ISSN 0036-0244, Russian Journal of Physical Chemistry A, 2009, Vol. 83, No. 2, pp. 318–323. © Pleiades Publishing, Ltd., 2009. Original Russian Text © L.F. Atyaksheva, B.N. Tarasevich, E.S. Chukhrai, O.M. Poltorak, 2009, published in Zhurnal Fizicheskoi Khimii, 2009, Vol. 83, No. 2, pp. 391–396.

BIOPHYSICAL CHEMISTRY

Thermal Inactivation of Alkali Phosphatases under Various Conditions L. F. Atyaksheva, B. N. Tarasevich, E. S. Chukhrai, and O. M. Poltorak† Faculty of Chemistry, Moscow State University, Moscow, Russia e-mail: [email protected] Received December 10, 2007

Abstract—The thermal inactivation of alkali phosphatases from bacteria Escherichia coli (ECAP), bovine intestines (bovine IAP), and chicken intestines (chicken IAP) was studied in different buffer solutions and in the solid state. The conclusion was made that these enzymes had maximum stability in the solid state, and, in a carbonate buffer solution, their activity decreased most rapidly. It was found that the bacterial enzyme was more stable than animal phosphatases. It was noted that, for ECAP, four intermediate stages preceded the loss of enzyme activity, and, for bovine and chicken IAPs, three intermediate stages were observed. The activation energy of thermal inactivation of ECAP over the range 25–70°C was determined to be 80 kJ/mol; it corresponded to the dissociation of active dimers into inactive monomers. Higher activation energies (~200 kJ/mol) observed at the initial stage of thermal inactivation of animal phosphatases resulted from the simultaneous loss of enzyme activity caused by dimer dissociation and denaturation. It was shown that the activation energy of denaturation of monomeric animal alkali phosphatases ranged from 330 to 380 kJ/mol depending on buffer media. It was concluded that the inactivation of solid samples of alkali phosphatases at 95°C was accompanied by an about twofold decrease in the content of β structures in protein molecules. DOI: 10.1134/S0036024409020307

INTRODUCTION Alkaline phosphatases (EC 3.1.3.1) are present in almost all living organisms [1]. According to the data of X-ray diffraction analysis, they are dimeric enzymes [2–5]. Catalytic activity is also typical of tetrameric forms of alkaline phosphatases, whereas their monomers are inactive. A kinetic analysis of the thermal inactivation of alkaline phosphatase from chicken intestines showed that the dissociative mechanism with hidden stages before activity loss could be one of the possible process mechanisms [6]. When this mechanism operates at the first stages of the process, stable protein complexes unable to dissociate transform into labile oligomers retaining catalytic activity and capable of dissociation. In this case, enzyme activity remains constant and the induction period is observed. At the next stage, labile oilgomers dissociate into subunits. At these stages, all processes are reversible; the denaturation of inactive subunit occurs at the last (irreversible) stage. For dimeric enzymes like alkaline phosphatases, the process can be represented as n

E2

kn k–n

…

k2 k–2

(Active forms)

E2

k1 k–1

2E 1

kd

2E d .

(1)

(Inactive forms)

Depending on enzyme properties and conditions of thermal inactivation, only part of the above scheme can † Deceased.

be realized. The goal of this work was to compare the basic rules governing the thermal inactivation of alkaline phosphatases of various origins to establish the general features of and differences between these processes and determine the number and conditions of the appearance of hidden stages not accompanied by the loss of enzyme catalytic activity. EXPERIMENTAL In experiments, lyophilized preparations of alkaline phosphatases from Escherichia coli (ECAP, Sigma, 50% protein), bovine intestines (bovine IAP, Sigma, 25% protein), and chicken intestines (chicken IAP, Reanal, 12% protein) were used. The protein content was determined by the Bradford method. The catalytic activity of alkaline phosphatases was estimated with 4-nitrophenylphosphate by spectrophotometrically recording the product of its hydrolysis, 4-nitrophenol, at 400 nm. Saturated solutions of the substrate in a tris-HCl buffer (pH 9.0, optimum catalytic activity) were used. Thermal inactivation was performed for solid enzyme samples and their solutions in the following buffer systems (pH 8.5): tris(oxymethyl)aminomethane–HCl (tris), NaHCO3–Na2CO3 (carbonate), and Na4B4O7–HCl (borate). Enzyme concentrations in solution ranged from 0.01 to 1 mg/ml. The solution was placed into a thermostat at the required temperature,

318

THERMAL INACTIVATION OF ALKALI PHOSPHATASES UNDER VARIOUS CONDITIONS V/V0 1.0

V/V0 1.0

0.8

0.8 1

0.6

0.6

0.4

0.4

0.2

0

1

1

0.2

2

3

2

3

4

5

6 τ, h

0

Fig. 1. Kinetic curves of thermal inactivation of alkali phosphatases from (1) bacteria E. coli, (2) bovine intestines, and (3) chicken intestines in carbonate buffer solution, pH 8.5, at 50°ë.

