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Stabilization and re-activation of trapped enzyme by immobilized heat shock protein and molecular chaperones
Biosensors and Bioelectronics 18 (2003) 311 /317 www.elsevier.com/locate/bios
Stabilization and re-activation of trapped enzyme by immobilized heat shock protein and molecular chaperones Yunhui Yang 1, Jiang Zeng, Chunguang Gao 2, Ulrich J. Krull Department of Chemistry, Chemical Sensors Group, University of Toronto at Mississauga, 3359 Mississauga Road North, Mississauga, ON, Canada L5L 1C6 Received 31 October 2001; received in revised form 9 April 2002; accepted 15 July 2002
Abstract The potential of using immobilized Heat Shock Protein 70 (HSP 70) in combination with other molecular chaperones to ameliorate problems of enzyme denaturation was investigated. Firefly luciferase was used as a model enzyme due to its sensitivity to thermal denaturation, and the availability of a sensitive chemiluminescent assay method for determination of relative activity of this enzyme. Control experiments and development of effective combinations of HSP with other chaperones involved re-activation of enzyme in bulk solution. A combination of HSP 70, a-crystallin and reticulocyte lysate (RL) in bulk solution were found to reactivate soluble firefly luciferase to about 60% of the initial activity after the enzyme activity had been reduced to less than 2% by thermal denaturation. HSP 70 that was covalently immobilized onto glass surfaces was also able to re-activate denatured enzyme that was in bulk solution. Over 30% of the initial activity could be regained from heat denatured enzyme when using immobilized HSP in the presence of other chaperones. The activity of soluble enzyme decayed to negligible values in a period of days when stored at room temperature. In the presence of immobilized HSP and chaperones, activity stabilized at about 10% of the initial activity even after many weeks. The results suggest that immobilized molecular chaperones such as HSP 70 may provide some potential for stabilization and re-activation of enzymes that are trapped in thin aqueous films for applications in biosensors and reactors. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Molecular chaperones; HSP 70; a-Crystallin; Immobilization; Re-activation; Firefly luciferase; Heat shock protein; Reticulocyte lysate
1. Introduction A problem that is commonly encountered in biosensor development has been the denaturation of entrapped and immobilized enzymes. Denaturation of active proteins is also a problem encountered by any living organism, and a number of natural remediation mechanisms and pathways have been identified (Kiang and Tsokos, 1998; Santoro, 2000). Molecular chaperones are
Corresponding author E-mail address: [email protected] (U.J. Krull). 1 Present address: Department of Chemistry, Yunnan Normal University, Kunming 650091, PR China. 2 Present address: Department of Chemistry, Shanxi University, Taiyuan 030006, PR China.
cellular proteins widely present in prokaryotic and eukaryotic cells that bind and stabilize the structure of other proteins and polypeptides to facilitate their biological functions. Heat Shock Protein 70 (HSP 70, also known as DnaK) belongs to a family of molecular chaperones that are responsible for preventing damage to proteins that is induced by environmental stress such as heating. HSP can also refolding damaged proteins to regenerate active conformations. HSP 70 has been shown to have such functions both in vivo (Souren et al., 1999; Nollen et al., 1999) and in vitro (Lee and Vierling, 2000). HSP 70 can direct correct folding by binding to nascent or unfolded polypeptides and/or the folding intermediates, to prevent improper polypeptide chain interactions that lead to aggregation (Hartl, 1996).
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HSP 70 is perhaps the best studied example of molecular chaperones. It represents a family of highly conserved ATPases of relative molecular mass about 70,000 Da. They are structurally subdivided into an Nterminal ATPase domain of /45 kDa, a /18 kDa portion containing the peptide-binding site, and a more variable /10 kDa segment. HSP 70 has essential roles in protein metabolism under both stress and non-stress conditions. This regulatory ability results from the binding and release of hydrophobic segments of an unfolding polypeptide chain in an ATP-hydrolytic reaction cycle. The quantitative detection of HSP has been proposed as an indicator of the level of stress that an organism experiences (Dunlap and Matsumura, 1997; Karouna-Renier and Zehr, 1999; Van Overmeire et al., 2001). Correct folding of proteins in vitro often requires the assistance of other molecular chaperones (Schroder et al., 1993). For example, reticulocyte lysate (RL) from rabbit has been reported to assist HSP 70 in repairing denatured firefly luciferase. RL contains cellular components necessary for protein synthesis (tRNA, ribosomes, amino acids, initiation, elongation and termination factors), and an extensive endogenous chaperone network, where those assisting the function of HSP 70 have not been fully defined (Minami et al., 2000). a-crystallin, a major eye lens protein, along with homologous heat shock protein 25 (HSP 25), are members of another small HSP family. Recent in vitro data indicates that small HSPs act to bind non-native protein folding intermediates, preventing their aggregation so as to maintain them in a state that is available for ATP-dependent refolding by other chaperones (Carver et al., 1995). This new study investigates the use of an immobilized HSP for re-activation of soluble denatured enzyme that is trapped in a small aqueous volume. The primary chaperone, HSP 70, was covalently immobilized to a glass surface. Initial investigations were done using HSP 70 in solution so as to confirm observations of reactivation, and then to develop some optimization of the effectiveness of HSP 70 in mixture with other chaperones. Firefly luciferase (Luc), was chosen as a model enzyme for this study. Due to the availability of a highly sensitive assay for Luc activity, Luc has been used extensively as a model for protein folding in vitro. The fact that Luc is inactivated at moderate temperatures (39 /42 8C) makes it an excellent model protein for chaperone studies (Blum et al., 1985). The enzyme produces yellow green light (562 nm) in the presence of ATP, luciferin (D-LH2), Mg2 and molecular oxygen, with a quantum efficiency of as high as 0.88 according to the following reaction: LucLH2 ATP 0 LucLH2 AMPPPi
LucLH2 AMPO2 0 LucoxyluciferinAMP CO2 light
2. Materials and methods 2.1. Reagents Adenosine 5?-triphosphate (ATP), luciferin, firefly luciferase, a-crystallin, dithiothreitol (DTT), human HSP 70 (recombinant expressed in Escherichia coli ), 3aminopropyltriethoxy-silane (APTES), glutaraldehyde, and bovine serum albumin (BSA) were obtained from Sigma Canada (Oakville, Canada). Reticulocyte lysate from rabbit and Luciferase Assay Mix were from Promega (Whitby, CA). The inner bottom surfaces of 5 ml glass vials (Pierce Reacti-Vial, Rockford, IL) were used to immobilize HSP 70. 2.2. Instrumentation A water bath with temperature control of 9/0.1 8C (Lauda, Germany) was used to thermally denature Luc. A liquid scintillation counter (Model 1409, Wallac, Finland) was used to measure the light emission catalyzed by Luc. 2.3. Cleaning of glass surfaces Glass surfaces were cleaned using a 1:1:5 mixture of 25% ammonia, 30% H2O2 and distilled water at 80 8C with stirring for 5 min. After washing with water the glass samples were treated with a 1:1:5 mixture of 15 M HCl, 30% H2O2 and distilled water, at 80 8C for 5 min with stirring. Finally, the samples were rinsed twice with 15 ml portions of H2O, 25 ml methanol, 20 ml CH2Cl2, and 30 ml diethyl ether. Any residual diethyl ether was removed under reduced pressure. 2.4. Evaporation silanization Glass samples were rinsed with 2% APTES solution in dry chloroform, and heated to 60 8C until dry. They were then washed with chloroform and cured at 120 8C in an oven for 24 h, followed by storage it a vacuum desiccator. 2.5. Activation of silane with glutaraldehyde Coupling buffer was prepared using 0.015 ml of 25% glutaraldehyde and 0.285 ml of 10 mM phosphate buffer (pH 7.5), and contained 2 mM DTT. Glass surfaces were covered with this solution while being gently stirred for 90 min at room temperature. The activated glass
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surfaces were washed with coupling buffer four times (Raman Suri and Misshra, 1996).
