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Covalent protein immobilization on glass surfaces: Application to alkaline phosphatase
Journal of Biotechnology 118 (2005) 265–269
Covalent protein immobilization on glass surfaces: Application to alkaline phosphatase Russell H. Taylor a,c , Sylvie M. Fournier c , Brigitte L. Simons b,c , Harvey Kaplan b , Mary Alice Hefford a,c,∗ a
Department of Biochemistry, University of Ottawa, Canada K1H 8M5 Department of Chemistry, University of Ottawa, Canada K1W 6N5 Center for Biologics Research, Biologics and Genetic Therapies Directorate, Health Canada, Ottawa, Ont., Canada K1A 0L2 b
c
Received 23 September 2004; received in revised form 21 April 2005; accepted 2 May 2005
Abstract Lyophilized alkaline phosphatase (ALPase) was immobilized on aminated glass surfaces using the in vacuo cross-linking process [Simons, B.L., King, M.C., Cyr, T., Hefford, M.A., Kaplan, H., 2002. Zero-length cross-linking of lyophilized proteins. Protein Sci. 11, 1558–1564]. In this procedure, amide bonds were formed between carboxyl groups on the protein and amino groups on the glass surface. After the non-covalently attached enzyme was removed the immobilized ALPase not only retained its activity but could also be used, washed and reused at least six times without significant loss of activity. An average of 1.4 ± 0.6 mg of reusable ALPase per gram of glass fibre was immobilized based on the activity of the soluble equivalent. © 2005 Elsevier B.V. All rights reserved. Keywords: Protein immobilization; Alkaline phosphatase; Cross-linking; Amide bond; Glass; Enzymatic activity
1. Introduction Immobilization of enzymes has many theoretical and practical applications. Traditionally, enzyme immobilization is carried out by activating a functional ∗ Corresponding author at: Center for Biologics Research, Biologics and Genetic Therapies Directorate, Health Canada, Ottawa, Ont., Canada K1A 0L2. Tel.: +1 613 957 3627; fax: +1 613 941 8933. E-mail address: mary [email protected] (M.A. Hefford).
0168-1656/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2005.05.007
group, either on the protein surface or on the solid support, with chemical reagents (Lunblad, 1995). However, this strategy is not without significant disadvantage. For example chemical modification of proteins often irreversibly alters the protein and reduces activity (Immoto and Yamada, 1989). In addition, trace amounts of “activation” chemicals remaining after the cross-linking reaction compromise pharmaceutical applications. Finally, immobilization under aqueous conditions requires relatively large
266
R.H. Taylor et al. / Journal of Biotechnology 118 (2005) 265–269
amounts of activating reagents that must be employed with great care and, as a result, their use in large-scale immobilization is inefficient. In contrast, the in vacuo cross-linking method, recently developed by Simons et al. (2002), provides a facile means of cross-linking proteins without the use of chemical reagents. The latter method is based on the observation that interacting ammonium and carboxylate ions readily form an amide linkage under vacuum. This methodology can be used to take advantage of the interactions between ammonium ions attached to a glass surface and pre-existing carboxylate ions on proteins in the lyophilized state to covalently immobilize active enzymes on glass surfaces. The present communication demonstrates the application to alkaline phosphatase (ALPase).
2. Materials and methods 2.1. Activation of silica filter disks Glass fibre filter disks (2.4 cm in diameter) were purchased from Millipore (APFC02500) and refluxed with 12 N HCl for 2 h. Disks were then washed with excess dH2 O using a Buchner funnel and oven dried at 80 ◦ C for 30 min. A 10% (v/v) solution of 3-aminopropyltrimethoxysilane (Aldrich 28,177-8) in toluene was used to aminate the glass surface as described by Stark and Holmberg (1989). The filter disks were added to this solution and refluxed for 18 h. Finally, disks were rinsed three times each with toluene, acetone, and then dH2 O. 2.2. Immobilization of alkaline phosphatase A 10 mg/ml aqueous stock of bovine intestinal mucosa alkaline phosphatase (Sigma P-7640 Lot. 121K7078) was prepared and adjusted to pH 7.0 with 1 N NaOH. Activated glass filter disks were soaked in 200 l of the stock solution until saturated (approximately 10 s). These moist filter disks were flash frozen with liquid nitrogen and lyophilized. Next, these dried ALPase-disks were sealed under vacuum (∼50 mTorr) and incubated at 80 ◦ C for 96 h as described by Simons et al. (2002). Disks with ALPase immobilized by crosslinking were rinsed in a Buchner funnel with excess 0.1 M NaCl then dH2 O, lyophilized, and stored at 4 ◦ C.
