laboratory Manual. Cold Spring Harbour, NY: Cold Spring Harbour Laboratory, 1982: 133-134. 2. Harris MR. Biotechnol Adv 1991; 9:185-196. 3. Bronstein I, Voyta JC, Thorpe GH et al. Clin Chem 1989; 35:144 l- 1446. 4. Gray CH. Clinical Chemical Pathology, 7th edn. London: Edward Arnold, 1974:110. 5. Femley HN. In: Boyer PD, ed. The Enzymes vol 4. NY: Academic Press, 1971: 417-447. 6. Tietz NW, Rinker AD, Shaw LM. J Clin Chem Clin Biochem 1983; 21:731-748. 7. Pace CN, Shirley BA, Thompson A. In: Creighton TE, ed. Protein Structure: a Practical Approach. Oxford: IRL Press, 1989:311-329. 8. Franks F. Trends Biotechnol 1994; 12" 114-117. 9. Yang B, Viscidi R, Yolken R. Anal Bioehem 1993; 213" 422-425. 10. Nakagami S, Matsunaga H, Oka Net al. Anal Bioehem 1991; 198" 75-79. 11. de la Foumiere L, Nosjean O, Buchet R et al. Bioehim Biophys Acta 1995; 1248: 186192. 12. Chakrabarti A, Basak S. Eur J Biochem 1996; 241" 462-467. 13. Czekay C, Bauer K. Biochem J 1993; 290: 921-926. 14. Satoh I. Biosensors Bioelectron 1991; 6: 375-379. 15. Khmelnitsky YuL, Mozhaev VV, Belova ABet al. Eur J Biochem 1991; 198: 31-41. 16. O F~gain C. Stabilizing Protein Function. Berlin: Springer Verlag, 1997. 17. Boivin P, Kobos RK, Papa SL et al. Biotechnol Appl Biochem 1991; 14: 155-169.
47
Stability Properties o f C a l f Intestinal Alkaline P h o s p h a t a s e Ultan McKEON, Brendan O'CONNOR & Ciarhn 0'F/kG.~qq" School of Biological Sciences, Dublin City University, Dublin 9, Ireland.
Inactivation of alkaline phosphatase (APase) in the range 48-65~ fits a first order decay; an Arrhenius plot gives estimates of low-temperature half-lives. APase tolerates urea but not GdC1. Micromolar amounts EDTA lead to inactivation, as do millimolar amounts 1,10phenanthroline. APase is sensitive to SDS but retains significant activity in presence of the solvents DMSO, DMF or THF (up to 60% (v/v) in buffer).
1. INTRODUCTION Alkaline phosphatase (Orthophosphate monoester phosphohydrolase; EC 3.1.3.1; APase) is used to prevent self-annealing of DNA by cleavage of 3'-terminal phosphate groups (1). Its ability to utilize luminescent substrates has led to its use as a reporter enzyme in nonradioactive DNA probes (2) and in sensitive enzyme immunoassays (3). Its levels in serum can be diagnostic in clinical chemistry (4) and it is included in commercial control sera. Long-term persistence of enzyme activity can be a critical parameter in these various types of diagnostic or molecular biology products. It would be useful to have some indication of the baseline stability characteristics of APase and of its ability to withstand various kinds of inactivating influences. The tetrameric, zinc-containing calf intestinal APase (5) is widely used. Here we explore the intrinsic stability properties and limitations of this enzyme and we report on its tolerances of heat, denaturants, chelating agents, detergent and water-miscible organic solvents.
2. EXPERIMENTAL Boehringer Mannheim calf intestinal APase (product # 108 138) was diluted 1/1500 in 2amino-2-methyl-1-propanol/metal ion buffer (6) for use and assayed by the method of IFCC [intemational Federation of Clinical Chemistry] (6). 4-nitrophenyl phosphate and other assay reagents were from Sigma, as were urea, guanidine chloride [GdC1], EDTA, 1,10phenanthroline and SDS. Aldrich supplied 1,7- and 4,7-phenanthrolines.