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buffer (total 250 ml). This solution was heated with immobilized HSP 70 for 8 min at 40 8C and cooled in an ice bath for 30 s.
2.6. Immobilization of HSP 70 A solution of 20 mL 14.3 mM HSP 70 in 0.23 ml coupling buffer was prepared. The activated glass surfaces were covered with this solution and were incubated for 5 h at room temperature. After incubation, the glass samples were washed with ice-cold coupling buffer containing 1 mol/l NaCl to wash away loosely bound/absorbed protein (Riberio et al., 1998), and were then finally washed with coupling buffer alone. The protein-coated glass surfaces were covered with 0.5 mg/ml NaBH4 solution for 40 min at 4 8C to stabilize the coupling. This was followed by coverage of the surfaces with 1.0 mg/ml BSA solution for 2 h to minimize subsequent non-selective binding. 2.7. Co-immobilization of the complex of HSP 70 and Luc A volume of 104 ml of 2.68 mM Luc and 20 ml 14.3 mM HSP 70 were added into 126 ml of coupling buffer. The remaining steps were as stated for covalent immobilization of HSP 70. 2.8. Luc activity assay The assay solution was composed of 1.2 mM ATP, 0.4 mM luciferin, 5 mM MgSO4, 0.5 mM EDTA, 0.5 mM DTT, 0.1 mg/ml BSA, in 25 mM Tricine at pH 7.8. A volume of 2.5 ml of denatured or re-activated Luc samples was added to 50 ml of the assay solution. Alternatively, 50 ml of commercial Luciferase Assay Mix was used to replace the prepared assay solution for measuring the Luc activity. After mixing, the vial was placed into a scintillation counter for photon counting. Photon emission was measured for a period of 1 min. 2.9. Thermal denaturation of Luc One micromolar Luc was heated for 8 min at 39.5 8C in pH 7.5 denaturing buffer (25 mM HEPES, 5 mM MgSO4, 5 mM DTT, 150 mM KCl) containing 1 mM acrystallin and 1 mM HSP 70 but no ATP (total volume 50 ml). This was sufficient to denature the Luc to less than 2% of its original activity. After heating, samples were immediately cooled in an ice bath for 30 s. Similar experiments were done where complexes of HSP 70 were activated by addition of 1.64 mM ATP during the heating step, and/or after denaturation. When studying the effectiveness of re-activation by immobilized HSP 70, the solution that was used to cover the immobilized HSP 70 consisted of 1 mM Luc, 1 mM acrystallin, with or without 1.64 mM ATP, in denaturing
2.10. Re-activation of thermally inactivated Luc by free HSP 70 After denaturation of Luc, refolding was initiated by adding 1 ml of the denaturation solution to 40 ml of refolding buffer containing 7.5 mM BSA, 50 mM KCl and 2 mM ATP, with or without 50% (v/v) RL and/or 1.5 mM HSP 70, and/or 1 mM a-crystallin. Refolding was allowed to proceed by incubation at 30 8C for up to 2 h. Luc activity was measured and expressed as percentages relative to that of an equivalent amount of native Luc that had not been heat denatured. 2.11. Re-activation of thermally inactivated Luc by immobilized HSP 70 After the denaturation solution containing Luc was heated, the sample was incubated at 30 8C using refolding solution as was done for HSP 70 when in bulk solution. Luc activity was measured at time intervals by taking 2.5 ul of solution, and adding 50 ul prepared assay solution to this sample. Luc activity was expressed as percentages relative to that of an equivalent amount of native Luc that had not been thermally denatured. Control experiments used immobilized HSP 70 that was denatured in solution at 90 8C for 10 min. The long-term stability of immobilized HSP 70 was examined by re-using surfaces that were coated with immobilized protein, and daily repetition of the denaturation/re-activation experiments were done using freshly prepared solutions.