1

2

3

4 τ, h

Fig. 2. Kinetic curves of thermal inactivation of alkali phosphatase from chicken intestines in carbonate buffer, pH 8.5, at (1) 45, (2) 48, (3) 50, (4) 52, and (5) 60°ë.

and samples for the determination of enzymatic activity were taken at certain time intervals. The duration of thermal treatment was varied from 15 min to 10 h depending on the process rate. When solid samples were used, weighed enzyme portions in sealed vessels were placed into a thermostat and held at the required temperature for a certain period of time. After cooling, the sample was dissolved in a buffer solution and its activity was determined by the standard method. The V/V0 ratio was a measure of enzyme inactivation, where V is the initial rate of the catalytic reaction for a sample withdrawn at a definite time instant and V0 is the initial rate of the catalytic reaction for the initial sample. The IR spectra were recorded on an IR200 ThermoNicolet FTIR spectrometer over the range 400– 4000 cm–1. The samples were prepared as pellets with KBr. RESULTS AND DISCUSSION The results of our study showed that the mechanism of the inactivation of alkaline phosphatases was determined by the temperature, properties and state of the enzyme and composition of buffer solution. As was shown previously [7], the rate of inactivation of animal alkaline phosphatase depended also on medium pH, and the maxima of pH activity and pH stability of the enzyme did not coincide. Let us consider the basic characteristics of the thermal inactivation of alkaline phosphatases at constant pH. The kinetic mechanism of thermal inactivation with hidden stages, which are not accompanied by the loss of catalytic activity (the initial stages of scheme 1), is valid for all alkaline phosphatases studied in this work, but under different conditions. Figure 1 shows the kinetic curves of thermal inactivation of ECAP, bovine IAP, and chicken IAP in a carbonate buffer solution at RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A

3

4

5 2

319

50°ë. ECAP is most stable to the action of temperature: enzyme activity remains constant during thermal treatment for 3.5 h, then the enzyme is inactivated (keff = 5.3 × 10–5 s–1). Under these conditions, the induction period for the thermal inactivation of bovine IAP is as short as 0.5 h, and, for chicken IAP, is absent at all. The initial effective rate constants for inactivation of animal alkaline phosphatases are close to each other and are of (3.5–3.7) × 10–4 s–1, which exceeds keff for ECAP by a factor of 1.5. An induction period appears in the kinetic curves of thermal inactivation of chicken IAP in a carbonate buffer solution at 45°ë (Fig. 2). During thermal treatment of alkaline phosphatases in the solid state, the induction period in kinetic curves is retained at much higher temperatures: for chicken IAP, at 85°ë and, for bovine IAP, at 95°ë (Fig. 3). The temperature of the appearance of the induction period in the kinetic curves of thermal inactivation and its length depend also on the composition of buffer solutions. Among the systems studied, the highest temperature of the appearance of the induction period and its maximum duration at a constant temperature is characteristic of borate buffer, and these values are minimum for thermal inactivation in carbonate buffer (Table 1). The presence of the induction period in the kinetic curves can be caused by the multistep character of the initial stage of the rupture of interdomain bonds. During the induction period, hidden structural changes occur in the contact region of the oligomer, and, according to Scheme (1), a catalytically active but less stable (capable of reversible dissociation) oligomer appears. A kinetic analysis of thermal inactivation curves with induction periods [8] can be used to estimate the smallest number of stages n not accompanied by the loss of enzyme activity during the multistage degradation of the conformation lock. The determination of parameter

Vol. 83

No. 2

2009

320

ATYAKSHEVA et al.

V/V0

–ln (V/V0)

(‡)

0 1.0

1

1

2

0.5

3

0.6 2 3 4

0.2

4

1.0 5 1.5

0

60

120

180

240

300 τ, min

6 2.0 0

Fig. 3. Kinetic curves of thermal inactivation of solid samples of alkali phosphatase from (1, 2, 4) chicken intestines and (3) bovine intestines at (1) 75, (2) 85, and (3, 4) 95°C. The activity was determined in carbonate buffer, pH 8.5. The y-intercept (δ) is related to the number of stages without activity loss n as n = 0.13 + δ/0.13 – 0.05δ [8]. In this case, δ = 0.11, n ≈ 2.

