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Thermal inactivation of protective antigen of Bacillus anthracis and its prevention by polyol osmolytes
BBRC Biochemical and Biophysical Research Communications 322 (2004) 1029–1037 www.elsevier.com/locate/ybbrc
Thermal inactivation of protective antigen of Bacillus anthracis and its prevention by polyol osmolytes Samer Singh, Aparna Singh, Mohd. Azhar Aziz, Syed Mohsin Waheed, Rajiv Bhat*, Rakesh Bhatnagar* Centre for Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India Received 26 July 2004
Abstract Protective antigen (PA) of Bacillus anthracis is the main immunogen of all anthrax vaccines. It is a highly thermolabile molecule and loses its activity rapidly when exposed to higher temperatures. Earlier some cosolvents had been used to stabilize PA with variable success but no study has been done to find out the primary cause of PA thermal inactivation. This study aims at elucidating the predominant cause of thermal inactivation of PA in order to develop more effective strategies for its thermostabilization. The prime cause for the loss of biological activity of PA at high temperature was its aggregation and an inverse correlation between PA activity and its aggregation on heating was observed. Inactivation of the protein by autolysis did not occur. This paper reports the use of a series of polyol osmolytes to stabilize PA. Different polyols stabilized PA to a different extent against thermal inactivation in a concentration dependent manner, with glycerol stabilizing to the maximum extent. Addition of NaCl to glycerol solution further enhanced the thermal stability of PA. An increase in the T1/2 value, the temperature at which 50% of the activity is retained during short-term incubation, of more than 20 °C was observed. The half-life (t1/2) of PA thermal inactivation at 40 °C increased by more than 6 times in the presence of the mixture of glycerol and NaCl as compared to control. This study demonstrates for the first time that aggregation of the PA molecule is the predominant cause of its thermal inactivation, and can be very effectively prevented by the use of glycerol and other polyols to increase the shelf life of the recombinant vaccine against anthrax. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Anthrax; Aggregation; Glycerol; Osmolytes
Protective antigen (PA) is the most important molecule among the tripartite anthrax toxin complex molecules i.e., protective antigen (PA: 83 kDa), edema factor (89 kDa), and lethal factor (LF: 90 kDa), produced by B. anthracis. It plays a pivotal role in anthrax pathogenesis as it effects transport of LF and EF into the cytoplasm [1]. PA is also the main immunogenic molecule that confers protection against anthrax making it a major constituent of all the present-day vaccines. Even though all the available vaccines against anthrax *
Corresponding authors. Fax: +91 11 26717040. E-mail addresses: [email protected] (R. Bhat), rakbhat01@ yahoo.com, [email protected] (R. Bhatnagar). 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.08.020
are quite effective, the associated side effects due to the presence of LF, EF, and cellular debris in trace amounts have led to endeavors to develop better and safer alternatives such as recombinant vaccine based on PA [2–4]. We have shown earlier that PA is a highly thermolabile molecule [5] and hence accidental exposure of PA to higher temperatures during transportation or storage could compromise the quality and efficacy of the vaccine. The deterioration of protein pharmaceuticals on storage has been a well-documented fact and a cause of great concern. The instability in a protein stems from its inherent physical and chemical properties, which are also modulated by the extrinsic factors like pH, temperature, ionic strength, presence of other compounds, etc.
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Various investigations have been carried out to characterize the primary cause of deterioration in protein stability and its prevention so that the overall quality of the proteins could be improved [6]. The most common form of physical instability encountered is the aggregation of protein molecules, e.g., human growth hormone or hGH, insulin and human relaxin [7–9], etc., whereas the common forms of chemical instability encountered are hydrolysis or autolysis, deamidation, disulfide bond breakage and rearrangement, succinimidation, and isomerization [6,10]. There have been many endeavors to stabilize protein molecules using the approaches of protein engineering [11–14] and cosolvent engineering [5,6,15,16] with variable success rates. The identification of predominant factors of inactivation is the first step towards generating comprehensive strategies for the stabilization of a protein molecule. To elucidate the possible mechanism of thermal inactivation of PA, we performed different in vitro experiments in which PA was exposed to higher temperature for different durations and tested for changes in its physical characteristics as well as residual activity. We investigated possible autolysis and the aggregation behavior of PA on exposure to higher temperatures. This study demonstrates for the first time that heat inactivation of PA was primarily caused by aggregation, while autolysis apparently did not play any role. Furthermore, we investigated the effect of a series of polyols, like adonitol, erythritol, xylitol, sorbitol, and glycerol, one of the best-studied osmolyte series, on the prevention of thermal inactivation of PA. Among the various excipients used, glycerol was found to be the best stabilizer that increased the T1/2, the temperature at which 50% activity is retained on short-term incubation to the maximum extent (>25 °C). Glycerol was able to prevent aggregation of PA as well as helped in extending the half-life (t1/2) of PA thermal inactivation by more than 6 times upon incubation at 40 °C, which is 1.5 times more than that reported earlier with other osmolytes [5,17]. This study demonstrates that aggregation is the primary cause of the thermal inactivation of PA and cosolvents of the polyol series such as glycerol can be successfully employed to extend the shelf life of the recombinant anthrax vaccine.
Materials and methods Reagents and supplies. Escherichia coli strain DH5a and RAW264.7, a macrophage like cell line, were obtained from ATCC (American Type Culture Collection; USA). Growth media components for E. coli were from Hi Media Laboratories (India). Cell culture plastic ware was obtained from Corning (USA). Fetal calf serum (FCS) was from Biological Industries (Israel). RPMI 1640, 3-(4,5-dimethylthiazol-3-yl)-5-diphenyltetrazolium bromide (MTT), phenyl methylsulfonyl fluoride (PMSF), Hepes, NaCl, adonitol, erythritol, sorbitol, xylitol, and other chemicals were purchased from Sigma