3. Results and discussion The biochemical cascade that is associated with the function of HSP 70 has been previously modeled as a multi-step cycle as shown in Fig. 1 (Santoro, 2000; Hartl, 1996; Schroder et al., 1993). This is a relatively complicated cascade that has not yet been completely characterized. The active reagents can be obtained by extraction from natural sources, but these preparations are also not completely characterized. From the cascade scheme, the rate of recovery of enzyme activity, when assisted by HSP 70 and other chaperones, is anticipated to be dependent on the presence of ATP. It has been reported that ATP plays a role in the protection and refolding of denatured proteins by HSP 70 (Lee and Vierling, 2000). The chaperone activities of HSP 70 are usually ATPdependent (Hartl, 1996). Our experiments confirmed that if ATP was present during the course of the heat
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with RL was used for re-activation. Recovery of activity was at least 10% higher again when ATP was present. Small HSPs are known to be particularly effective in preventing thermal aggregation of other proteins and maintain them in a state that is suitable for ATPdependent refolding by other chaperones. In this study, a-crystallin was examined as a small HSP that might assist in the recovery of the enzymatic activity of Luc. When Luc was heated in the presence of a mixture of acrystallin, RL, HSP 70 and ATP, about 60% recovery of the activity of Luc was observed over a period of less than 2 h (Fig. 2). The same experiment without ATP addition provided a recovery of activity of only about 40%. The same experiment with ATP added, but without a-crystallin provided activity recovery of only about 30%. The results demonstrate that restoration of Luc activity can be observed, but is relatively small, if only HSP 70 is present. Significant gains can be achieved if ATP is present, and if other chaperones or chaperonelike proteins such as a-crystallin are available. 3.2. Re-activation of Luc using covalently immobilized HSP 70 Fig. 1. A model indicating the cycle of function of HSP 70 and some co-factors.
denaturation and re-activation of Luc, a significantly improved rate of recovery of activity could be achieved in comparison to the case where ATP was not added. 3.1. Re-activation of Luc using HSP 70 in solution Thermally denatured Luc did not recover more than 5% of its original activity, and usually indicated no measurable recovery at all. In the presence of HSP 70 alone, only a small degree of re-activation of denatured Luc (to about 10% activity) could be observed in solution (Fig. 2). It is known that the efficiency of refolding of Luc by HSP 70 can be assisted by other molecular chaperones and cofactors such as DnaJ and GrpE. RL contains an extensive endogenous chaperone network which can help HSP 70 to reactivate denatured Luc (Yonehara and Minami, 1996). Only about 10% of enzyme activity was recovered when RL was used alone to reactivate denatured Luc. In the presence of both HSP 70 and RL, the reactivation of enzyme became more efficient. This result was maximized when denaturation was done in the presence of HSP 70. It is likely the case that HSP 70 prevented irreversible denaturation of Luc and maintained it in a folding-competent state during thermal denaturing. About 40% recovery of the Luc activity was observed in situations where denaturation was done in the presence of HSP 70, and a combination of HSP 70
The capability of using immobilized HSP in achieving protection and renaturation of enzymes may provide a wider range of opportunities for applications in areas such as biosensor preparation. It was not clear whether HSP 70 could be immobilized with substantial retention of its functional properties. It is clear in the literature that some forms of HSP exist at cell surfaces, and are functional in terms of protection and re-activation of proteins that are in solution (Hoffman and Garduno, 1999; Kiang and Tsokos, 1998). A report about covalent immobilzation of HSP 90 to carboxymethyl-dextran surface-plasmon resonance (SPR) chips indicated that some activity was retained for binding to denatured proteins, and Kd values for binding interactions were reported (Csermely et al., 1997). A report about crosslinking of a-crystallin indicated that a substantial reduction of chaperone-like activity was noted (Shridas et al., 2001). A typical non-specific, non-orienting protein immobilization protocol was selected for our preliminary studies, and immobilization was based on use of surface activation using silane, followed by glutaraldehyde reaction. Glass vials were treated to obtain the following three surface modifications: (a) Native HSP 70 was immobilized onto glass surfaces; (b) HSP 70 was treated by heating at 90 8C for 10 min, and then was immobilized onto glass surfaces; and (c) surfaces were treated with BSA solution. The supporting solution contained Luc, a-crystallin, ATP and denaturing buffer, as these provided good recoveries of activity in experiments
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Fig. 2. Some representative results of percentage of re-activation of thermally denatured Luc based on initial activities without thermal denaturation. One micromolar Luc was heated for 8 min at 39.5 8C at pH 7.5 in denaturing buffer (25 mM HEPES, 5 mM MgSO4, 5 mM DTT, 150 mM KCl) with or without additives, followed by re-activation, with or without additives: Sample 1: Heating, no additives; Re-activation, no additives. Sample 2: Heating, a-crystallin; Re-activation, RL. Sample 3: Heating, HSP 70, ATP; Re-activation, RL, HSP 70. Sample 4: Heating, a-crystallin, HSP 70; Re-activation, RL, HSP 70. Sample 5: Heating, a-crystallin, HSP 70; Re-activation, HSP 70. Sample 6: Heating, a-crystallin, HSP 70, ATP; Reactivation, RL, HSP 70.
that were done in bulk solution. RL was not used in the experiments that involved surface immobilized HSP 70 because the complexity of the RL solution was such that it was not possible to begin to understand issues of nonselective adsorption and co-factor availability.
In all three cases, the activity of Luc was reduced to less than 2% after heating at 40 8C for 8 min in the vials. The denatured Luc was left in the vials for incubation at 30 8C, and the activity was observed to gradually recover (Fig. 3). Over a period of 200 min, the
Fig. 3. Time dependence of re-activation using immobilized HSP 70. Glass vials were treated to obtain the following three surface modifications: (a) HSP 70 was immobilized onto glass surfaces ("); (b) HSP 70 was treated by heating at 90 8C for 10 min, and then was immobilized onto glass surfaces (control, j); Surfaces were treated with BSA solution (control, '). The supporting solution in all experiments contained Luc, a-crystallin, ATP and denaturing buffer. Luc was denatured in each case at 40 8C for 8 min. The sample was then incubated in denaturing solution at 30 8C. Luc activity was measured at various time intervals and expressed as percentages relative to that of an equivalent amount of native Luc measured prior to the heating step.