2

4

–ln(V/V0)

6 (b)

8 τ, h

0 1

0.5

2 n gives information about enzyme properties, because 1.0 n is a special characteristic of the state of the interprotein contact in the oligomeric enzyme. During storage of enzymes, initial stages of inactivation can occur 1.5 which have no noticeable effect on the catalytic activity 3 of preparations but influence enzyme stability. The parameter n and induction period duration in 2.0 0 2 4 6 the thermal inactivation of three alkaline phosphatases τ, h under various conditions are listed in Table 1. A maximum number of intermediate stages not accompanied Fig. 4. Kinetic dependences of thermal inactivation in carby the loss of catalytic activity is observed for the therbonate buffer, pH 8.5, of alkali phosphatases from (a) mal inactivation of ECAP (n = 4). During the thermal bovine intestines and (b) bacteria E. coli in the coordinates inactivation of alkaline phosphatases of the animal oriof a first-order equation; (a) (1) 45, (2) 48, (3) 50, (4) 52, gin (bovine IAP and chicken IAP) in buffer solutions (5) 55, and (6) 60°ë; (b) (1) 50, (2) 60, and (3) 70°ë. The number of intermediate stages without activity loss n was (pH 8.5), n = 3, and, for solid samples of these determined from y-intercepts: ln(1 + δ) = 0.204, δ = 0.226, enzymes, n = 2. n = 0.13 + δ/0.13 – 0.05δ = 3. Let us consider the stages of active ECAP dimer transformation that can precede its dissociation into inactive monomers (see Scheme (1)). The structure of monomers appears. Thus, the number of hidden stages the ECAP dimer is stabilized by the “conformation is equal to four, which corresponds to the experimental lock” formed by three spatially separated contacts, two value n = 4. of which are peripheral and identical in their properties, The structures of interprotein contacts in alkaline and the third one is situated near the active center [9]. phosphatases of animal and bacterial origin are someThe interprotein bond between monomers with spa- what different, and animal alkaline phosphatases must tially separated contacts admits its multistage break- be less stable [10], which is observed experimentally. down. The presence of an induction period in the kinetic curve As the conformation lock in the ECAP dimer is of thermal inactivation of solid chicken and bovine IAP formed by three complementary elements, the dimer at 75–95°ë suggests that, in solid samples, not simultawith three intact contact elements is the most stable. neous degradation of all contact fragments in a protein When one of contacts is broken, a less stable interme- globule can also occur. The number of stages preceding diate with two bonding elements is formed. Next, an inactivation then decreases to two. intermediate with one strong bonding element is The experimental kinetic curves at times τ > τind are formed. Once all three elements are broken, a labile linearized in the coordinates of a first-order equation dimer capable of reversible dissociation into inactive (Fig. 4). For animal alkaline phosphatases over the RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A

Vol. 83

No. 2

2009

THERMAL INACTIVATION OF ALKALI PHOSPHATASES UNDER VARIOUS CONDITIONS Table 1. The duration of induction period τ and the minimum number of stages n preceding activity loss caused by thermal inactivation of alkali phosphatases from (I) bacteria E. coli, (II) chicken intestines, and (III) bovine intestines under various conditions t, °C 95 85 75 70 60 55 52 50

48 45

τ, h

n

0.25 0.25 2.0 2.5 3.0 0.15 0.25 3.5 1.25 0.5 0.75 1.25 4.5 3.25 2.5 0.75

2 2 2 4 4 3 3 4 3 3 3 3 3 3 3 3

Enzyme Conditions III II II I I III III I III III III III III III III II

A A A B B C D B C B D B C D B B

range 48–55°ë, there is a break of kinetic curves (Fig. 4a); the process is characterized by two effective inactivation rate constants (k1eff and k2eff), which can be determined from the slopes of lines before and after the break. During the thermal inactivation of ECAP over the temperature range 50–70°ë (Fig. 4b) and bovine IAP (Fig. 4a) and chicken IAP at 60°ë, kinetic curves with breaks were not observed, and the process was characterized by the effective rate constant keff. One of the origins of breaks in kinetic curves is the dissociative mechanism of inactivation [8]. For dimeric enzymes, it is described by the scheme k1 k–1

2E 1

kd

2E d ,

(2)

where E1 is the inactive monomer capable of the reversible formation of the catalytically active dimer E2, Ed is the denaturated monomer, which cannot form the E2 complex under the given kinetic experiment conditions; kd is the rate constant for monomer denaturation, k1 is the rate constant for the dissociation of the dimer, and k1/k–1 = Kdis is the equilibrium constant of dissociation. The effective dissociative inactivation rate constants k1eff and k2eff can be used to calculate k1 and kd (Scheme (2)) [8]. Figure 5 shows the temperature dependences of the effective inactivation rate constants (k1eff or k2eff) in the RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A

Table 2. Activation energies (E1 and E2 , kJ/mol) of thermal inactivation of alkali phosphatases Enzyme

Conditions

E1

I II III

B B A A B D C

80 200 170 180 200 190 160

E2 380

360 350 330

Note: The activation energies were determined from the temperature dependence of the initial effective inactivation rate constant k1eff (E1) and denaturation rate constant kd (E2). Designations are given in Table 1.