Chemical (USA). Glycerol was from USB (USA). The Ni–NTA agarose was obtained from Qiagen (Germany). The Centricon concentrators of 30 kDa cut-off limit were from Millipore (USA). Expression and purification of PA. Recombinant PA was purified from E. coli DH5a transformed with pMW1 [4] as described earlier [16]. E. coli DH5a carrying pMW1 was grown at 37 °C (250 rpm) in 100 ml LB medium containing 100 lg/ml ampicillin. It was allowed to grow upto A600 1.5–2.0. Cells were harvested by centrifugation at 4000g for 10 mins. The pellet was resuspended in 10 ml lysis buffer (8 M urea, 0.1 M sodium phosphate, and 350 mM NaCl, pH 7.8) and the cells were stirred at 37 °C for 1 h. The lysate was centrifuged at 12,000g for 30 min. The supernatant was loaded on a Ni–NTA column pre-equilibrated with the lysis buffer. The PA bound to the Ni–NTA column was refolded by gradual removal of urea by passing a gradient of 8.0–0.0 M urea in buffer containing 0.1 M sodium phosphate, 350 mM NaCl, pH 7.8. The recombinant protein bound to the column was eluted with 250 mM imidazole in 0.1 M sodium phosphate, 350 mM NaCl, pH 7.4. PA fractions obtained after Ni–NTA affinity chromatography of >90% purity (as determined by densitometry of Coomassie brilliant blue stained 12% SDS–PAGE gel) were taken and the buffer was exchanged to 20 mM Hepes with 250 mM NaCl, pH 7.5, using Centricon concentrators of 30 kDa cut-off limit. The protein samples were stored at 4 °C for further use. Cell culture and cytotoxicity assay. Macrophage-like cell line RAW264.7 was maintained in RPMI 1640 medium containing 10% heat inactivated FCS, 25 mM Hepes, 100 U/ml penicillin, and 200 lg/ ml streptomycin under humidified environment supplemented with 5% CO2 at 37 °C [16]. For biological assay, a 96-well culture plate was prepared with 90% cell confluency. PA samples incubated at different temperatures were added to the cells at a final concentration of 0.8 lg/ ml along with LF at a final concentration of 0.3 lg/ml, unless mentioned otherwise, and incubated for 4 h at 37 °C. PA stored at 4 °C and unexposed to any higher temperature was used along with LF as a positive control. The same amount of additive (cosolvent) along with LF but without PA was used as a negative control. Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) dye. MTT dissolved in RPMI was added to each well at a final concentration of 0.5 mg/ml and incubated for another 45 min at 37 °C to allow uptake and reduction of the dye by viable cells. The spent medium was discarded and after drying the plate, 100 ll of 25 mM HCl in 90% isopropyl alcohol was added to each well to dissolve the cells and formazone crystals formed as a result of reduction of the MTT dye by viable cells. The plate was vortexed and the absorption of the solutions was read at 540 nm using a microplate reader (Biorad) to determine the cell viability. Experiments for monitoring loss of PA activity. PA (100 lg/ml in 20 mM Hepes, 25 mM NaCl, pH 7.4) was subjected to heating at 55 °C for 10 min. The sample was mixed with SDS loading buffer and analyzed by 12% SDS–PAGE to find out the presence of extra bands as an indication of autolysis. Untreated control PA kept at 4 °C was processed in a similar manner and loaded alongside the exposed sample on SDS–PAGE for comparison. To determine the denaturation/aggregation behavior of PA on heating, both spectrophotometeric and PAGE studies were carried out. PA sample (50 lg/ml in 20 mM Hepes with 25 mM NaCl, pH 7.4) was heated with a scan rate of 1 °C/min and increase in absorbance at different wavelengths, i.e., 278, 340, 440, 600, and 700 nm, was monitored in a Shimadzu UV–visible spectrophotometer to which a temperature programmer was attached. The loss of soluble form of PA on heating was further analyzed by non-denaturing and denaturing PAGE. PA protein samples (50 lg/ml in 20 mM Hepes, 25 mM NaCl, pH 7.4, and 40 mM Hepes containing 2.7 M glycerol + 500 mM NaCl, pH 7.9) were incubated for 10–20 min at 55 °C and subjected to native PAGE (4–15%) and SDS–PAGE (12%). For SDS–PAGE the heattreated PA samples were centrifuged at 12,000g for 10 min and the supernatant was separated from the pellet. The supernatant and pellet of the samples were then analyzed by SDS–PAGE to confirm the loss
S. Singh et al. / Biochemical and Biophysical Research Communications 322 (2004) 1029–1037 of the soluble PA form on heating. PA sample that had not undergone any heat treatment was included in both the gels as control. In order to correlate the loss of activity with the aggregation of PA (40 lg/ml) in 20 mM Hepes, 25 mM NaCl, pH 7.4, and 40 mM Hepes with 2.7 M glycerol + 500 mM NaCl, pH 7.9, was incubated in a thermal cuvette at 46 and 58 °C and the increase in absorbance at 340 nm (A340) with time was measured to assess the extent of aggregation. At regular interval of time samples were withdrawn from the cuvette and stored at 4 °C and the residual activity was assessed by cytotoxicity assay to correlate the loss of activity of PA with aggregation. To determine the effect of the concentration of protein as well as polyols on thermal inactivation of PA, the PA stock (20 mM Hepes with 250 mM NaCl, pH 7.5) was diluted in a suitable buffer/cosolvent solution to achieve different concentrations of PA or cosolvent. The PA samples at a final concentration of 8 lg/ml (unless mentioned otherwise) were incubated at 40–75 °C for 20 min and their residual activity was determined by cytotoxicity assay on RAW 264.7 cell lines. PA at a final concentration of 4, 8, 16, and 32 lg/ml was used to study the effect of protein concentration on the thermal inactivation of the protein while the polyol concentration ranged from 0.9 to 2.7 M. Temperature at which 50% activity was retained after 20 min of incubation (T1/2) was determined for comparison of the ability of different cosolvents to stabilize PA. The most stabilizing cosolvent was then evaluated for its efficacy to increase the half-life of thermal inactivation (t1/2) of PA (8 lg/ml) at 40 °C for 0–120 h. All the experiments were done in triplicate and there was ±5% error in measurements/variation in values.
Results and discussion Inactivation of PA by temperature Autolysis Autolysis of a sensitive protein results in cleavage into fragments of smaller length/size, e.g., recombinant human macrophage colony stimulating factor (rhMCSF), insulin, subtilisin J, neutral proteases from Bacillus subtilis [6,18,19], etc. To examine the possibility of autolysis of PA as the cause of loss of its activity at higher temperatures, PA (100 lg/ml) was incubated at 55 °C for 10 min and analyzed by SDS–PAGE. It is clear from SDS–PAGE results that the band corresponding to PA remains intact without the appearance of extra or smaller size bands after heating at 55 °C for 10 min (Fig. 1). This result suggested that autolysis is not responsible for the accelerated loss of activity of PA when incubated at temperatures as high as 55 °C. Temperature-induced aggregation of PA Denaturation of proteins can be easily monitored by a spectrophotometer as it is accompanied by a change in the spectral properties of the protein. These changes are mostly centered around a narrow wavelength band due to changes in the environment of some residues (e.g., Trp, Tyr) while in the case of aggregation, scattering of the incident light occurs which is inversely related to the wavelength of incident light. The scattering of light results in increase in attenuation of the incident
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Fig. 1. A 12% SDS–PAGE of PA sample (100 lg/ml in 20 mM Hepes with 25 mM NaCl, pH 7.4) incubated at 55 °C for 10 min.
beam (increase in absorbance) over a wide range of wavelengths and the observed change in absorbance due to scattering is more at shorter wavelengths than at longer wavelengths [20]. Thus, the pattern of the change in the absorbance at different wavelengths should be different in the case of denaturation and aggregation. PA (50 lg/ml in 20 mM Hepes, 25 mM NaCl, pH 7.4) was heated from 25 to 85 °C at a sweep rate of 1 °C/min and the change in absorbance per degree rise in temperature at the wavelengths 278, 340, 440, 600, and 700 nm was monitored as a function of temperature (Fig. 2). The increase in absorbance starts around 40 °C and reaches a maximum at 49 °C. The increase in absorbance is smooth which starts at the same temperature for all the wavelengths and is observed over the entire range of wavelengths used, indicating that PA is getting aggregated.
Fig. 2. Absorbance increase at different wavelengths with increase in temperature. The PA (50 lg/ml in 20 mM Hepes, 25 mM NaCl, pH 7.4) was heated at a sweep rate of 1 °C/min. The increase in absorbance per degree rise in temperature (DA) was monitored at different wavelengths and plotted as a function of temperature. DA: (absorbance at given temperature T °C) (absorbance at T 1 °C).