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Fig. 4. The ability of immobilized HSP 70 to re-activate Luc after various periods of time. The same vials that were coated with HSP 70 were used on a daily basis to assess the ability to re-activate Luc.
activity was recovered to about 30% of the initial value before thermal denaturation. However, when the Luc was incubated with immobilized, 90 8C heat treated HSP 70, only about 5% of the activity recovery was observed (Fig. 3). Luc incubated in the BSA treated vial did not recover more than 2% of the initial activity. Immobilized HSP 70 was found to be able to reactivate Luc for a period of weeks after immobilization. The ability to re-activate the denatured enzyme remained relatively high for the first 2 days, and then reduced, with only about one-third of the initial reactivation being noted 6 days after immobilization (Fig. 4). However, the ability to re-activate to a level of about 10% of initial activity was available for weeks. Enzyme stored in aqueous buffer solution in glass vials at room temperature effectively lost all of its activity with a few days. Enzyme that was stored in the treated vials in the presence of the chaperones suffered a reduction in activity over a period of a week, but stabilized at an activity level of about 10%, and this remained constant for weeks. A preliminary experiment considered whether recovery of activity of chemically denatured enzyme could be achieved. The recovery of activity of chemically denatured firefly luciferase (Rungeling et al., 1999), and carbonic anhydrase (Nam and Walsh, 2002) has been shown to be possible by the action of molecular chaperones including HSP 70. In these reports, it was noted that chemical denaturation induced by materials such as guanidinium hydrochloride was not as readily reversed by chaperones (68% reported activity recovery) as was the case for re-activation after thermal denaturation (97% activity recovered). Our initial experiments
indicated that chemically denatured enzyme was reactivated by the immobilzed HSP system, and that activity levels of up to about 15/20% could be observed. An investigation was completed to determine whether co-immobilized HSP 70/Luc could improve the lifetime of the activity of Luc. The experimental conditions that were used included addition of a-crystallin and ATP after the immobilization. The HSP 70 and Luc were immobilized concurrently on the activated glass surface, with the expectation that they likely complexed in solution, and that the complexes might survive attachment to the surface. A control experiment was done using BSA to replace HSP 70. The results show that there is no significant difference in enzyme activity over time between immobilized HSP 70/Luc and BSA/Luc (typical enzyme activity decay where most of the initial activity is lost within days, data not shown). A further investigation was completed to determine whether co-immobilized HSP 70 and Luc could increase the thermal endurance of Luc. After immobilization, the samples were heated in the presence of a-crystallin and ATP at 40 8C for 8 min, using co-immobilized BSA/Luc as a control. The results indicate that co-immobilization of HSP 70/Luc does not increase the thermal endurance of Luc (data not shown).
4. Conclusions Luciferase re-activation after thermal denaturation could be achieved with regeneration of up to 60% of the initial enzyme activity by appropriate selection of Heat Shock Protein, molecular chaperones and co-factors.
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The protection procedure is at least partially ATPdependent, although some re-activation can be achieved in the absence of ATP. Covalently immobilized HSP 70 can re-activate thermally denatured Luc to a level of about 5 /10% activity, indicating that at least some significant quantity of the chaperone is in an active binding form. An improvement is observed when a-crystallin and ATP are introduced into the solution phase. The improvement provides activity recovery to a level of about 30%, which is only marginally smaller than what was observed in comparable experiments that were done using free HSP 70 and a-crystallin in solution (about 40% activity). After 6 days, immobilized HSP 70 in combination with acrystallin was still able to provide some re-activation of thermally denatured enzyme, and retained about onethird of its original activation ability. Similar levels of re-activation were noted for some weeks. The reduction in the ability of immobilized HSP 70 to provide reactivation after some days suggests that denaturation of the protein takes place on the glass surface. While the results of this work suggest that HSP 70 and Luc can form a complex in solution, it would appear that co-immobilization of HSP 70 and Luc likely causes inactivation of the important structural features of this complex that are responsible for maintaining activity of the enzyme. The immobilization method that was used in this work likely prevents the enzyme from undergoing the action of protein folding that is caused by the HSP 70 and co-factors. These preliminary results suggest that protein mobility is important, so that advantageous use of molecular chaperones might best be achieved for systems that use immobilized HSP and free or entrapped enzymes.
Acknowledgements We are grateful to the Natural Sciences and Engineering Council of Canada for financial support of this work. Y.Y. extends her thanks to the China Scholarship Council program, and C.G. extends his thanks to the government of Shanxi Province, for fellowship support as visiting scholars.
References Blum, L.J., Coulet, P.R., Gautheron, D.C., 1985. Collagen strip with immobilized luciferase for ATP bioluminescence determination. Biotech. Bioeng. 27, 232 /245. Carver, J.A., Guerreiro, N., Nicholls, K.A., Truscott, R.J.W., 1995. On the interaction of a-crystallin with unfolded proteins. Biochim. Biophys. Acta 1252, 251 /260.