2.3. Immobilized alkaline phosphatase activity assay The ALPase activity was determined using standard methods with p-nitrophenylphosphate (pNPP) as a substrate (Walter and Sch¨utt, 1974). Briefly, a stock solution of 350 M pNPP (Sigma N-6260, Lot 091K5315) in 25 mM glycine was prepared and adjusted to pH 9.6 with NaOH. The glass fibre filter disk containing the immobilized ALPase was tethered, using 2 mm plastic tubing, below the midline of a 25 mm × 150 mm culture tube. At time 0, 20.0 ml of pNPP solution was added to the culture tube with constant stirring. Every minute thereafter, a 90 l aliquot was removed from the mix and placed in a well of a microtiter plate containing 10 l of a stop solution composed of 0.1 M NaOH and 0.1 M EDTA. The absorbance at 405 nm of the ensuing solution in each well was measured using a Tecan GENios spectrophotometer (Tecan Systems Inc, San Jose, CA). The activity of immobilized ALPase was calculated using the formula: acitvity =
A405 (Vreaction /Valiquot ) β
(1)
where A405 is the absorbance at 405 nm, Valiquot the volume of the aliquot read (0.1 mL), Vreaction the volume of the reaction (20 mL) and β the extinction coefficient for para-nitrophenol (pNP (18.5 at 405 nm)). 2.4. Quantification of immobilized enzyme as a soluble equivalent A standard activity curve was generated using 20 mL of the 350 M pNPP substrate and free, soluble ALPase. At time 0, the substrate was stirred vigorously and 0.5, 1, 5, 20, 100 or 400 g of soluble ALPase were added. Aliquots were taken as described in Section 2.3 and the change in absorbance at 405 nm was calculated. Five different concentrations of soluble ALPase were assayed, each in duplicate, and the mean values plotted on a standard curve. This standard curve was used to estimate the amount of immobilized ALPase. 2.5. Washing and reuse of filter disks Between each trial the filter disk containing the immobilized ALPase was rinsed three times with excess (approximately 3× 10 mL) 1 M NaCl then
R.H. Taylor et al. / Journal of Biotechnology 118 (2005) 265–269
267
dH2 O by placing the disk in a Buchner funnel attached to a water aspirator. 2.6. Quantification of total immobilized protein The amount of protein immobilized on each filter disk was quantified by reaction with dansyl chloride, measurement of the total fluorescence of the sample (after acid hydrolysis and reconstitution) and interpolation using a standard curve. The standard fluorescence curve was made by weighing 5.00 mg ALPase, dissolving it in a solution of 6 M urea and 0.1 M sodium bicarbonate (pH 8.3) and reacting it with dansyl chloride. A 10% molar excess of dansyl chloride (Sigma D-2625 Lot 11K2620) in dimethyl formate was added per mole of lysine residue in ALPase. The reaction was allowed to proceed, with agitation, at room temperature for 30 min before termination by the addition of 100 mg of glycine. The sample was then transferred to 12-14000 MW cut-off dialysis tubing and thoroughly dialyzed against water. The dansylated-ALPase was acid hydrolysed in 6 N HCl in an evacuated, sealed tube (vacuum at 75 mTorr) by heating to 110 ◦ C for 28 h. After hydrolysis, HCl was removed by lyophilization and the dried sample was reconstituted to a concentration of 5.00 mg/mL hydrolysate in 0.05 N NaOH. This hydrolysate was used to create a dilution series. Each diluted sample was excited at 340 nm and the fluorescence emission at 465 nm was measured. ALPase immobilized on filter disks was dansylated in a solution of 6 M urea and 0.1 M sodium bicarbonate at pH 8.3. An excess (∼100-fold) of dansyl chloride in DMF was added drop-wise and reacted for 30 min as described above. Each filter disk was rinsed with MeOH and then dH2 O to remove unreacted reagent and dansyl sulphonic acid. Acid hydrolysis of dansylated, immobilized ALPase was carried out as described above. After reconstitution, total fluorescence was measured and the amount of protein determined from the standard curve. An activated disk without immobilized protein was also used as a control.