Note: A is the solid sample, B is the solution in carbonate buffer, C is the solution in borate buffer, and D is the solution in tris-HCl buffer.

E2

321

coordinates of the Arrhenius equation; the activation energies are given in Table 2. The activation energy of ECAP thermal inactivation is 80 kJ/mol in the carbonate buffer over the temperature range 25–70°ë; it corresponds to the dissociation of active dimers into inactive monomers. The experimental data showed that, at the initial stages of thermal inactivation over the temperature range specified, the process was reversible. According to the results obtained in [11], the inactivation of ECAP over this temperature range is completely reversible even when the enzyme loses 50% of its activity. When ECAP solutions are heated above 70°ë, intramolecular dynamics is intensified, and partial unfolding of protein globules occurs. The irreversible conformational transition characterized by globule unfolding, which is a high-effectiveness transition, and the loss of enzyme activity occur over the range 97– 100°ë [11]. –lnkeff 6 1 2 3 4

8

10

12

Vol. 83

2.9

3.0

3.1

3.2

3.3 3.4 103/T, ä–1

Fig. 5. The dependences of effective rate constants of inactivation of alkali phosphatases from (1) chicken intestines, (2, 3) bovine intestines, and (4) bacteria E. coli in (1, 2, 4) carbonate and (3) tris-HCl buffer solutions, pH 8.5, in the coordinates of the Arrhenius equation. No. 2

2009

322

ATYAKSHEVA et al.

A

isolated. The inactivation of labile monomeric alkaline phosphatases of the animal origin can then be described by the scheme

(‡)

0.4

E 2d

0.2

0 1500 A

1600

1700 ν, Òm–1

(b)

0.6

k2d

E2

k1 k–1

2E 1

kd

2E d ,

(3)

where E2 is the active dimer, E2d is the denaturated dimer, E1 is the inactive monomer capable of reversibly forming the catalytically active dimer E2, Ed is the denaturated monomer; k2d is the rate constant for dimer denaturation, kd is the rate constant for monomer denaturation, and k1 is the rate constant for the dissociation of the enzyme dimer. At the initial stage of inactivation characterized by the effective rate constant k1eff, all three processes, including the reversible dissociation of the dimer and the irreversible denaturation of the dimer and monomer, occur simultaneously. If k1 > k2d, the rate of dimer denaturation becomes negligibly low as time passes. The equation obtained for dissociative Scheme (2) to calculate the monomer denaturation rate [8] can then be used k 2eff ( V 0 + V τ ) k d = --------------------------------. 2(V 0 – V τ)

0.4

0.2

0 1500

1600

1700 ν, Òm–1

Fig. 6. IR spectrum in the region of Amide I and Amide II bands with the decomposition of the complex overlapping contour into Gaussian components for alkali phosphatase from (a) bovine intestines and (b) the same sample after thermal treatment in the solid state; 95°ë, treatment duration 5 h.

During thermal inactivation of animal alkaline phosphatases in various buffer solutions, the temperature coefficient of the process corresponds to the activation energy 190–200 kJ/mol (Fig. 5, Table 2). The same value was obtained for the thermal inactivation of alkaline phosphatase present in natural cow milk at 50– 80°ë [12]. This exceeds significantly the activation energy of dissociation, but is lower than for the denaturation of various proteins (usually 300–600 kJ/mol) [13]. It can be suggested that, at the first stage of inactivation characterized by the effective rate constant k1eff, the reversible dissociation of active dimer into inactive monomers and irreversible denaturation of dimeric enzyme can occur simultaneously. This suggestion is corroborated by the data presented in [14], where it was shown that the inactivated ECAP was a monomer, whereas, for bovine IAP, also inactive dimers were

(4)