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Csermely, P., Miyata, Y., Soti, C., Yahara, I., 1997. Binding affinity of proteins to HSP 90 correlates with both hydrophobicity and positive charges. A surface plasmon resonance study. Life Sci. 63, 411 /418. Dunlap, D.Y., Matsumura, F., 1997. Development of broad spectrum antibodies to heat shock protein 70s as biomarkers for detection of multiple stress by pollutants and environmental factors. Ecotoxicol. Environ. Saf. 37, 238 /244. Hartl, F.U., 1996. Molecular chaperones in cellular protein folding. Nature 381, 571 /578. Hoffman, P.S., Garduno, R.A., 1999. Surface-associated heat shock proteins of Legionella pneumophila and Helicobacter pylori : roles in pathogenesis and immunity. Infect. Dis. Obstetr. Gynecol. 7, 58 / 63. Karouna-Renier, N.K., Zehr, J.P., 1999. Ecological implications of molecular biomarkers: assaying sub-lethal stress in the midge Chironomus tentans using heat shock protein 70 (HSP-70) expression. Hydrobiologia 401, 255 /264. Kiang, J.G., Tsokos, G.C., 1998. Heat shock protein 70 kDa: molecular biology, biochemistry and physiology. Pharmacol. Ther. 80, 183 /201. Lee, G.J., Vierling, E., 2000. A small heat shock protein cooperates with heat shock protein 70 systems to reactivate a heat-denatured protein. Plant Physiol. 122, 189 /198. Minami, Y., Kawasaki, H., Minami, M., Tanahashi, N., Tanaka, K., Tahara, I., 2000. A critical role for the proteasome activator PA28 in the HSP 90-dependent protein refolding. J. Biol. Chem. 275 (12), 9055 /9061. Nam, S.-H., Walsh, M.K., 2002. Affinity purification and characterization of the Escherichia coli molecular chaperones. Protein Expr. Purif. 24, 282 /291. Nollen, E.A.A., Brunsting, J.F., Roelofsen, H., Weber, L.A., Kampinga, H.H., 1999. In vivo chaperone activity of heat shock protein 70 and thermotolerance. Mol. Cell Biol. 19 (3), 2069 /2079. Raman Suri, C., Misshra, G.C., 1996. Activating piezoelectric crystal surface by silanization for microgravimetric immunobiosensor application. Biosens. Bioelectron. 11 (12), 1199 /1205. Riberio, A.R., Santos, R.M., Rosario, L.M., Gil, M.H., 1998. Immobilization of luciferase from a firefly lantern extract on glass strips as an alternative strategy for luminescent detection of ATP. J. Biolumin. Chemilumin. 13, 371 /375. Rungeling, E., Laufen, T., Bahl, H., 1999. Functional characterisation of the chaperones Dna K, Dna J and Grp E from Clostridium acetobutylicum . FEMS Microbiol. Lett. 170, 119 /123. Santoro, M.G., 2000. Heat shock factors and the control of the stress response. Biochem. Pharmacol. 59, 55 /63. Schroder, H., Langer, T., Hartl, F.-U., Bukau, B., 1993. DnaK, DnaJ and GrpE from a cellular chaperone machinery capable of repairing heat-induced protein damage. EMBO J. Biol. Chem. 12 (11), 4137 /4145. Shridas, P., Sharma, Y., Balasubramanian, D., 2001. Transglutaminase-mediated cross-linking of a-crystallin: structural and functional consequnces. FEBS Lett. 499, 245 /250. Souren, J.E.M., Wiegant, F.A.C., Van Wijk, R., 1999. The role of hsp70 in protection and repair of luciferase activity in vivo; experimental data and mathematical modeling. Cell. Mol. Life Sci. 55, 799 /811. Van Overmeire, I., Clark, G.C., Brown, D.J., Chu, M.D., Cooke, W.M., Denison, M.S., Baeyens, W., Srebrnik, S., Goeyens, L., 2001. Trace contamination with dioxin-like chemicals: evaluation of bioassay-based TEQ determination for hazard assessment and regulatory responses. Environ. Sci. Policy 4, 345 /357. Yonehara, M., Minami, Y., 1996. Heat-induced chaperone activity of HSP90. J. Biol. Chem. 271 (5), 2641 /2645.
Stabilization and re-activation of trapped enzyme by immobilized heat shock protein and molecular chaperones Yunhui Yang 1, Jiang Zeng, Chunguang Gao 2, Ulrich J. Krull Department of Chemistry, Chemical Sensors Group, University of Toronto at Mississauga, 3359 Mississauga Road North, Mississauga, ON, Canada L5L 1C6 Received 31 October 2001; received in revised form 9 April 2002; accepted 15 July 2002
Abstract The potential of using immobilized Heat Shock Protein 70 (HSP 70) in combination with other molecular chaperones to ameliorate problems of enzyme denaturation was investigated. Firefly luciferase was used as a model enzyme due to its sensitivity to thermal denaturation, and the availability of a sensitive chemiluminescent assay method for determination of relative activity of this enzyme. Control experiments and development of effective combinations of HSP with other chaperones involved re-activation of enzyme in bulk solution. A combination of HSP 70, a-crystallin and reticulocyte lysate (RL) in bulk solution were found to reactivate soluble firefly luciferase to about 60% of the initial activity after the enzyme activity had been reduced to less than 2% by thermal denaturation. HSP 70 that was covalently immobilized onto glass surfaces was also able to re-activate denatured enzyme that was in bulk solution. Over 30% of the initial activity could be regained from heat denatured enzyme when using immobilized HSP in the presence of other chaperones. The activity of soluble enzyme decayed to negligible values in a period of days when stored at room temperature. In the presence of immobilized HSP and chaperones, activity stabilized at about 10% of the initial activity even after many weeks. The results suggest that immobilized molecular chaperones such as HSP 70 may provide some potential for stabilization and re-activation of enzymes that are trapped in thin aqueous films for applications in biosensors and reactors. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Molecular chaperones; HSP 70; a-Crystallin; Immobilization; Re-activation; Firefly luciferase; Heat shock protein; Reticulocyte lysate
1. Introduction A problem that is commonly encountered in biosensor development has been the denaturation of entrapped and immobilized enzymes. Denaturation of active proteins is also a problem encountered by any living organism, and a number of natural remediation mechanisms and pathways have been identified (Kiang and Tsokos, 1998; Santoro, 2000). Molecular chaperones are
Corresponding author E-mail address: [email protected] (U.J. Krull). 1 Present address: Department of Chemistry, Yunnan Normal University, Kunming 650091, PR China. 2 Present address: Department of Chemistry, Shanxi University, Taiyuan 030006, PR China.
cellular proteins widely present in prokaryotic and eukaryotic cells that bind and stabilize the structure of other proteins and polypeptides to facilitate their biological functions. Heat Shock Protein 70 (HSP 70, also known as DnaK) belongs to a family of molecular chaperones that are responsible for preventing damage to proteins that is induced by environmental stress such as heating. HSP can also refolding damaged proteins to regenerate active conformations. HSP 70 has been shown to have such functions both in vivo (Souren et al., 1999; Nollen et al., 1999) and in vitro (Lee and Vierling, 2000). HSP 70 can direct correct folding by binding to nascent or unfolded polypeptides and/or the folding intermediates, to prevent improper polypeptide chain interactions that lead to aggregation (Hartl, 1996).