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Loss of soluble form of PA on heating Aggregation has been shown to be the main cause of inactivation in a number of proteins, e.g., human growth hormone [7], insulin [8], human relaxin [9], recombinant human keratinocyte growth factor [21], porcine growth hormone [22], etc. Aggregation of proteins results in the formation of large visible aggregates or small soluble oligomers. In both the cases, the size of the molecules/cluster increases which can be analyzed using native PAGE. The heat-treated samples of PA (100 lg/ml in 20 mM Hepes with 25 mM NaCl, pH 7.4, and 40 mM Hepes with 2.7 M glycerol + 500 mM NaCl, pH 7.9) were analyzed by non-denaturing PAGE and SDS–PAGE (Figs. 3A and B). Results of native or non-denaturing PAGE indicated that heat treatment of the control samples resulted in aggregation, forming large aggregates of PA that were unable to enter into the gel (Fig. 3A). This was further corroborated by SDS–PAGE of the heat-treated samples after centrifugation (Fig. 3B), which showed presence of PA in the pellet but not in the supernatant. This can be explained only by assuming that heat treatment of PA caused the formation of such large aggregates that were unable to enter in the native gel and got separated in the form of pellet on centrifugation. The presence of 2.7 M glycerol with 500 mM NaCl (the best condition able to prevent thermal inactivation) was able to prevent aggregation of PA and hence PA remained in its soluble (Fig. 3B) and monomeric forms (Fig. 3A).
Fig. 3. (A) Five to fifteen percentage non-denaturing PAGE. (B) Twelve percentage SDS–PAGE showing loss of soluble PA (Cont.) on heating and its prevention by 2.7 M glycerol + 500 mM NaCl (Glyl.) when incubated at 55 °C for 10 and 20 min. (Sup., supernatant of the sample; Cont., PA sample in 20 mM Hepes with 25 mM NaCl, pH 7.4; and Glyl., PA sample in 40 mM Hepes with 2.7 M glycerol + 500 mM, pH 7.9).
Correlation of loss of activity with aggregation The correlation between aggregation and loss of PA activity was determined by measuring the residual activity of PA with the progression of aggregation. The loss in PA activity closely followed aggregation (Fig. 4). Control PA sample (40 lg/ml in 20 mM Hepes buffer with 25 mM NaCl, pH 7.4) rapidly lost activity with the progression of aggregation (i.e., increase in absorbance at 340 nm) and plummeted to zero within 6 min. at 46 °C. The PA protein in 2.7 M glycerol with 500 mM NaCl in 40 mM Hepes, pH 7.9, the best condition able to prevent thermal inactivation neither showed aggregation even after 30 min of incubation at 46 or 58 °C incubation nor resulted in any loss of activity. These findings conclusively prove that aggregation is the primary cause of the thermal inactivation of PA at moderately high temperatures (46 or 58 °C) and the prevention of aggregation could help prevent its thermal inactivation. Effect of PA concentration on the thermal stability of PA Protein concentration is known to affect the protein aggregation behavior [23,24] and causes deterioration in the quality of a number of proteins [7– 9,21,22]. The protein concentration has also been shown to affect the transition temperature of unfolding (Tm) as well as initial unfolding temperature in a number of cases [6]. In the case of interferon-b-1a a decrease in initial unfolding temperature of about 9 °C (from 77 to 68 °C) has been reported when the protein concentration increased from 10 lg to 100 lg/ml [25]. We studied the effect of protein concentration on T1/2 of PA, the temperature at which 50% activity was retained after 20 min of incubation. To examine the effect of protein concentration on thermal inactivation of PA, protein solutions containing varying concentrations of PA viz., 4, 8, 16, and 32 lg/ml (40 mM Hepes, 25 mM NaCl, pH 7.9) were incubated at 40–52 °C for 20 min and tested for the residual activity by cytotoxicity assays. The loss of PA activity on heating was highly sensitive to protein concentration (Fig. 5). The sample with 4 lg/ml final PA protein concentration retained 95% activity even after 20 min incubation at 49 °C while under the same conditions sample with 8 lg/ml PA retained only 60% activity, and the samples having 16 and 32 lg/ml of final PA protein concentration completely lost the activity. This shows an inverse relationship between T1/2 and PA concentration. It is clear from Fig. 5 that a variation in the concentration of PA in the range of 4–32 lg/ ml resulted in a decrease in the T1/2 by 6 °C from 51 to 45 °C. Therefore, it can be safely inferred from the results that thermal inactivation of PA is highly sensitive to protein concentration.
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Fig. 4. Correlation of loss of activity with aggregation: PA (40 lg/ml) was incubated at (A) 46 °C in 20 mM Hepes with 25 mM NaCl, pH 7.4, (C) 46 °C in 40 mM Hepes with 2.7 M glycerol + 500 mM NaCl, pH 7.9, and (E) 58 °C in 40 mM Hepes with 2.7 M glycerol + 500 mM NaCl, pH 7.9, and increase in absorbance was monitored at 340 nm; (B,D, and F) are residual activity in (A,C, and E), respectively, during incubation. Residual activity (RA) was determined by cytotoxicity assay.
Fig. 5. The dependence of PA thermostability on protein concentration. Different concentrations of PA in 40 mM Hepes with 25 mM NaCl, pH 7.9, were incubated at different temperatures for 2 min and the residual activity was determined by cytotoxicity assay.
Effect of polyols on the thermal stability of PA Polyols are one of the best-studied classes of osmolytes, which are produced during various denaturing stresses in microorganisms, insects, and plants [26–28]. Polyols along with certain sugars are known to stabilize the native state of proteins [15,29–37]. The protein stabilizing ability of polyols is a result of their preferential exclusion from the protein surface [35–38], leading to the preferential hydration of the protein molecule. The solvophobic nature of the peptide backbone that promotes folding and compaction of the protein molecule in the presence of polyols pushes the native () denatured/unfolded equilibrium between the native and the unfolded protein in favor of the native state and stabilizes the protein molecule [39–42]. The effect of various polyols on the thermal stability of PA was investigated by incubating PA with varying concentrations of polyols with and without 500 mM
NaCl at different temperatures (46–75 °C) for 20 min and then comparing the residual activity as determined by the cytotoxicity assay (Fig. 6). The effect of 0.9, 1.8, and 2.7 M polyol on the thermal stability of PA is shown in Figs. 6A–C, respectively. The T1/2 values for different polyols with and without 500 mM NaCl have also been determined and presented in Table 1. In general, the stabilizing effect of different polyols increased with an increase in their concentration but differed greatly in magnitude from each other. Glycerol increased the T1/2 for PA to the maximum extent (>25 °C) compared to control, which was more than 17 °C higher compared to T1/2 achieved earlier using glycine [17]. In general at any given molarity, the ability of polyols to stabilize PA followed the order glycerol > sorbitol > xylitol > adonitol > erythritol. It was observed that on addition of 500 mM NaCl to polyols the stability of PA invariably increased and the magnitude was always greater than in the presence of polyol or NaCl alone (Fig. 6D. It suggests that the mechanism of stabilization of PA is different in the case of polyols and salts. In the presence of 500 mM NaCl, the order of stabilization of PA changed to glycerol > xylitol > sorbitol > adonitol > erythritol. Although polyols and NaCl act in tandem and stabilize the protein, yet the presence of NaCl affected the interaction of polyol with the protein in some subtle way, which was different for each polyol. Salts stabilize proteins by the screening of unfavorable or repulsive charge–charge interactions [43,44] or by favorable electrostatic binding of ions [43–45] while polyols are known to increase the surface tension of water [15,35] and strengthen hydrophobic interactions [39–42].