Fig. 1. Activity of immobilized alkaline phosphatase: micromoles of pNPP hydrolysed in aliquots taken over time from a solution containing immobilized alkaline phosphatase for a typical filter disk. For each trial 20 mL of 350 uM pNPP was used. A 90 L aliquot was taken every minute, for 25 min and placed in 10 L of stopping solution.
activity of the immobilized enzyme immediately after preparation and initial washing is depicted as “trial 1”. Trials 2, 3, 4, 5 and 6 represent subsequent washing, rinsing and reusing of the same filter disk. The process was repeated with ALPase immobilized on four separate glass filter disks to give an average enzymatic activity. Fig. 2 shows that, after the first trial, the disk loses approximately 60% of its activity. In subsequent washing cycles, however, the losses are minimal and over 90% of the activity present after the first trial remains after additional washings.
3. Results Fig. 1 shows the rate of pNPP dephosphorylation by ALPase immobilized on a typical glass fibre filter disk treated using the in vacuo procedure. The enzymatic
Fig. 2. Average activity, with standard deviation, of immobilized alkaline phosphatase on disks (n = 4). Each disk was assayed four times and rinsed between successive trials.
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The amount of enzymatically active protein remaining on the disk was estimated as the soluble equivalent, i.e. the amount of soluble enzyme that would give the same activity as the immobilized enzyme in Fig. 1. On average (n = 4), it was found that an equivalent of 185 ± 30 g of active ALPase protein was present during the first trial and 35 ± 15 g for the second trial. Based on this result it was found that there was a soluble equivalent of 1.4 ± 0.6 mg of reusable immobilized alkaline phosphatase per g of glass in the second and subsequent trials. After the sixth trial, the total amount of protein remaining on the disk was quantified by comparing its fluorescence (see Section 2.6) to that of a similarly treated sample of soluble enzyme used to make the standard curve (data not shown). The result indicates that 52.7 g of protein can be bound to a 24 mg filter disk with a diameter of 2.4 cm. This represents 2.16 mg of total protein remaining immobilized per gram of silica fibre. A detectable fluorescence was evident in the negative control (which consisted of a dansylated glass fibre filter with no protein attached), and is presumed to arise due to shearing of dansyl-labelled silica during hydrolysis. When estimating the amount of protein immobilized, this background fluorescence was subtracted.