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HSP 70 is perhaps the best studied example of molecular chaperones. It represents a family of highly conserved ATPases of relative molecular mass about 70,000 Da. They are structurally subdivided into an Nterminal ATPase domain of /45 kDa, a /18 kDa portion containing the peptide-binding site, and a more variable /10 kDa segment. HSP 70 has essential roles in protein metabolism under both stress and non-stress conditions. This regulatory ability results from the binding and release of hydrophobic segments of an unfolding polypeptide chain in an ATP-hydrolytic reaction cycle. The quantitative detection of HSP has been proposed as an indicator of the level of stress that an organism experiences (Dunlap and Matsumura, 1997; Karouna-Renier and Zehr, 1999; Van Overmeire et al., 2001). Correct folding of proteins in vitro often requires the assistance of other molecular chaperones (Schroder et al., 1993). For example, reticulocyte lysate (RL) from rabbit has been reported to assist HSP 70 in repairing denatured firefly luciferase. RL contains cellular components necessary for protein synthesis (tRNA, ribosomes, amino acids, initiation, elongation and termination factors), and an extensive endogenous chaperone network, where those assisting the function of HSP 70 have not been fully defined (Minami et al., 2000). a-crystallin, a major eye lens protein, along with homologous heat shock protein 25 (HSP 25), are members of another small HSP family. Recent in vitro data indicates that small HSPs act to bind non-native protein folding intermediates, preventing their aggregation so as to maintain them in a state that is available for ATP-dependent refolding by other chaperones (Carver et al., 1995). This new study investigates the use of an immobilized HSP for re-activation of soluble denatured enzyme that is trapped in a small aqueous volume. The primary chaperone, HSP 70, was covalently immobilized to a glass surface. Initial investigations were done using HSP 70 in solution so as to confirm observations of reactivation, and then to develop some optimization of the effectiveness of HSP 70 in mixture with other chaperones. Firefly luciferase (Luc), was chosen as a model enzyme for this study. Due to the availability of a highly sensitive assay for Luc activity, Luc has been used extensively as a model for protein folding in vitro. The fact that Luc is inactivated at moderate temperatures (39 /42 8C) makes it an excellent model protein for chaperone studies (Blum et al., 1985). The enzyme produces yellow green light (562 nm) in the presence of ATP, luciferin (D-LH2), Mg2 and molecular oxygen, with a quantum efficiency of as high as 0.88 according to the following reaction: LucLH2 ATP 0 LucLH2 AMPPPi
LucLH2 AMPO2 0 LucoxyluciferinAMP CO2 light
2. Materials and methods 2.1. Reagents Adenosine 5?-triphosphate (ATP), luciferin, firefly luciferase, a-crystallin, dithiothreitol (DTT), human HSP 70 (recombinant expressed in Escherichia coli ), 3aminopropyltriethoxy-silane (APTES), glutaraldehyde, and bovine serum albumin (BSA) were obtained from Sigma Canada (Oakville, Canada). Reticulocyte lysate from rabbit and Luciferase Assay Mix were from Promega (Whitby, CA). The inner bottom surfaces of 5 ml glass vials (Pierce Reacti-Vial, Rockford, IL) were used to immobilize HSP 70. 2.2. Instrumentation A water bath with temperature control of 9/0.1 8C (Lauda, Germany) was used to thermally denature Luc. A liquid scintillation counter (Model 1409, Wallac, Finland) was used to measure the light emission catalyzed by Luc. 2.3. Cleaning of glass surfaces Glass surfaces were cleaned using a 1:1:5 mixture of 25% ammonia, 30% H2O2 and distilled water at 80 8C with stirring for 5 min. After washing with water the glass samples were treated with a 1:1:5 mixture of 15 M HCl, 30% H2O2 and distilled water, at 80 8C for 5 min with stirring. Finally, the samples were rinsed twice with 15 ml portions of H2O, 25 ml methanol, 20 ml CH2Cl2, and 30 ml diethyl ether. Any residual diethyl ether was removed under reduced pressure. 2.4. Evaporation silanization Glass samples were rinsed with 2% APTES solution in dry chloroform, and heated to 60 8C until dry. They were then washed with chloroform and cured at 120 8C in an oven for 24 h, followed by storage it a vacuum desiccator. 2.5. Activation of silane with glutaraldehyde Coupling buffer was prepared using 0.015 ml of 25% glutaraldehyde and 0.285 ml of 10 mM phosphate buffer (pH 7.5), and contained 2 mM DTT. Glass surfaces were covered with this solution while being gently stirred for 90 min at room temperature. The activated glass
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surfaces were washed with coupling buffer four times (Raman Suri and Misshra, 1996).
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buffer (total 250 ml). This solution was heated with immobilized HSP 70 for 8 min at 40 8C and cooled in an ice bath for 30 s.
2.6. Immobilization of HSP 70 A solution of 20 mL 14.3 mM HSP 70 in 0.23 ml coupling buffer was prepared. The activated glass surfaces were covered with this solution and were incubated for 5 h at room temperature. After incubation, the glass samples were washed with ice-cold coupling buffer containing 1 mol/l NaCl to wash away loosely bound/absorbed protein (Riberio et al., 1998), and were then finally washed with coupling buffer alone. The protein-coated glass surfaces were covered with 0.5 mg/ml NaBH4 solution for 40 min at 4 8C to stabilize the coupling. This was followed by coverage of the surfaces with 1.0 mg/ml BSA solution for 2 h to minimize subsequent non-selective binding. 2.7. Co-immobilization of the complex of HSP 70 and Luc A volume of 104 ml of 2.68 mM Luc and 20 ml 14.3 mM HSP 70 were added into 126 ml of coupling buffer. The remaining steps were as stated for covalent immobilization of HSP 70. 2.8. Luc activity assay The assay solution was composed of 1.2 mM ATP, 0.4 mM luciferin, 5 mM MgSO4, 0.5 mM EDTA, 0.5 mM DTT, 0.1 mg/ml BSA, in 25 mM Tricine at pH 7.8. A volume of 2.5 ml of denatured or re-activated Luc samples was added to 50 ml of the assay solution. Alternatively, 50 ml of commercial Luciferase Assay Mix was used to replace the prepared assay solution for measuring the Luc activity. After mixing, the vial was placed into a scintillation counter for photon counting. Photon emission was measured for a period of 1 min. 2.9. Thermal denaturation of Luc One micromolar Luc was heated for 8 min at 39.5 8C in pH 7.5 denaturing buffer (25 mM HEPES, 5 mM MgSO4, 5 mM DTT, 150 mM KCl) containing 1 mM acrystallin and 1 mM HSP 70 but no ATP (total volume 50 ml). This was sufficient to denature the Luc to less than 2% of its original activity. After heating, samples were immediately cooled in an ice bath for 30 s. Similar experiments were done where complexes of HSP 70 were activated by addition of 1.64 mM ATP during the heating step, and/or after denaturation. When studying the effectiveness of re-activation by immobilized HSP 70, the solution that was used to cover the immobilized HSP 70 consisted of 1 mM Luc, 1 mM acrystallin, with or without 1.64 mM ATP, in denaturing
2.10. Re-activation of thermally inactivated Luc by free HSP 70 After denaturation of Luc, refolding was initiated by adding 1 ml of the denaturation solution to 40 ml of refolding buffer containing 7.5 mM BSA, 50 mM KCl and 2 mM ATP, with or without 50% (v/v) RL and/or 1.5 mM HSP 70, and/or 1 mM a-crystallin. Refolding was allowed to proceed by incubation at 30 8C for up to 2 h. Luc activity was measured and expressed as percentages relative to that of an equivalent amount of native Luc that had not been heat denatured. 2.11. Re-activation of thermally inactivated Luc by immobilized HSP 70 After the denaturation solution containing Luc was heated, the sample was incubated at 30 8C using refolding solution as was done for HSP 70 when in bulk solution. Luc activity was measured at time intervals by taking 2.5 ul of solution, and adding 50 ul prepared assay solution to this sample. Luc activity was expressed as percentages relative to that of an equivalent amount of native Luc that had not been thermally denatured. Control experiments used immobilized HSP 70 that was denatured in solution at 90 8C for 10 min. The long-term stability of immobilized HSP 70 was examined by re-using surfaces that were coated with immobilized protein, and daily repetition of the denaturation/re-activation experiments were done using freshly prepared solutions.