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Fig. 6. Effect of polyols on the stability of PA: (A) 0.9 M; (B) 1.8 M; (C) 2.7 M; (D) 0.9 M polyol + 500 mM NaCl; and (E) 2.7 M glycerol + 500 mM NaCl and 2.7 M glycerol only. PA (8 lg/ml) in 40 mM Hepes, pH 7.9, was incubated with different concentrations of polyols with and without 500 mM NaCl at different temperatures for 20 min. (A–D) or longer (E) and residual activity was determined by cytotoxicity assay. (Ado, adonitol; Xyl, xylitol; Ery, erythritol; Glyl, glycerol; Sor, sorbitol; Cont, PA in 25 mM NaCl; and N, 500 mM NaCl unless mentioned otherwise).
Some interesting observations were made during this investigation. For example, erythritol destabilized PA at lower concentrations while with increase in its concentration its ability to stabilize PA increased. T1/2 for PA at 0.9, 1.8, and 2.7 M erythritol were 43.7, 46.3, and 51.5 °C, respectively (Table 1), while that of control was 50.3 °C. This could be because of strong specific destabilizing interactions of erythritol with PA at lower concentrations which get overwhelmed by the weak, but large, stabilizing interactions at higher concentration as suggested earlier in the case of stabilization of BSA by glycine [46]. Adonitol and xylitol, which are epimers, stabilized PA to different levels. At 0.9 M concentration adonitol behaved like control but increase in the concentration increased the stabilization of PA, while xylitol behaved like a good stabilizer among the polyols studied. Different behavior of epimers has been reported earlier in the case of collagen. The differential effect of epimers towards stabilizing collagen molecule has been
attributed to differences in the solvent organization property of the solute molecules because of variation in the stereochemistry of the hydroxyl groups [47]. Effect of glycerol towards stabilization of PA was highly concentration dependent. At 0.9 M it was similar to that of sorbitol with a T1/2 of 55.7 °C, but with increasing concentration its effect deviated from that of sorbitol (Fig. 6C). An interesting behavior was observed when 500 mM NaCl was present along with 0.9 M glycerol (Fig. 6D). The residual activity obtained on incubation of PA for 20 min. in the range of 58–70 °C was 60– 75%, which deviated from the usual pattern of decrease in the activity of PA in the presence of other polyols in the same range of temperatures. It could be due to glycerol assisted refolding of the heat-denatured PA. Glycerol has been shown to increase the refolding yield of a number of proteins, e.g., rhodanese, rabbit muscle creatine kinase, porcine pancreatic elastase [48–50], etc. Earlier, it has been shown that polyols also affect the
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Table 1 Effect of polyols on T1/2 of PA (Fig. 6) Co-solvent
Concentration (M)
T1/2 (20 min) in °C
Control (25 mM NaCl) Glycerol Glycerol Glycerol Glycerol Erythritol Erythritol Erythritol Erythritol Adonitol Adonitol Adonitol Adonitol Sorbitol Sorbitol Sorbitol Sorbitol Xylitol Xylitol Xylitol
0 0.9 1.8 2.7 0.9 M + 500 mM 0.9 1.8 2.7 0.9 M + 500 mM 0.9 1.8 2.7 0.9 M + 500 mM 0.9 1.8 2.7 0.9 M + 500 mM 0.9 1.8 0.9 M + 500 mM
50.3 55.7 61.9 >75 >70 43.7 46.3 51.5 <55 50.2 53.2 57.0 59.7 55.3 58.4 62.5 60.2 53.5 55.1 64.5
NaCl
NaCl
NaCl
NaCl
NaCl
Note. PA was incubated at a final concentration of 8 lg/ml in the presence of various polyols for thermostability studies. PA was used at a final concentration of 0.8 lg/ml along with 0.3 lg/ml of LF for cytotoxicity assay on RAW264.7 cell line.
refolding of heat-denatured proteins to varying extents, e.g., chymotrypsinogen, lysozyme, ribonuclease, and staphylococcal nuclease [31–33,51]. The presence of NaCl along with polyols invariably resulted in the enhancement of residual activity obtained at any given temperature as compared to the residual activity obtained when only polyols were used (Fig. 6D and Table 1). This observation indicates that the mechanisms operational in the stabilization of the PA by polyols and NaCl are different and additive. The better stabilization achieved in the presence of glycerol in comparison to sorbitol can be due to better refolding of the heat-denatured PA on cooling. Stabilization of PA by xylitol and sorbitol invariably increased with addition of NaCl but the order of the overall stabilization achieved differed. At 0.9 M concentration, sorbitol stabilized PA better than xylitol but addition of NaCl reversed the trend and xylitol along with NaCl led to a greater stabilization of PA as compared to sorbitol along with NaCl (Fig. 6D and Table 1). This observation indicates that though the mechanisms operational in the stabilization of PA by polyols and NaCl are different in nature, they can interfere with each otherÕs interactions with the protein molecule in a subtle manner. When PA was incubated at 58–75 °C in the presence of 2.7 M glycerol + 500 mM NaCl for 0–160 min, the rate of loss of activity was initially slow followed by a fast phase (Fig. 6E). It was found that loss of PA activity in the presence of 2.7 M glycerol on incubation at 64 °C follows approximately the same profile as 2.7 M glyc-
Fig. 7. Effect of 2.7 M glycerol + 500 mM NaCl on stability of PA on extended incubation at 40 °C: PA (8 lg/ml) in 40 mM Hepes, 25 mM NaCl and 40 mM Hepes, and 2.7 M glycerol + 500 mM NaCl were incubated at 40 °C for 0–120 h and the residual activity was determined by cytotoxicity assay. (2.7 M Glyl + 500 mM NaCl: 2.7 M glycerol with 500 mM NaCl; Cont: PA in 25 mM NaCl only).
erol + 500 mM NaCl follows on incubation at 75 °C, which strongly supports the notion that mechanisms operating for the stabilization of PA by polyols and salts are different in nature (Figs. 6A, D, and E). Feasibility of using 2.7 M glycerol with 500 mM NaCl to increase the half-life of thermal inactivation of PA at 40 °C was examined by incubating PA for up to 120 h. The 2.7 M glycerol along with 500 mM NaCl was able to extend the half-life (t1/2) of thermal inactivation of PA at 40 °C by more than 6 times over the control, i.e., from 7 h. to more than 42 h (Fig. 7). This increase in t1/2 is 1.5 times higher as compared to the previously reported result using glycine as a stabilizer [17]. To conclude, this study suggests that thermal inactivation of PA at moderately high temperatures is primarily caused by aggregation and other processes such as autolysis do not have any significant role in its inactivation mechanisms. The ability of polyols to stabilize the PA molecule against thermal inactivation varied widely. While erythritol destabilized PA molecule at lower concentrations on one hand, 2.7 M glycerol along with 500 mM NaCl was able to lead to 100% retention of PA activity on shorter incubation even at 70 °C on the other hand. The combination of 2.7 M glycerol and 500 mM NaCl also extended the half-life of thermal inactivation of PA at 40 °C by more than 6 times over the control. This study besides elucidating the primary cause of thermal inactivation of PA helped to identify conditions to considerably increase the half-life of PA against thermal inactivation at moderately high temperatures which would further help in improving the shelflife of the recombinant PA vaccine.