"Author for correspondence.Tel +35317045288; Fax +35317045412; Email [email protected]
48 Dimethylsulphoxide [DMSO] was from BDH while acetone, dimethylformamide [DMF] and tetrahydrofuran [THF] were from Labsean (Dublin, Ireland). For the temperature profile, aliquots of APase were placed (after preheating to 35-40~ for 10 min at temperatures in the range 35-70~ then removed onto ice to prevent further denaturation. Residual activity was later determined under optimal conditions. APase was heated continuously at 48~ 50~ 55~ 60~ and 650C to obtain thermoinactivation curves. Residual activities of samples, removed at intervals and stored on ice, were assayed. An exponential decay curve of remaining activity against time was plotted to estimate the first order rate constant, k, at each temperature. Urea and GdC1 stock solutions (5M) were prepared according to (7), but using Tris, pH 8.3, instead of MOPS buffer. Incubation of the enzyme in the denaturant took place at 30~ for 2 h, after which time enzyme activity was determined. The effects of EDTA (pH 8.3), 1,7-phenanthroline, 1,10-phenanthroline and 4,7phenanthroline on activity were determined. After incubation at 370C for 4h, samples were extensively dialysed (40C) against 10mM Tris-HC1, pH 8.3. Activities were then measured. Enzyme was treated with various v/v concentrations of THF, DMF and DMSO and assayed after 90 min at room temperature. APase was treated with various w/v concentrations of SDS. After 2h at 30~ catalytic activity was measured.
3. RESULTS & DISCUSSION
Thermostability Studies. The temperature profile experiment gave 57~ as halfinactivation temperature, Tso. All thermoinactivation curves in the range 48~176 fitted a first order decay curve and gave linear semi-log replots. Values for the first-order rate constant, k, and half lives (t~t2= 0.693/k) are shown in Table 1. Table 1 Values of first-order rate constant, k, for APase thermoinactivation Temperature
48~
50~
55~
60~
65~
k, min"
0.021+ 0.001
0.028+ 0.001
0.063+ 0.002
0.143+ 0.008
0.30+ 0.04
An Arrhenius plot of these data yielded a value of 142 kJ.mol-! for the inactivating event under the conditions used. Extrapolation of the Arrhenius plot permits estimation of k (and of likely half lives) at temperatures of interest - see Table 2. Note that the value for 0~ takes no account of possible freezing effects. The values obtained under our particular experimental conditions may not be generally applicable. Franks (8) has discussed strengths and weaknesses of accelerated stability testing.
49 Table 2 APase half-lives estimated from Arrhenius plot. Temperature
0~
4~
20~
25~
37~
Half-life, h
6360
2568
88.8
33.6
3.6
Half-life, days
265
107
3.7
1.4
0.15
The DNA dephosphorylation protocol of Maniatis et al. (1) used calf intestinal APase for 1 h at 37~ followed by inactivation at 68~ for 15 min in SDS. The present results indicate that the enzyme remains active throughout the 37~ incubation and will quickly undergo inactivation at 68~ In the measurement of PCR products, Yang et al. (9) incubated APase at 37 ~ for 30 min in an enzyme-substrate reaction. Nakagami et al. (10) used APase in an enzyme-conjugated DNA probe system; they carried out hybridization of probe with target DNA at 42~ and found that a 30 min hybridization period gave the greatest sensitivity, with decreased sensitivities at longer times. These protocols appear to make good use of the intrinsic thermal stability characteristics of APase. de la Fourniere and colleagues (11) studied thermal and pH stabilities of bovine intestinal APase by Fourier transform infrared spectroscopy [FTIR]. At temperatures up to 70~ (or at pH values down to 5.4), activity loss was not accompanied by significant FTIR changes; i.e. there were no notable conformational alterations. At 80~ (or at pH 3.4), however, APase began to unfold after it had completely lost activity. FTIR results were confirmed by H/2H exchange and circular dichroism. Denaturant Stability. Fig. 1 shows the effects of increasing concentrations of urea and GdCI on APase activity. Urea up to 3M exerts a slight activation effect and approximately 80% activity is retained even at 5M urea. GdC1 has a similar activating effect up to 0.5M but APase activity declines steeply at GdC1 concentrations above 0.5M and is almost absent at 2M GdC1. GdC1 is a more potent denaturant than urea (7). At low concentrations, both denaturants slightly activate APase, perhaps by altering its conformation slightly to favour catalysis. 0.1M GdC1 caused tertiary structural changes in the monomeric horseradish peroxidase without affecting activity (12). Nakagami et al. (10) noted that APase retained 40% activity after 3 h in 6M urea. Effect of EDTA and Phenanthrolines. Micromolar EDTA concentrations have a potent adverse effect on APase (Fig. 2), as one would expect for a Zn-containing enzyme. This is most likely due to chelation of the catalytically-essential Zn 2§ ion and is confirmed by Fig. 3, which shows the effects of phenanthrolines on APase activity. The chelating 1,10phenanthroline adversely affects APase (but at millimolar concentrations) while the nonchelating 4,7- and 1,7-phenanthrolines (13) have little or no effect. Note that exhaustive dialysis of EDTA-containing samples took place prior to assay to prevent EDTA interference in the assay mix. Femley (5) has stated that the kinetics of EDTA inhibition are complicated and depend on both time and substrate. Satoh (14) has used the immobilized apoenzyme [Zn-free APase] for microanalysis of Zn(II) ions. This system could be used at least 120 times; treatment with a chelating agent regenerated the reactor following each measurement. Maniatis et al. (1) used EDTA together with high temperature to terminate the dephosphorylation reaction.