3. Results and discussion The biochemical cascade that is associated with the function of HSP 70 has been previously modeled as a multi-step cycle as shown in Fig. 1 (Santoro, 2000; Hartl, 1996; Schroder et al., 1993). This is a relatively complicated cascade that has not yet been completely characterized. The active reagents can be obtained by extraction from natural sources, but these preparations are also not completely characterized. From the cascade scheme, the rate of recovery of enzyme activity, when assisted by HSP 70 and other chaperones, is anticipated to be dependent on the presence of ATP. It has been reported that ATP plays a role in the protection and refolding of denatured proteins by HSP 70 (Lee and Vierling, 2000). The chaperone activities of HSP 70 are usually ATPdependent (Hartl, 1996). Our experiments confirmed that if ATP was present during the course of the heat
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with RL was used for re-activation. Recovery of activity was at least 10% higher again when ATP was present. Small HSPs are known to be particularly effective in preventing thermal aggregation of other proteins and maintain them in a state that is suitable for ATPdependent refolding by other chaperones. In this study, a-crystallin was examined as a small HSP that might assist in the recovery of the enzymatic activity of Luc. When Luc was heated in the presence of a mixture of acrystallin, RL, HSP 70 and ATP, about 60% recovery of the activity of Luc was observed over a period of less than 2 h (Fig. 2). The same experiment without ATP addition provided a recovery of activity of only about 40%. The same experiment with ATP added, but without a-crystallin provided activity recovery of only about 30%. The results demonstrate that restoration of Luc activity can be observed, but is relatively small, if only HSP 70 is present. Significant gains can be achieved if ATP is present, and if other chaperones or chaperonelike proteins such as a-crystallin are available. 3.2. Re-activation of Luc using covalently immobilized HSP 70 Fig. 1. A model indicating the cycle of function of HSP 70 and some co-factors.
denaturation and re-activation of Luc, a significantly improved rate of recovery of activity could be achieved in comparison to the case where ATP was not added. 3.1. Re-activation of Luc using HSP 70 in solution Thermally denatured Luc did not recover more than 5% of its original activity, and usually indicated no measurable recovery at all. In the presence of HSP 70 alone, only a small degree of re-activation of denatured Luc (to about 10% activity) could be observed in solution (Fig. 2). It is known that the efficiency of refolding of Luc by HSP 70 can be assisted by other molecular chaperones and cofactors such as DnaJ and GrpE. RL contains an extensive endogenous chaperone network which can help HSP 70 to reactivate denatured Luc (Yonehara and Minami, 1996). Only about 10% of enzyme activity was recovered when RL was used alone to reactivate denatured Luc. In the presence of both HSP 70 and RL, the reactivation of enzyme became more efficient. This result was maximized when denaturation was done in the presence of HSP 70. It is likely the case that HSP 70 prevented irreversible denaturation of Luc and maintained it in a folding-competent state during thermal denaturing. About 40% recovery of the Luc activity was observed in situations where denaturation was done in the presence of HSP 70, and a combination of HSP 70
The capability of using immobilized HSP in achieving protection and renaturation of enzymes may provide a wider range of opportunities for applications in areas such as biosensor preparation. It was not clear whether HSP 70 could be immobilized with substantial retention of its functional properties. It is clear in the literature that some forms of HSP exist at cell surfaces, and are functional in terms of protection and re-activation of proteins that are in solution (Hoffman and Garduno, 1999; Kiang and Tsokos, 1998). A report about covalent immobilzation of HSP 90 to carboxymethyl-dextran surface-plasmon resonance (SPR) chips indicated that some activity was retained for binding to denatured proteins, and Kd values for binding interactions were reported (Csermely et al., 1997). A report about crosslinking of a-crystallin indicated that a substantial reduction of chaperone-like activity was noted (Shridas et al., 2001). A typical non-specific, non-orienting protein immobilization protocol was selected for our preliminary studies, and immobilization was based on use of surface activation using silane, followed by glutaraldehyde reaction. Glass vials were treated to obtain the following three surface modifications: (a) Native HSP 70 was immobilized onto glass surfaces; (b) HSP 70 was treated by heating at 90 8C for 10 min, and then was immobilized onto glass surfaces; and (c) surfaces were treated with BSA solution. The supporting solution contained Luc, a-crystallin, ATP and denaturing buffer, as these provided good recoveries of activity in experiments
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Fig. 2. Some representative results of percentage of re-activation of thermally denatured Luc based on initial activities without thermal denaturation. One micromolar Luc was heated for 8 min at 39.5 8C at pH 7.5 in denaturing buffer (25 mM HEPES, 5 mM MgSO4, 5 mM DTT, 150 mM KCl) with or without additives, followed by re-activation, with or without additives: Sample 1: Heating, no additives; Re-activation, no additives. Sample 2: Heating, a-crystallin; Re-activation, RL. Sample 3: Heating, HSP 70, ATP; Re-activation, RL, HSP 70. Sample 4: Heating, a-crystallin, HSP 70; Re-activation, RL, HSP 70. Sample 5: Heating, a-crystallin, HSP 70; Re-activation, HSP 70. Sample 6: Heating, a-crystallin, HSP 70, ATP; Reactivation, RL, HSP 70.
that were done in bulk solution. RL was not used in the experiments that involved surface immobilized HSP 70 because the complexity of the RL solution was such that it was not possible to begin to understand issues of nonselective adsorption and co-factor availability.