Acknowledgment The work was supported by the grant from World Bank assisted National Agricultural Technology Project (NATP), ICAR, New Delhi.
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Thermal inactivation of protective antigen of Bacillus anthracis and its prevention by polyol osmolytes Samer Singh, Aparna Singh, Mohd. Azhar Aziz, Syed Mohsin Waheed, Rajiv Bhat*, Rakesh Bhatnagar* Centre for Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India Received 26 July 2004
Abstract Protective antigen (PA) of Bacillus anthracis is the main immunogen of all anthrax vaccines. It is a highly thermolabile molecule and loses its activity rapidly when exposed to higher temperatures. Earlier some cosolvents had been used to stabilize PA with variable success but no study has been done to find out the primary cause of PA thermal inactivation. This study aims at elucidating the predominant cause of thermal inactivation of PA in order to develop more effective strategies for its thermostabilization. The prime cause for the loss of biological activity of PA at high temperature was its aggregation and an inverse correlation between PA activity and its aggregation on heating was observed. Inactivation of the protein by autolysis did not occur. This paper reports the use of a series of polyol osmolytes to stabilize PA. Different polyols stabilized PA to a different extent against thermal inactivation in a concentration dependent manner, with glycerol stabilizing to the maximum extent. Addition of NaCl to glycerol solution further enhanced the thermal stability of PA. An increase in the T1/2 value, the temperature at which 50% of the activity is retained during short-term incubation, of more than 20 °C was observed. The half-life (t1/2) of PA thermal inactivation at 40 °C increased by more than 6 times in the presence of the mixture of glycerol and NaCl as compared to control. This study demonstrates for the first time that aggregation of the PA molecule is the predominant cause of its thermal inactivation, and can be very effectively prevented by the use of glycerol and other polyols to increase the shelf life of the recombinant vaccine against anthrax. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Anthrax; Aggregation; Glycerol; Osmolytes
Protective antigen (PA) is the most important molecule among the tripartite anthrax toxin complex molecules i.e., protective antigen (PA: 83 kDa), edema factor (89 kDa), and lethal factor (LF: 90 kDa), produced by B. anthracis. It plays a pivotal role in anthrax pathogenesis as it effects transport of LF and EF into the cytoplasm [1]. PA is also the main immunogenic molecule that confers protection against anthrax making it a major constituent of all the present-day vaccines. Even though all the available vaccines against anthrax *
Corresponding authors. Fax: +91 11 26717040. E-mail addresses: [email protected] (R. Bhat), rakbhat01@ yahoo.com, [email protected] (R. Bhatnagar). 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.08.020
are quite effective, the associated side effects due to the presence of LF, EF, and cellular debris in trace amounts have led to endeavors to develop better and safer alternatives such as recombinant vaccine based on PA [2–4]. We have shown earlier that PA is a highly thermolabile molecule [5] and hence accidental exposure of PA to higher temperatures during transportation or storage could compromise the quality and efficacy of the vaccine. The deterioration of protein pharmaceuticals on storage has been a well-documented fact and a cause of great concern. The instability in a protein stems from its inherent physical and chemical properties, which are also modulated by the extrinsic factors like pH, temperature, ionic strength, presence of other compounds, etc.
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Various investigations have been carried out to characterize the primary cause of deterioration in protein stability and its prevention so that the overall quality of the proteins could be improved [6]. The most common form of physical instability encountered is the aggregation of protein molecules, e.g., human growth hormone or hGH, insulin and human relaxin [7–9], etc., whereas the common forms of chemical instability encountered are hydrolysis or autolysis, deamidation, disulfide bond breakage and rearrangement, succinimidation, and isomerization [6,10]. There have been many endeavors to stabilize protein molecules using the approaches of protein engineering [11–14] and cosolvent engineering [5,6,15,16] with variable success rates. The identification of predominant factors of inactivation is the first step towards generating comprehensive strategies for the stabilization of a protein molecule. To elucidate the possible mechanism of thermal inactivation of PA, we performed different in vitro experiments in which PA was exposed to higher temperature for different durations and tested for changes in its physical characteristics as well as residual activity. We investigated possible autolysis and the aggregation behavior of PA on exposure to higher temperatures. This study demonstrates for the first time that heat inactivation of PA was primarily caused by aggregation, while autolysis apparently did not play any role. Furthermore, we investigated the effect of a series of polyols, like adonitol, erythritol, xylitol, sorbitol, and glycerol, one of the best-studied osmolyte series, on the prevention of thermal inactivation of PA. Among the various excipients used, glycerol was found to be the best stabilizer that increased the T1/2, the temperature at which 50% activity is retained on short-term incubation to the maximum extent (>25 °C). Glycerol was able to prevent aggregation of PA as well as helped in extending the half-life (t1/2) of PA thermal inactivation by more than 6 times upon incubation at 40 °C, which is 1.5 times more than that reported earlier with other osmolytes [5,17]. This study demonstrates that aggregation is the primary cause of the thermal inactivation of PA and cosolvents of the polyol series such as glycerol can be successfully employed to extend the shelf life of the recombinant anthrax vaccine.
Materials and methods Reagents and supplies. Escherichia coli strain DH5a and RAW264.7, a macrophage like cell line, were obtained from ATCC (American Type Culture Collection; USA). Growth media components for E. coli were from Hi Media Laboratories (India). Cell culture plastic ware was obtained from Corning (USA). Fetal calf serum (FCS) was from Biological Industries (Israel). RPMI 1640, 3-(4,5-dimethylthiazol-3-yl)-5-diphenyltetrazolium bromide (MTT), phenyl methylsulfonyl fluoride (PMSF), Hepes, NaCl, adonitol, erythritol, sorbitol, xylitol, and other chemicals were purchased from Sigma