50 140 120 100 9.,..4
p. 80
0
<
60
.,.,.1
o
GdCI
o
Urea
l,.,,,d
40 20 I
I--
1
2
I
I
I
3
4
5
C oncentration(M
)
Fig. 1. Effects of denaturant on APase activity.
100
p.
80
0
< r .!.=,
60
40
:
"--....
20 0
10
20
30
EDTA(~tM)
Fig. 2. Effects ofEDTA on APasr activity.
40
50
60
70
51 120 100
80
~-
e A
0
<
t~
1,10 Phenanthroline 4,7 P h e n a n t h r o l i n e
60
".=. 40
0
5
10
15
C oncentration(m
20
M )
Fig. 3. Effects of Phenantrolines on APase activity.
120
9-~
80
<
60
A m
,ab
100
o.,,t
,..,
o
40
THF
m
DMF
A
20
DMSO
A
0
20
40
%
60
v/v
Fig. 4. Effects of Solvents on APase activity.
solvent
80
100
52
Effect of Solvents. APase showed great tolerance of the solvents DMF, DMSO and THF as their concentrations (% v/v) increased (Fig. 4). APase completely withstands up to 60% (v/v) DMSO (with a slight activation effect); above 63%, activity declines in a threshold manner and is absent at 66%. Similarly, APase is completely tolerant of DMF up to 66% (v/v). Here also, a steep threshold effect occurs and activity is virtually absent at 80% DMF. THF has no effect on activity up to 82% (v/v) and approximately 30% activity is retained in 100% THF. This last result is remarkable in view of THF's high denaturation capacity value for enzymes (15). Effect of SDS. Low SDS concentrations adversely affect APase; however, more than 50% of initial activity remains at 1% (w/v) SDS and approximately 30% activity is retained at 10% (w/v). Clearly, APase retains significant activity at the 0.5% level of SDS used by Nakagami et al. (10) in their prehybridization mixture. This study helps to define tolerable storage and reaction conditions for native APase; use beyond these limits will likely require the implementation of one or more stabilization strategies (reviewed in 16). Boivin et al. (17) have described the stabilization of calf intestinal APase by immobilization and chemical modification. Acknowledgement. We thank Mr Damien O'Brien for help in preparing this manuscript.
REFERENCES 1. Maniatis T, Fritsch EF, Sambrook J. Molecular Cloning: a Laboratory Manual. Cold Spring Harbour, NY: Cold Spring Harbour Laboratory, 1982: 133-134. 2. Harris MR. Biotechnol Adv 1991; 9:185-196. 3. Bronstein I, Voyta JC, Thorpe GH et al. Clin Chem 1989; 35:144 l- 1446. 4. Gray CH. Clinical Chemical Pathology, 7th edn. London: Edward Arnold, 1974:110. 5. Femley HN. In: Boyer PD, ed. The Enzymes vol 4. NY: Academic Press, 1971: 417-447. 6. Tietz NW, Rinker AD, Shaw LM. J Clin Chem Clin Biochem 1983; 21:731-748. 7. Pace CN, Shirley BA, Thompson A. In: Creighton TE, ed. Protein Structure: a Practical Approach. Oxford: IRL Press, 1989:311-329. 8. Franks F. Trends Biotechnol 1994; 12" 114-117. 9. Yang B, Viscidi R, Yolken R. Anal Bioehem 1993; 213" 422-425. 10. Nakagami S, Matsunaga H, Oka Net al. Anal Bioehem 1991; 198" 75-79. 11. de la Foumiere L, Nosjean O, Buchet R et al. Bioehim Biophys Acta 1995; 1248: 186192. 12. Chakrabarti A, Basak S. Eur J Biochem 1996; 241" 462-467. 13. Czekay C, Bauer K. Biochem J 1993; 290: 921-926. 14. Satoh I. Biosensors Bioelectron 1991; 6: 375-379. 15. Khmelnitsky YuL, Mozhaev VV, Belova ABet al. Eur J Biochem 1991; 198: 31-41. 16. O F~gain C. Stabilizing Protein Function. Berlin: Springer Verlag, 1997. 17. Boivin P, Kobos RK, Papa SL et al. Biotechnol Appl Biochem 1991; 14: 155-169.