In all three cases, the activity of Luc was reduced to less than 2% after heating at 40 8C for 8 min in the vials. The denatured Luc was left in the vials for incubation at 30 8C, and the activity was observed to gradually recover (Fig. 3). Over a period of 200 min, the
Fig. 3. Time dependence of re-activation using immobilized HSP 70. Glass vials were treated to obtain the following three surface modifications: (a) HSP 70 was immobilized onto glass surfaces ("); (b) HSP 70 was treated by heating at 90 8C for 10 min, and then was immobilized onto glass surfaces (control, j); Surfaces were treated with BSA solution (control, '). The supporting solution in all experiments contained Luc, a-crystallin, ATP and denaturing buffer. Luc was denatured in each case at 40 8C for 8 min. The sample was then incubated in denaturing solution at 30 8C. Luc activity was measured at various time intervals and expressed as percentages relative to that of an equivalent amount of native Luc measured prior to the heating step.
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Fig. 4. The ability of immobilized HSP 70 to re-activate Luc after various periods of time. The same vials that were coated with HSP 70 were used on a daily basis to assess the ability to re-activate Luc.
activity was recovered to about 30% of the initial value before thermal denaturation. However, when the Luc was incubated with immobilized, 90 8C heat treated HSP 70, only about 5% of the activity recovery was observed (Fig. 3). Luc incubated in the BSA treated vial did not recover more than 2% of the initial activity. Immobilized HSP 70 was found to be able to reactivate Luc for a period of weeks after immobilization. The ability to re-activate the denatured enzyme remained relatively high for the first 2 days, and then reduced, with only about one-third of the initial reactivation being noted 6 days after immobilization (Fig. 4). However, the ability to re-activate to a level of about 10% of initial activity was available for weeks. Enzyme stored in aqueous buffer solution in glass vials at room temperature effectively lost all of its activity with a few days. Enzyme that was stored in the treated vials in the presence of the chaperones suffered a reduction in activity over a period of a week, but stabilized at an activity level of about 10%, and this remained constant for weeks. A preliminary experiment considered whether recovery of activity of chemically denatured enzyme could be achieved. The recovery of activity of chemically denatured firefly luciferase (Rungeling et al., 1999), and carbonic anhydrase (Nam and Walsh, 2002) has been shown to be possible by the action of molecular chaperones including HSP 70. In these reports, it was noted that chemical denaturation induced by materials such as guanidinium hydrochloride was not as readily reversed by chaperones (68% reported activity recovery) as was the case for re-activation after thermal denaturation (97% activity recovered). Our initial experiments
indicated that chemically denatured enzyme was reactivated by the immobilzed HSP system, and that activity levels of up to about 15/20% could be observed. An investigation was completed to determine whether co-immobilized HSP 70/Luc could improve the lifetime of the activity of Luc. The experimental conditions that were used included addition of a-crystallin and ATP after the immobilization. The HSP 70 and Luc were immobilized concurrently on the activated glass surface, with the expectation that they likely complexed in solution, and that the complexes might survive attachment to the surface. A control experiment was done using BSA to replace HSP 70. The results show that there is no significant difference in enzyme activity over time between immobilized HSP 70/Luc and BSA/Luc (typical enzyme activity decay where most of the initial activity is lost within days, data not shown). A further investigation was completed to determine whether co-immobilized HSP 70 and Luc could increase the thermal endurance of Luc. After immobilization, the samples were heated in the presence of a-crystallin and ATP at 40 8C for 8 min, using co-immobilized BSA/Luc as a control. The results indicate that co-immobilization of HSP 70/Luc does not increase the thermal endurance of Luc (data not shown).
4. Conclusions Luciferase re-activation after thermal denaturation could be achieved with regeneration of up to 60% of the initial enzyme activity by appropriate selection of Heat Shock Protein, molecular chaperones and co-factors.
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The protection procedure is at least partially ATPdependent, although some re-activation can be achieved in the absence of ATP. Covalently immobilized HSP 70 can re-activate thermally denatured Luc to a level of about 5 /10% activity, indicating that at least some significant quantity of the chaperone is in an active binding form. An improvement is observed when a-crystallin and ATP are introduced into the solution phase. The improvement provides activity recovery to a level of about 30%, which is only marginally smaller than what was observed in comparable experiments that were done using free HSP 70 and a-crystallin in solution (about 40% activity). After 6 days, immobilized HSP 70 in combination with acrystallin was still able to provide some re-activation of thermally denatured enzyme, and retained about onethird of its original activation ability. Similar levels of re-activation were noted for some weeks. The reduction in the ability of immobilized HSP 70 to provide reactivation after some days suggests that denaturation of the protein takes place on the glass surface. While the results of this work suggest that HSP 70 and Luc can form a complex in solution, it would appear that co-immobilization of HSP 70 and Luc likely causes inactivation of the important structural features of this complex that are responsible for maintaining activity of the enzyme. The immobilization method that was used in this work likely prevents the enzyme from undergoing the action of protein folding that is caused by the HSP 70 and co-factors. These preliminary results suggest that protein mobility is important, so that advantageous use of molecular chaperones might best be achieved for systems that use immobilized HSP and free or entrapped enzymes.
Acknowledgements We are grateful to the Natural Sciences and Engineering Council of Canada for financial support of this work. Y.Y. extends her thanks to the China Scholarship Council program, and C.G. extends his thanks to the government of Shanxi Province, for fellowship support as visiting scholars.
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