Chemical (USA). Glycerol was from USB (USA). The Ni–NTA agarose was obtained from Qiagen (Germany). The Centricon concentrators of 30 kDa cut-off limit were from Millipore (USA). Expression and purification of PA. Recombinant PA was purified from E. coli DH5a transformed with pMW1 [4] as described earlier [16]. E. coli DH5a carrying pMW1 was grown at 37 °C (250 rpm) in 100 ml LB medium containing 100 lg/ml ampicillin. It was allowed to grow upto A600 1.5–2.0. Cells were harvested by centrifugation at 4000g for 10 mins. The pellet was resuspended in 10 ml lysis buffer (8 M urea, 0.1 M sodium phosphate, and 350 mM NaCl, pH 7.8) and the cells were stirred at 37 °C for 1 h. The lysate was centrifuged at 12,000g for 30 min. The supernatant was loaded on a Ni–NTA column pre-equilibrated with the lysis buffer. The PA bound to the Ni–NTA column was refolded by gradual removal of urea by passing a gradient of 8.0–0.0 M urea in buffer containing 0.1 M sodium phosphate, 350 mM NaCl, pH 7.8. The recombinant protein bound to the column was eluted with 250 mM imidazole in 0.1 M sodium phosphate, 350 mM NaCl, pH 7.4. PA fractions obtained after Ni–NTA affinity chromatography of >90% purity (as determined by densitometry of Coomassie brilliant blue stained 12% SDS–PAGE gel) were taken and the buffer was exchanged to 20 mM Hepes with 250 mM NaCl, pH 7.5, using Centricon concentrators of 30 kDa cut-off limit. The protein samples were stored at 4 °C for further use. Cell culture and cytotoxicity assay. Macrophage-like cell line RAW264.7 was maintained in RPMI 1640 medium containing 10% heat inactivated FCS, 25 mM Hepes, 100 U/ml penicillin, and 200 lg/ ml streptomycin under humidified environment supplemented with 5% CO2 at 37 °C [16]. For biological assay, a 96-well culture plate was prepared with 90% cell confluency. PA samples incubated at different temperatures were added to the cells at a final concentration of 0.8 lg/ ml along with LF at a final concentration of 0.3 lg/ml, unless mentioned otherwise, and incubated for 4 h at 37 °C. PA stored at 4 °C and unexposed to any higher temperature was used along with LF as a positive control. The same amount of additive (cosolvent) along with LF but without PA was used as a negative control. Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) dye. MTT dissolved in RPMI was added to each well at a final concentration of 0.5 mg/ml and incubated for another 45 min at 37 °C to allow uptake and reduction of the dye by viable cells. The spent medium was discarded and after drying the plate, 100 ll of 25 mM HCl in 90% isopropyl alcohol was added to each well to dissolve the cells and formazone crystals formed as a result of reduction of the MTT dye by viable cells. The plate was vortexed and the absorption of the solutions was read at 540 nm using a microplate reader (Biorad) to determine the cell viability. Experiments for monitoring loss of PA activity. PA (100 lg/ml in 20 mM Hepes, 25 mM NaCl, pH 7.4) was subjected to heating at 55 °C for 10 min. The sample was mixed with SDS loading buffer and analyzed by 12% SDS–PAGE to find out the presence of extra bands as an indication of autolysis. Untreated control PA kept at 4 °C was processed in a similar manner and loaded alongside the exposed sample on SDS–PAGE for comparison. To determine the denaturation/aggregation behavior of PA on heating, both spectrophotometeric and PAGE studies were carried out. PA sample (50 lg/ml in 20 mM Hepes with 25 mM NaCl, pH 7.4) was heated with a scan rate of 1 °C/min and increase in absorbance at different wavelengths, i.e., 278, 340, 440, 600, and 700 nm, was monitored in a Shimadzu UV–visible spectrophotometer to which a temperature programmer was attached. The loss of soluble form of PA on heating was further analyzed by non-denaturing and denaturing PAGE. PA protein samples (50 lg/ml in 20 mM Hepes, 25 mM NaCl, pH 7.4, and 40 mM Hepes containing 2.7 M glycerol + 500 mM NaCl, pH 7.9) were incubated for 10–20 min at 55 °C and subjected to native PAGE (4–15%) and SDS–PAGE (12%). For SDS–PAGE the heattreated PA samples were centrifuged at 12,000g for 10 min and the supernatant was separated from the pellet. The supernatant and pellet of the samples were then analyzed by SDS–PAGE to confirm the loss
S. Singh et al. / Biochemical and Biophysical Research Communications 322 (2004) 1029–1037 of the soluble PA form on heating. PA sample that had not undergone any heat treatment was included in both the gels as control. In order to correlate the loss of activity with the aggregation of PA (40 lg/ml) in 20 mM Hepes, 25 mM NaCl, pH 7.4, and 40 mM Hepes with 2.7 M glycerol + 500 mM NaCl, pH 7.9, was incubated in a thermal cuvette at 46 and 58 °C and the increase in absorbance at 340 nm (A340) with time was measured to assess the extent of aggregation. At regular interval of time samples were withdrawn from the cuvette and stored at 4 °C and the residual activity was assessed by cytotoxicity assay to correlate the loss of activity of PA with aggregation. To determine the effect of the concentration of protein as well as polyols on thermal inactivation of PA, the PA stock (20 mM Hepes with 250 mM NaCl, pH 7.5) was diluted in a suitable buffer/cosolvent solution to achieve different concentrations of PA or cosolvent. The PA samples at a final concentration of 8 lg/ml (unless mentioned otherwise) were incubated at 40–75 °C for 20 min and their residual activity was determined by cytotoxicity assay on RAW 264.7 cell lines. PA at a final concentration of 4, 8, 16, and 32 lg/ml was used to study the effect of protein concentration on the thermal inactivation of the protein while the polyol concentration ranged from 0.9 to 2.7 M. Temperature at which 50% activity was retained after 20 min of incubation (T1/2) was determined for comparison of the ability of different cosolvents to stabilize PA. The most stabilizing cosolvent was then evaluated for its efficacy to increase the half-life of thermal inactivation (t1/2) of PA (8 lg/ml) at 40 °C for 0–120 h. All the experiments were done in triplicate and there was ±5% error in measurements/variation in values.
Results and discussion Inactivation of PA by temperature Autolysis Autolysis of a sensitive protein results in cleavage into fragments of smaller length/size, e.g., recombinant human macrophage colony stimulating factor (rhMCSF), insulin, subtilisin J, neutral proteases from Bacillus subtilis [6,18,19], etc. To examine the possibility of autolysis of PA as the cause of loss of its activity at higher temperatures, PA (100 lg/ml) was incubated at 55 °C for 10 min and analyzed by SDS–PAGE. It is clear from SDS–PAGE results that the band corresponding to PA remains intact without the appearance of extra or smaller size bands after heating at 55 °C for 10 min (Fig. 1). This result suggested that autolysis is not responsible for the accelerated loss of activity of PA when incubated at temperatures as high as 55 °C. Temperature-induced aggregation of PA Denaturation of proteins can be easily monitored by a spectrophotometer as it is accompanied by a change in the spectral properties of the protein. These changes are mostly centered around a narrow wavelength band due to changes in the environment of some residues (e.g., Trp, Tyr) while in the case of aggregation, scattering of the incident light occurs which is inversely related to the wavelength of incident light. The scattering of light results in increase in attenuation of the incident
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Fig. 1. A 12% SDS–PAGE of PA sample (100 lg/ml in 20 mM Hepes with 25 mM NaCl, pH 7.4) incubated at 55 °C for 10 min.
beam (increase in absorbance) over a wide range of wavelengths and the observed change in absorbance due to scattering is more at shorter wavelengths than at longer wavelengths [20]. Thus, the pattern of the change in the absorbance at different wavelengths should be different in the case of denaturation and aggregation. PA (50 lg/ml in 20 mM Hepes, 25 mM NaCl, pH 7.4) was heated from 25 to 85 °C at a sweep rate of 1 °C/min and the change in absorbance per degree rise in temperature at the wavelengths 278, 340, 440, 600, and 700 nm was monitored as a function of temperature (Fig. 2). The increase in absorbance starts around 40 °C and reaches a maximum at 49 °C. The increase in absorbance is smooth which starts at the same temperature for all the wavelengths and is observed over the entire range of wavelengths used, indicating that PA is getting aggregated.
Fig. 2. Absorbance increase at different wavelengths with increase in temperature. The PA (50 lg/ml in 20 mM Hepes, 25 mM NaCl, pH 7.4) was heated at a sweep rate of 1 °C/min. The increase in absorbance per degree rise in temperature (DA) was monitored at different wavelengths and plotted as a function of temperature. DA: (absorbance at given temperature T °C) (absorbance at T 1 °C).