4. Discussion Alkaline phosphatase has been immobilized on a variety of surfaces (Surinenaite et al., 1996; Wiley et al., 2001; Filmon et al., 2002) but, to our knowledge, there is only one previous report of immobilization on glass (Weetall, 1969). In 1969, Weetall reported that 0.74 mg of ALPase per gram of glass could be immobilized by adding the enzyme to glass beads chemically activated with diazo groups. Our results show a larger amount of active immobilized enzyme but, given the uncertainties in the relative densities of the glass used in the two studies, this difference may not be significant. Our results do demonstrate, however, that the extent of covalent immobilization achieved by the in vacuo process is at least comparable to that obtained by the immobilization processes using conventional chemical activation. We, like Weetall, report the amount of immobilized enzyme as the soluble equivalent—that is, the
amount of soluble ALPase that gives the same enzymatic activity. In practice, the soluble enzyme equivalent is indicative of the reaction conditions (amount of immobilized enzyme, time of reaction, etc.) necessary to achieve substrate hydrolysis. It is, therefore, a more useful value than, for example, the absolute amount of enzyme immobilized. Inhomogeneity of the reaction mixture necessarily results when one component of the enzyme–substrate is immobilized on a solid support, and some reduction in the apparent specific activity (enzymatic activity under a given set of reaction conditions per absolute amount of protein immobilized) is both expected and routinely observed (Zingaro and Uziel, 1970). However, since enzyme immobilization has the potential to physically alter the ability of individual enzyme molecules to catalyze reactions when they do come into contact with substrate, we also determined the total amount of protein immobilized per gram of glass fibre. Comparison of this value (2.16 mg protein per gram of fibre remaining immobilized after six uses) to the soluble equivalent of reusable immobilized ALPase (1.4 mg per gram of fibre) indicates that approximately 67% of the apparent specific activity of the soluble enzyme can be retained after immobilization and several reuses. This value compares favourably with examples of ALPase immobilized on other surfaces (1% of specific activity retained on an ethylene-maleic anhydride co-polymer (Zingaro and Uziel, 1970) and 56–72% on various vanacryls (Brown and Joyeau, 1974) as well as to values obtained for acid phosphatases immobilized on plain and glycerol coated glass (4 and 14%, respectively (Van Hekken et al., 1990)). Despite the caveats in interpretation of the specific activity of inhomogeneous mixtures, these data suggest that the in vacuo immobilization process is at least as effective as more traditional chemical methods of immobilization in preserving the activity of the enzyme molecules immobilized on the glass. Our results also indicate that filter disks containing immobilized enzymes have the potential for repeated use without significant loss of activity. Approximately 60% of the ALPase activity was removed after the first trial but there was virtually no further loss of activity in subsequent trials. It was expected that the washing steps in the process before trial 1 would remove any non-covalently associated protein. Nevertheless, a large decrease in activity between trial 1 and trial 2 was consistently observed. While the source of this
R.H. Taylor et al. / Journal of Biotechnology 118 (2005) 265–269
loss of activity is not clear, it may be that, despite the attempts to remove any non-covalently bound enzyme using 0.1 M NaCl, some still remains associated with the glass matrix during the first trial. Regardless of the mechanism, the covalently immobilized enzyme remaining after the first trial and washing was stable; the losses of activity with five subsequent washes of the disk were minimal. In summary, the successful covalent immobilization of alkaline phosphatase without the use of activating chemicals demonstrates that the in vacuo method is a practical and efficient process for immobilizing proteins especially small amounts of valuable proteins. The success with ALPase demonstrates that this immobilization process should be applicable to all soluble proteins containing carboxyl groups and other solid supports with free amino groups.
References Brown, E., Joyeau, R., 1974. Immobilized enzymes. 7. Use of vanacryls in preparation of immobilized arginase and alkaline phosphatase. Polymer 15, 546–552. Filmon, R., Grizon, F., Basl´e, M.F., Chappaard, D., 2002. Effects of negatively charged groups (carboxymethyl) on the calcification of poly(2-hydroxyethyl methacrylate). Biomaterials 23, 3053–3059.