Here, V0 and Vτ are the rates at t = 0 and t = τ (the “break point” in kinetic curve), respectively, and k2eff is the effective rate constant determined from the slope of the kinetic curve in semilogarithmic coordinates at t ≥ τ. The activation energies (E2) calculated from the temperature dependences of the rate constants for the denaturation of monomeric animal phosphatases are listed in Table 2. These activation energies show that, for animal phosphatases at the initial stages, the dissociation of dimers and their denaturation can occur simultaneously. At subsequent stages, activity loss characterized by higher activation energies (330–380 kJ/mol), however, becomes a determining process. The basic reason for the loss of catalytic activity by bacterial enzyme over the temperature range 25–70°ë is the dissociation of active dimers into inactive monomers (E‡ = 80 kJ/mol). The animal alkaline phosphatases in solution are unstable to heating: at 50°ë, they lose half of their activity within 0.5–1 h, and, at 60°ë, within several minutes (Figs. 1, 2). In the solid state, these enzymes are much more stable to the action of temperature. The heating of the samples of bovine IAP and chicken IAP at 60°ë for 5 h is not accompanied by a decrease in their catalytic activity. If solid samples of these enzymes are heated at 95°ë, their enzyme activity decreases by a factor of two within 1 h (chicken IAP) or 2.5 h (bovine IAP). This is easy to explain if it is taken into account that the presence of water facilitates the breakdown of hydrophobic contacts in protein molecules. The samples of alkaline phosphatases subjected to temperature treatment in the solid state were studied by

RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A

Vol. 83

No. 2

2009

THERMAL INACTIVATION OF ALKALI PHOSPHATASES UNDER VARIOUS CONDITIONS

IR spectroscopy. Figure 6 shows IR spectrum fragments in the region of Amide I and Amide II bands (1470–1700 cm–1) for the initial sample of bovine IAP and the same sample treated at 95°ë for 5 h. The complex contour of bands in these spectra is caused by overlapping of several individual bands, which are usually assigned to different types of the protein secondary structure [15, 16]. The decomposition component with a maximum at 1659 cm–1 was assigned to β structures, and the components at 1635 and 1620 cm–1, to α-helical segments of the enzyme molecule. A comparison of the spectra showed that, in the sample treated at 95°ë for 5 h, the relative content of β structures decreased noticeably (approximately by a factor of two), and the content of α structures remained almost unchanged. Thus, the inactivation of solid samples of alkaline phosphatases at 95°ë was accompanied by changes in the secondary structure of protein molecules. REFERENCES 1. R. B. McComb, G. N. Bowers, and S. Posen, Alkaline Phosphatases (Plenum, New York, 1979). 2. E. E. Kim and H. W. Wyckoff, Clin. Chim. Acta 186, 175 (1990). 3. M. H. le Du, T. Stigbrand, M. J. Taussig, et al., J. Biol. Chem. 276, 9158 (2001).

RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A

323

4. M. de Backer, S. McSweeney, H. B. Rasmussen, et al., J. Mol. Biol. 318, 1265 (2002). 5. E. Wang, D. Koutsioulis, H.-K. S. Leiros, et al., J. Mol. Biol. 366, 1318 (2007). 6. O. M. Poltorak and E. S. Chukhrai, Zh. Fiz. Khim. 69 (2), 330 (1995). 7. L. F. Atyaksheva, O. M. Poltorak, E. S. Chukhrai, and S. A. Fedosov, Zh. Fiz. Khim. 80 (4), 733 (2006) [Russ. J. Phys. Chem. 80 (4), 630 (2006)]. 8. O. M. Poltorak and E. S. Chukhrai, Itogi Nauki Tekh., Ser. Biotekhnol. 5, 50 (1986). 9. O. M. Poltorak, E. S. Chukhray, I. Y. Torshin, et al., J. Mol. Catal. B: Enzym. 7, 165 (1999). 10. O. M. Poltorak, E. S. Chukhray, A. A. Kozlenkov, et al., J. Mol. Catal. B: Enzym. 7, 157 (1999). 11. S. Fadiloglu, O. Erkmen, and G. Sekeroglu, J. Food Processing Preservation 30 (3), 258 (2006). 12. A. E. Lyubarev and B. I. Kurganov, Usp. Biol. Khim. 40, 43 (2000). 13. L. Zhang, R. Buchet, and G. Azzar, Biochem. J. 392, 407 (2005). 14. V. M. Mazhul’ and S. Zh. Kananovich, Biofizika 51 (3), 418 (2006) [Biophysics 51 (3), 364 (2006)]. 15. D. M. Byler and H. Susi, Biopolymers 25, 469 (1986). 16. L. de la Fourniere, O. Nosjean, R. Buchet, and B. Roux, Biochim. Biophys. Acta 1248, 186 (1995).

Vol. 83

No. 2

2009