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Loss of soluble form of PA on heating Aggregation has been shown to be the main cause of inactivation in a number of proteins, e.g., human growth hormone [7], insulin [8], human relaxin [9], recombinant human keratinocyte growth factor [21], porcine growth hormone [22], etc. Aggregation of proteins results in the formation of large visible aggregates or small soluble oligomers. In both the cases, the size of the molecules/cluster increases which can be analyzed using native PAGE. The heat-treated samples of PA (100 lg/ml in 20 mM Hepes with 25 mM NaCl, pH 7.4, and 40 mM Hepes with 2.7 M glycerol + 500 mM NaCl, pH 7.9) were analyzed by non-denaturing PAGE and SDS–PAGE (Figs. 3A and B). Results of native or non-denaturing PAGE indicated that heat treatment of the control samples resulted in aggregation, forming large aggregates of PA that were unable to enter into the gel (Fig. 3A). This was further corroborated by SDS–PAGE of the heat-treated samples after centrifugation (Fig. 3B), which showed presence of PA in the pellet but not in the supernatant. This can be explained only by assuming that heat treatment of PA caused the formation of such large aggregates that were unable to enter in the native gel and got separated in the form of pellet on centrifugation. The presence of 2.7 M glycerol with 500 mM NaCl (the best condition able to prevent thermal inactivation) was able to prevent aggregation of PA and hence PA remained in its soluble (Fig. 3B) and monomeric forms (Fig. 3A).
Fig. 3. (A) Five to fifteen percentage non-denaturing PAGE. (B) Twelve percentage SDS–PAGE showing loss of soluble PA (Cont.) on heating and its prevention by 2.7 M glycerol + 500 mM NaCl (Glyl.) when incubated at 55 °C for 10 and 20 min. (Sup., supernatant of the sample; Cont., PA sample in 20 mM Hepes with 25 mM NaCl, pH 7.4; and Glyl., PA sample in 40 mM Hepes with 2.7 M glycerol + 500 mM, pH 7.9).
Correlation of loss of activity with aggregation The correlation between aggregation and loss of PA activity was determined by measuring the residual activity of PA with the progression of aggregation. The loss in PA activity closely followed aggregation (Fig. 4). Control PA sample (40 lg/ml in 20 mM Hepes buffer with 25 mM NaCl, pH 7.4) rapidly lost activity with the progression of aggregation (i.e., increase in absorbance at 340 nm) and plummeted to zero within 6 min. at 46 °C. The PA protein in 2.7 M glycerol with 500 mM NaCl in 40 mM Hepes, pH 7.9, the best condition able to prevent thermal inactivation neither showed aggregation even after 30 min of incubation at 46 or 58 °C incubation nor resulted in any loss of activity. These findings conclusively prove that aggregation is the primary cause of the thermal inactivation of PA at moderately high temperatures (46 or 58 °C) and the prevention of aggregation could help prevent its thermal inactivation. Effect of PA concentration on the thermal stability of PA Protein concentration is known to affect the protein aggregation behavior [23,24] and causes deterioration in the quality of a number of proteins [7– 9,21,22]. The protein concentration has also been shown to affect the transition temperature of unfolding (Tm) as well as initial unfolding temperature in a number of cases [6]. In the case of interferon-b-1a a decrease in initial unfolding temperature of about 9 °C (from 77 to 68 °C) has been reported when the protein concentration increased from 10 lg to 100 lg/ml [25]. We studied the effect of protein concentration on T1/2 of PA, the temperature at which 50% activity was retained after 20 min of incubation. To examine the effect of protein concentration on thermal inactivation of PA, protein solutions containing varying concentrations of PA viz., 4, 8, 16, and 32 lg/ml (40 mM Hepes, 25 mM NaCl, pH 7.9) were incubated at 40–52 °C for 20 min and tested for the residual activity by cytotoxicity assays. The loss of PA activity on heating was highly sensitive to protein concentration (Fig. 5). The sample with 4 lg/ml final PA protein concentration retained 95% activity even after 20 min incubation at 49 °C while under the same conditions sample with 8 lg/ml PA retained only 60% activity, and the samples having 16 and 32 lg/ml of final PA protein concentration completely lost the activity. This shows an inverse relationship between T1/2 and PA concentration. It is clear from Fig. 5 that a variation in the concentration of PA in the range of 4–32 lg/ ml resulted in a decrease in the T1/2 by 6 °C from 51 to 45 °C. Therefore, it can be safely inferred from the results that thermal inactivation of PA is highly sensitive to protein concentration.
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Fig. 4. Correlation of loss of activity with aggregation: PA (40 lg/ml) was incubated at (A) 46 °C in 20 mM Hepes with 25 mM NaCl, pH 7.4, (C) 46 °C in 40 mM Hepes with 2.7 M glycerol + 500 mM NaCl, pH 7.9, and (E) 58 °C in 40 mM Hepes with 2.7 M glycerol + 500 mM NaCl, pH 7.9, and increase in absorbance was monitored at 340 nm; (B,D, and F) are residual activity in (A,C, and E), respectively, during incubation. Residual activity (RA) was determined by cytotoxicity assay.
Fig. 5. The dependence of PA thermostability on protein concentration. Different concentrations of PA in 40 mM Hepes with 25 mM NaCl, pH 7.9, were incubated at different temperatures for 2 min and the residual activity was determined by cytotoxicity assay.
Effect of polyols on the thermal stability of PA Polyols are one of the best-studied classes of osmolytes, which are produced during various denaturing stresses in microorganisms, insects, and plants [26–28]. Polyols along with certain sugars are known to stabilize the native state of proteins [15,29–37]. The protein stabilizing ability of polyols is a result of their preferential exclusion from the protein surface [35–38], leading to the preferential hydration of the protein molecule. The solvophobic nature of the peptide backbone that promotes folding and compaction of the protein molecule in the presence of polyols pushes the native () denatured/unfolded equilibrium between the native and the unfolded protein in favor of the native state and stabilizes the protein molecule [39–42]. The effect of various polyols on the thermal stability of PA was investigated by incubating PA with varying concentrations of polyols with and without 500 mM
NaCl at different temperatures (46–75 °C) for 20 min and then comparing the residual activity as determined by the cytotoxicity assay (Fig. 6). The effect of 0.9, 1.8, and 2.7 M polyol on the thermal stability of PA is shown in Figs. 6A–C, respectively. The T1/2 values for different polyols with and without 500 mM NaCl have also been determined and presented in Table 1. In general, the stabilizing effect of different polyols increased with an increase in their concentration but differed greatly in magnitude from each other. Glycerol increased the T1/2 for PA to the maximum extent (>25 °C) compared to control, which was more than 17 °C higher compared to T1/2 achieved earlier using glycine [17]. In general at any given molarity, the ability of polyols to stabilize PA followed the order glycerol > sorbitol > xylitol > adonitol > erythritol. It was observed that on addition of 500 mM NaCl to polyols the stability of PA invariably increased and the magnitude was always greater than in the presence of polyol or NaCl alone (Fig. 6D. It suggests that the mechanism of stabilization of PA is different in the case of polyols and salts. In the presence of 500 mM NaCl, the order of stabilization of PA changed to glycerol > xylitol > sorbitol > adonitol > erythritol. Although polyols and NaCl act in tandem and stabilize the protein, yet the presence of NaCl affected the interaction of polyol with the protein in some subtle way, which was different for each polyol. Salts stabilize proteins by the screening of unfavorable or repulsive charge–charge interactions [43,44] or by favorable electrostatic binding of ions [43–45] while polyols are known to increase the surface tension of water [15,35] and strengthen hydrophobic interactions [39–42].