269
Immoto, T., Yamada, H., 1989. In: Creighton, T.E. (Ed.), Protein Function: A Practical Approach. Oxford University Press, Chapter 10, pp. 247–277. Lunblad, R.L., 1995. Techniques in Protein Modification. CRC Press, Boca Raton, FL, Chapter 15, pp. 249–262. Simons, B.L., King, M.C., Cyr, T., Hefford, M.A., Kaplan, H., 2002. Zero-length cross-linking of lyophilized proteins. Protein Sci. 11, 1558–1564. Stark, M.B., Holmberg, K., 1989. Covalent immobilization of lipase in organic solvents. Biotechnol. Bioeng. 34, 942–950. Surinenaite, B.R., Bendikene, V.G., Iuodka, B.A., 1996. Immobilization of enzymes on carriers with magnetic properties: the search for optimum conditions for immobilization of alkaline phosphatase from the chicken graft. Prikl. Biokhim. Mikrobiol. 32, 609–614. Walter, K., Sch¨utt, C., 1974. In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis, vol. II, 2nd ed. Academic Press, New York, pp. 860–864. Weetall, H.H., 1969. Alkaline phosphatase insolubilized by covalent linkage to porous glass. Nature 223, 959–960. Wiley, J.P., Hughes, K.A., Kaiser, R.J., Kesicki, E.A., Lund, K.P., Stolowitz, M.L., 2001. Phenylboronic acid-salicylhydroxamic acid bioconjugates. 2. Polyvalent immobilization of protein ligands for affinity chromatography. Bioconjug. Chem. 2, 240– 250. Van Hekken, D.L., Thompson, M.P., Strange, E.D., 1990. Immobilization of potato acid phosphatase on succinamidopropyl glass beads for the dephosporylation of bovine whole casein. J. Dairy Sci. 73, 2720–2730. Zingaro, R.A., Uziel, M., 1970. Preparation and properties of active, insoluble alkaline phosphatase. Biochem. Biophys. Acta 213, 371–379.
Covalent protein immobilization on glass surfaces: Application to alkaline phosphatase Russell H. Taylor a,c , Sylvie M. Fournier c , Brigitte L. Simons b,c , Harvey Kaplan b , Mary Alice Hefford a,c,∗ a
Department of Biochemistry, University of Ottawa, Canada K1H 8M5 Department of Chemistry, University of Ottawa, Canada K1W 6N5 Center for Biologics Research, Biologics and Genetic Therapies Directorate, Health Canada, Ottawa, Ont., Canada K1A 0L2 b
c
Received 23 September 2004; received in revised form 21 April 2005; accepted 2 May 2005
Abstract Lyophilized alkaline phosphatase (ALPase) was immobilized on aminated glass surfaces using the in vacuo cross-linking process [Simons, B.L., King, M.C., Cyr, T., Hefford, M.A., Kaplan, H., 2002. Zero-length cross-linking of lyophilized proteins. Protein Sci. 11, 1558–1564]. In this procedure, amide bonds were formed between carboxyl groups on the protein and amino groups on the glass surface. After the non-covalently attached enzyme was removed the immobilized ALPase not only retained its activity but could also be used, washed and reused at least six times without significant loss of activity. An average of 1.4 ± 0.6 mg of reusable ALPase per gram of glass fibre was immobilized based on the activity of the soluble equivalent. © 2005 Elsevier B.V. All rights reserved. Keywords: Protein immobilization; Alkaline phosphatase; Cross-linking; Amide bond; Glass; Enzymatic activity
1. Introduction Immobilization of enzymes has many theoretical and practical applications. Traditionally, enzyme immobilization is carried out by activating a functional ∗ Corresponding author at: Center for Biologics Research, Biologics and Genetic Therapies Directorate, Health Canada, Ottawa, Ont., Canada K1A 0L2. Tel.: +1 613 957 3627; fax: +1 613 941 8933. E-mail address: mary [email protected] (M.A. Hefford).
0168-1656/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2005.05.007
group, either on the protein surface or on the solid support, with chemical reagents (Lunblad, 1995). However, this strategy is not without significant disadvantage. For example chemical modification of proteins often irreversibly alters the protein and reduces activity (Immoto and Yamada, 1989). In addition, trace amounts of “activation” chemicals remaining after the cross-linking reaction compromise pharmaceutical applications. Finally, immobilization under aqueous conditions requires relatively large
266
R.H. Taylor et al. / Journal of Biotechnology 118 (2005) 265–269
amounts of activating reagents that must be employed with great care and, as a result, their use in large-scale immobilization is inefficient. In contrast, the in vacuo cross-linking method, recently developed by Simons et al. (2002), provides a facile means of cross-linking proteins without the use of chemical reagents. The latter method is based on the observation that interacting ammonium and carboxylate ions readily form an amide linkage under vacuum. This methodology can be used to take advantage of the interactions between ammonium ions attached to a glass surface and pre-existing carboxylate ions on proteins in the lyophilized state to covalently immobilize active enzymes on glass surfaces. The present communication demonstrates the application to alkaline phosphatase (ALPase).
2. Materials and methods 2.1. Activation of silica filter disks Glass fibre filter disks (2.4 cm in diameter) were purchased from Millipore (APFC02500) and refluxed with 12 N HCl for 2 h. Disks were then washed with excess dH2 O using a Buchner funnel and oven dried at 80 ◦ C for 30 min. A 10% (v/v) solution of 3-aminopropyltrimethoxysilane (Aldrich 28,177-8) in toluene was used to aminate the glass surface as described by Stark and Holmberg (1989). The filter disks were added to this solution and refluxed for 18 h. Finally, disks were rinsed three times each with toluene, acetone, and then dH2 O. 2.2. Immobilization of alkaline phosphatase A 10 mg/ml aqueous stock of bovine intestinal mucosa alkaline phosphatase (Sigma P-7640 Lot. 121K7078) was prepared and adjusted to pH 7.0 with 1 N NaOH. Activated glass filter disks were soaked in 200 l of the stock solution until saturated (approximately 10 s). These moist filter disks were flash frozen with liquid nitrogen and lyophilized. Next, these dried ALPase-disks were sealed under vacuum (∼50 mTorr) and incubated at 80 ◦ C for 96 h as described by Simons et al. (2002). Disks with ALPase immobilized by crosslinking were rinsed in a Buchner funnel with excess 0.1 M NaCl then dH2 O, lyophilized, and stored at 4 ◦ C.
2.3. Immobilized alkaline phosphatase activity assay The ALPase activity was determined using standard methods with p-nitrophenylphosphate (pNPP) as a substrate (Walter and Sch¨utt, 1974). Briefly, a stock solution of 350 M pNPP (Sigma N-6260, Lot 091K5315) in 25 mM glycine was prepared and adjusted to pH 9.6 with NaOH. The glass fibre filter disk containing the immobilized ALPase was tethered, using 2 mm plastic tubing, below the midline of a 25 mm × 150 mm culture tube. At time 0, 20.0 ml of pNPP solution was added to the culture tube with constant stirring. Every minute thereafter, a 90 l aliquot was removed from the mix and placed in a well of a microtiter plate containing 10 l of a stop solution composed of 0.1 M NaOH and 0.1 M EDTA. The absorbance at 405 nm of the ensuing solution in each well was measured using a Tecan GENios spectrophotometer (Tecan Systems Inc, San Jose, CA). The activity of immobilized ALPase was calculated using the formula: acitvity =
A405 (Vreaction /Valiquot ) β
(1)
where A405 is the absorbance at 405 nm, Valiquot the volume of the aliquot read (0.1 mL), Vreaction the volume of the reaction (20 mL) and β the extinction coefficient for para-nitrophenol (pNP (18.5 at 405 nm)). 2.4. Quantification of immobilized enzyme as a soluble equivalent A standard activity curve was generated using 20 mL of the 350 M pNPP substrate and free, soluble ALPase. At time 0, the substrate was stirred vigorously and 0.5, 1, 5, 20, 100 or 400 g of soluble ALPase were added. Aliquots were taken as described in Section 2.3 and the change in absorbance at 405 nm was calculated. Five different concentrations of soluble ALPase were assayed, each in duplicate, and the mean values plotted on a standard curve. This standard curve was used to estimate the amount of immobilized ALPase. 2.5. Washing and reuse of filter disks Between each trial the filter disk containing the immobilized ALPase was rinsed three times with excess (approximately 3× 10 mL) 1 M NaCl then
R.H. Taylor et al. / Journal of Biotechnology 118 (2005) 265–269
267
dH2 O by placing the disk in a Buchner funnel attached to a water aspirator. 