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Fig. 6. Effect of polyols on the stability of PA: (A) 0.9 M; (B) 1.8 M; (C) 2.7 M; (D) 0.9 M polyol + 500 mM NaCl; and (E) 2.7 M glycerol + 500 mM NaCl and 2.7 M glycerol only. PA (8 lg/ml) in 40 mM Hepes, pH 7.9, was incubated with different concentrations of polyols with and without 500 mM NaCl at different temperatures for 20 min. (A–D) or longer (E) and residual activity was determined by cytotoxicity assay. (Ado, adonitol; Xyl, xylitol; Ery, erythritol; Glyl, glycerol; Sor, sorbitol; Cont, PA in 25 mM NaCl; and N, 500 mM NaCl unless mentioned otherwise).
Some interesting observations were made during this investigation. For example, erythritol destabilized PA at lower concentrations while with increase in its concentration its ability to stabilize PA increased. T1/2 for PA at 0.9, 1.8, and 2.7 M erythritol were 43.7, 46.3, and 51.5 °C, respectively (Table 1), while that of control was 50.3 °C. This could be because of strong specific destabilizing interactions of erythritol with PA at lower concentrations which get overwhelmed by the weak, but large, stabilizing interactions at higher concentration as suggested earlier in the case of stabilization of BSA by glycine [46]. Adonitol and xylitol, which are epimers, stabilized PA to different levels. At 0.9 M concentration adonitol behaved like control but increase in the concentration increased the stabilization of PA, while xylitol behaved like a good stabilizer among the polyols studied. Different behavior of epimers has been reported earlier in the case of collagen. The differential effect of epimers towards stabilizing collagen molecule has been
attributed to differences in the solvent organization property of the solute molecules because of variation in the stereochemistry of the hydroxyl groups [47]. Effect of glycerol towards stabilization of PA was highly concentration dependent. At 0.9 M it was similar to that of sorbitol with a T1/2 of 55.7 °C, but with increasing concentration its effect deviated from that of sorbitol (Fig. 6C). An interesting behavior was observed when 500 mM NaCl was present along with 0.9 M glycerol (Fig. 6D). The residual activity obtained on incubation of PA for 20 min. in the range of 58–70 °C was 60– 75%, which deviated from the usual pattern of decrease in the activity of PA in the presence of other polyols in the same range of temperatures. It could be due to glycerol assisted refolding of the heat-denatured PA. Glycerol has been shown to increase the refolding yield of a number of proteins, e.g., rhodanese, rabbit muscle creatine kinase, porcine pancreatic elastase [48–50], etc. Earlier, it has been shown that polyols also affect the
S. Singh et al. / Biochemical and Biophysical Research Communications 322 (2004) 1029–1037
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Table 1 Effect of polyols on T1/2 of PA (Fig. 6) Co-solvent
Concentration (M)
T1/2 (20 min) in °C
Control (25 mM NaCl) Glycerol Glycerol Glycerol Glycerol Erythritol Erythritol Erythritol Erythritol Adonitol Adonitol Adonitol Adonitol Sorbitol Sorbitol Sorbitol Sorbitol Xylitol Xylitol Xylitol
0 0.9 1.8 2.7 0.9 M + 500 mM 0.9 1.8 2.7 0.9 M + 500 mM 0.9 1.8 2.7 0.9 M + 500 mM 0.9 1.8 2.7 0.9 M + 500 mM 0.9 1.8 0.9 M + 500 mM
50.3 55.7 61.9 >75 >70 43.7 46.3 51.5 <55 50.2 53.2 57.0 59.7 55.3 58.4 62.5 60.2 53.5 55.1 64.5
NaCl
NaCl
NaCl
NaCl
NaCl
Note. PA was incubated at a final concentration of 8 lg/ml in the presence of various polyols for thermostability studies. PA was used at a final concentration of 0.8 lg/ml along with 0.3 lg/ml of LF for cytotoxicity assay on RAW264.7 cell line.
refolding of heat-denatured proteins to varying extents, e.g., chymotrypsinogen, lysozyme, ribonuclease, and staphylococcal nuclease [31–33,51]. The presence of NaCl along with polyols invariably resulted in the enhancement of residual activity obtained at any given temperature as compared to the residual activity obtained when only polyols were used (Fig. 6D and Table 1). This observation indicates that the mechanisms operational in the stabilization of the PA by polyols and NaCl are different and additive. The better stabilization achieved in the presence of glycerol in comparison to sorbitol can be due to better refolding of the heat-denatured PA on cooling. Stabilization of PA by xylitol and sorbitol invariably increased with addition of NaCl but the order of the overall stabilization achieved differed. At 0.9 M concentration, sorbitol stabilized PA better than xylitol but addition of NaCl reversed the trend and xylitol along with NaCl led to a greater stabilization of PA as compared to sorbitol along with NaCl (Fig. 6D and Table 1). This observation indicates that though the mechanisms operational in the stabilization of PA by polyols and NaCl are different in nature, they can interfere with each otherÕs interactions with the protein molecule in a subtle manner. When PA was incubated at 58–75 °C in the presence of 2.7 M glycerol + 500 mM NaCl for 0–160 min, the rate of loss of activity was initially slow followed by a fast phase (Fig. 6E). It was found that loss of PA activity in the presence of 2.7 M glycerol on incubation at 64 °C follows approximately the same profile as 2.7 M glyc-
Fig. 7. Effect of 2.7 M glycerol + 500 mM NaCl on stability of PA on extended incubation at 40 °C: PA (8 lg/ml) in 40 mM Hepes, 25 mM NaCl and 40 mM Hepes, and 2.7 M glycerol + 500 mM NaCl were incubated at 40 °C for 0–120 h and the residual activity was determined by cytotoxicity assay. (2.7 M Glyl + 500 mM NaCl: 2.7 M glycerol with 500 mM NaCl; Cont: PA in 25 mM NaCl only).
erol + 500 mM NaCl follows on incubation at 75 °C, which strongly supports the notion that mechanisms operating for the stabilization of PA by polyols and salts are different in nature (Figs. 6A, D, and E). Feasibility of using 2.7 M glycerol with 500 mM NaCl to increase the half-life of thermal inactivation of PA at 40 °C was examined by incubating PA for up to 120 h. The 2.7 M glycerol along with 500 mM NaCl was able to extend the half-life (t1/2) of thermal inactivation of PA at 40 °C by more than 6 times over the control, i.e., from 7 h. to more than 42 h (Fig. 7). This increase in t1/2 is 1.5 times higher as compared to the previously reported result using glycine as a stabilizer [17]. To conclude, this study suggests that thermal inactivation of PA at moderately high temperatures is primarily caused by aggregation and other processes such as autolysis do not have any significant role in its inactivation mechanisms. The ability of polyols to stabilize the PA molecule against thermal inactivation varied widely. While erythritol destabilized PA molecule at lower concentrations on one hand, 2.7 M glycerol along with 500 mM NaCl was able to lead to 100% retention of PA activity on shorter incubation even at 70 °C on the other hand. The combination of 2.7 M glycerol and 500 mM NaCl also extended the half-life of thermal inactivation of PA at 40 °C by more than 6 times over the control. This study besides elucidating the primary cause of thermal inactivation of PA helped to identify conditions to considerably increase the half-life of PA against thermal inactivation at moderately high temperatures which would further help in improving the shelflife of the recombinant PA vaccine.
Acknowledgment The work was supported by the grant from World Bank assisted National Agricultural Technology Project (NATP), ICAR, New Delhi.
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