2.6. Quantification of total immobilized protein The amount of protein immobilized on each filter disk was quantified by reaction with dansyl chloride, measurement of the total fluorescence of the sample (after acid hydrolysis and reconstitution) and interpolation using a standard curve. The standard fluorescence curve was made by weighing 5.00 mg ALPase, dissolving it in a solution of 6 M urea and 0.1 M sodium bicarbonate (pH 8.3) and reacting it with dansyl chloride. A 10% molar excess of dansyl chloride (Sigma D-2625 Lot 11K2620) in dimethyl formate was added per mole of lysine residue in ALPase. The reaction was allowed to proceed, with agitation, at room temperature for 30 min before termination by the addition of 100 mg of glycine. The sample was then transferred to 12-14000 MW cut-off dialysis tubing and thoroughly dialyzed against water. The dansylated-ALPase was acid hydrolysed in 6 N HCl in an evacuated, sealed tube (vacuum at 75 mTorr) by heating to 110 ◦ C for 28 h. After hydrolysis, HCl was removed by lyophilization and the dried sample was reconstituted to a concentration of 5.00 mg/mL hydrolysate in 0.05 N NaOH. This hydrolysate was used to create a dilution series. Each diluted sample was excited at 340 nm and the fluorescence emission at 465 nm was measured. ALPase immobilized on filter disks was dansylated in a solution of 6 M urea and 0.1 M sodium bicarbonate at pH 8.3. An excess (∼100-fold) of dansyl chloride in DMF was added drop-wise and reacted for 30 min as described above. Each filter disk was rinsed with MeOH and then dH2 O to remove unreacted reagent and dansyl sulphonic acid. Acid hydrolysis of dansylated, immobilized ALPase was carried out as described above. After reconstitution, total fluorescence was measured and the amount of protein determined from the standard curve. An activated disk without immobilized protein was also used as a control.
Fig. 1. Activity of immobilized alkaline phosphatase: micromoles of pNPP hydrolysed in aliquots taken over time from a solution containing immobilized alkaline phosphatase for a typical filter disk. For each trial 20 mL of 350 uM pNPP was used. A 90 L aliquot was taken every minute, for 25 min and placed in 10 L of stopping solution.
activity of the immobilized enzyme immediately after preparation and initial washing is depicted as “trial 1”. Trials 2, 3, 4, 5 and 6 represent subsequent washing, rinsing and reusing of the same filter disk. The process was repeated with ALPase immobilized on four separate glass filter disks to give an average enzymatic activity. Fig. 2 shows that, after the first trial, the disk loses approximately 60% of its activity. In subsequent washing cycles, however, the losses are minimal and over 90% of the activity present after the first trial remains after additional washings.
3. Results Fig. 1 shows the rate of pNPP dephosphorylation by ALPase immobilized on a typical glass fibre filter disk treated using the in vacuo procedure. The enzymatic
Fig. 2. Average activity, with standard deviation, of immobilized alkaline phosphatase on disks (n = 4). Each disk was assayed four times and rinsed between successive trials.
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The amount of enzymatically active protein remaining on the disk was estimated as the soluble equivalent, i.e. the amount of soluble enzyme that would give the same activity as the immobilized enzyme in Fig. 1. On average (n = 4), it was found that an equivalent of 185 ± 30 g of active ALPase protein was present during the first trial and 35 ± 15 g for the second trial. Based on this result it was found that there was a soluble equivalent of 1.4 ± 0.6 mg of reusable immobilized alkaline phosphatase per g of glass in the second and subsequent trials. After the sixth trial, the total amount of protein remaining on the disk was quantified by comparing its fluorescence (see Section 2.6) to that of a similarly treated sample of soluble enzyme used to make the standard curve (data not shown). The result indicates that 52.7 g of protein can be bound to a 24 mg filter disk with a diameter of 2.4 cm. This represents 2.16 mg of total protein remaining immobilized per gram of silica fibre. A detectable fluorescence was evident in the negative control (which consisted of a dansylated glass fibre filter with no protein attached), and is presumed to arise due to shearing of dansyl-labelled silica during hydrolysis. When estimating the amount of protein immobilized, this background fluorescence was subtracted.
4. Discussion Alkaline phosphatase has been immobilized on a variety of surfaces (Surinenaite et al., 1996; Wiley et al., 2001; Filmon et al., 2002) but, to our knowledge, there is only one previous report of immobilization on glass (Weetall, 1969). In 1969, Weetall reported that 0.74 mg of ALPase per gram of glass could be immobilized by adding the enzyme to glass beads chemically activated with diazo groups. Our results show a larger amount of active immobilized enzyme but, given the uncertainties in the relative densities of the glass used in the two studies, this difference may not be significant. Our results do demonstrate, however, that the extent of covalent immobilization achieved by the in vacuo process is at least comparable to that obtained by the immobilization processes using conventional chemical activation. We, like Weetall, report the amount of immobilized enzyme as the soluble equivalent—that is, the
amount of soluble ALPase that gives the same enzymatic activity. In practice, the soluble enzyme equivalent is indicative of the reaction conditions (amount of immobilized enzyme, time of reaction, etc.) necessary to achieve substrate hydrolysis. It is, therefore, a more useful value than, for example, the absolute amount of enzyme immobilized. Inhomogeneity of the reaction mixture necessarily results when one component of the enzyme–substrate is immobilized on a solid support, and some reduction in the apparent specific activity (enzymatic activity under a given set of reaction conditions per absolute amount of protein immobilized) is both expected and routinely observed (Zingaro and Uziel, 1970). However, since enzyme immobilization has the potential to physically alter the ability of individual enzyme molecules to catalyze reactions when they do come into contact with substrate, we also determined the total amount of protein immobilized per gram of glass fibre. Comparison of this value (2.16 mg protein per gram of fibre remaining immobilized after six uses) to the soluble equivalent of reusable immobilized ALPase (1.4 mg per gram of fibre) indicates that approximately 67% of the apparent specific activity of the soluble enzyme can be retained after immobilization and several reuses. This value compares favourably with examples of ALPase immobilized on other surfaces (1% of specific activity retained on an ethylene-maleic anhydride co-polymer (Zingaro and Uziel, 1970) and 56–72% on various vanacryls (Brown and Joyeau, 1974) as well as to values obtained for acid phosphatases immobilized on plain and glycerol coated glass (4 and 14%, respectively (Van Hekken et al., 1990)). Despite the caveats in interpretation of the specific activity of inhomogeneous mixtures, these data suggest that the in vacuo immobilization process is at least as effective as more traditional chemical methods of immobilization in preserving the activity of the enzyme molecules immobilized on the glass. Our results also indicate that filter disks containing immobilized enzymes have the potential for repeated use without significant loss of activity. Approximately 60% of the ALPase activity was removed after the first trial but there was virtually no further loss of activity in subsequent trials. It was expected that the washing steps in the process before trial 1 would remove any non-covalently associated protein. Nevertheless, a large decrease in activity between trial 1 and trial 2 was consistently observed. While the source of this
R.H. Taylor et al. / Journal of Biotechnology 118 (2005) 265–269
loss of activity is not clear, it may be that, despite the attempts to remove any non-covalently bound enzyme using 0.1 M NaCl, some still remains associated with the glass matrix during the first trial. Regardless of the mechanism, the covalently immobilized enzyme remaining after the first trial and washing was stable; the losses of activity with five subsequent washes of the disk were minimal. In summary, the successful covalent immobilization of alkaline phosphatase without the use of activating chemicals demonstrates that the in vacuo method is a practical and efficient process for immobilizing proteins especially small amounts of valuable proteins. The success with ALPase demonstrates that this immobilization process should be applicable to all soluble proteins containing carboxyl groups and other solid supports with free amino groups.
References Brown, E., Joyeau, R., 1974. Immobilized enzymes. 7. Use of vanacryls in preparation of immobilized arginase and alkaline phosphatase. Polymer 15, 546–552. Filmon, R., Grizon, F., Basl´e, M.F., Chappaard, D., 2002. Effects of negatively charged groups (carboxymethyl) on the calcification of poly(2-hydroxyethyl methacrylate). Biomaterials 23, 3053–3059.
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