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Catalytic Effects of Galactose Oxidase on Micelle-Forming Galactolipids
Journal of Colloid and Interface Science 255, 260–264 (2002) doi:10.1006/jcis.2002.8676
Catalytic Effects of Galactose Oxidase on Micelle-Forming Galactolipids1 Hiromi Kitano,∗,2 Yukiko Ishino,∗ and Ali Hamed Al-Arifi† ∗ Department of Chemical and Biochemical Engineering, Toyama University, Toyama 930, Japan; and †Institute of Pharmaceutical Chemistry, University of Halle, D-06099, Germany Received January 25, 2002; accepted August 14, 2002
Catalytic effects of galactose oxidase on the oxidation of β-Dgalactose-carrying lipids with an oligo-ethylene glycol spacer (number of ethylene glycol units (n) = 1, 2, 3, 6, 9, 13, and 20) were examined. The affinity of galactose oxidase for the galactose residue in the amphiphile (estimated by the inverse of the Michaelis constant, Km ) was much higher than those for free D-galactose and small β-D-galactopyranosides, and dependent on the length of the ethylene glycol spacer. That is, both below and above the critical micellar concentration, the 1/Km values decreased with an increase in the n value. The effectiveness of the enzyme, which can be estimated by the kcat /Km value, showed the same tendency as the 1/Km value. These results could be attributed to the role of the nonpolar environment around the galactose residue in the binding by the enzyme. A significant enhancement of the enzymatic oxidation of galactose residue on the liposome surface was also observed. C 2002 Elsevier Science (USA) Key Words: galactolipid; galactose oxidase; CMC; liposome; spacer length; Michaelis constant.
INTRODUCTION
Recent studies on carbohydrate chains have been clarifying the importance of carbohydrate chains in vivo (e.g., immunological protection system, nervous system, virus infection, fertilization) (1–4). Based on these studies, glycoconjugates, which are carbohydrates linked to a variety of materials, including proteins, lipids, and synthetic polymers, have been practically applied in various fields (5, 6). For instance, techniques of medical treatment such as artificial organs or drug delivery systems (DDS) have been developed by taking advantage of the recognition of carbohydrates by cells in vivo and by investigating their therapeutic effects on diseases (7, 8). We have been studying the recognition between sugar-carrying liposomes and sugar receptors at liquid–lipid and solid–lipid interfaces (9, 10). Galactose oxidase catalyzes the oxidization of exposed primary hydroxyl groups in nonreducing, terminal galactose and
N -acetyl-D-galactosamine residues (11). A combination of galactose oxidase with NaB[3 H]4 is widely used as a technique to label cell-surface glycoconjugates (12–14). It was found that only a portion of glycolipids are available to galactose oxidase, and different glycolipids vary in their susceptibilities to the enzyme (15–18). The interaction of galactose oxidase with the major red-cell glycolipid (globoside) and GM1 ganglioside in liposome suspensions was also reported previously (17, 18). Recently, using a lipophilic radical initiator and galactosecarrying vinyl monomer, 2-methacryloyloxyethyl β-Dgalactopyranoside (MEGal), we prepared novel amphiphiles with many pendant galactose residues (galactolipids) (10). By incorporating the galactolipids into liposomes, we could examine the recognition of amino group-carrying liposomes by galactose-carrying liposomes, which had been treated with galactose oxidase beforehand, as a model system of induction of lymphocyte cytotoxicity (19). We also found that the affinity of galactose oxidase for the galactose residues in the polymer chains on the liposome surface was much stronger than that for free galactose (more than 104 times) (19, 20). In this report, to verify what happens in the catalytic reaction of enzyme with the carbohydrate chains on the colloid surfaces, galactose-carrying lipids with an oligo-ethylene glycol spacer, HD-Sn -Gal (Scheme 1), were prepared. Kinetic analyses of the catalytic behavior of galactose oxidase in the oxidation of galactose residues in lipids in various reaction fields including micelles and liposome surfaces were examined. Why galactose oxidase exhibits enhanced catalytic activity on the galactose residues on the colloid surfaces will be described below.
1 Presented at the 50th Annual Meeting of the Society of Polymer Science, Japan, at Osaka, in May 2001. 2 To whom correspondence should be addressed.
0021-9797/02 $35.00
C 2002 Elsevier Science (USA)
All rights reserved.
260
MATERIALS AND METHODS
1. Materials β-D-Galactolipids with hexadecyl groups and various oligoethyleneglycol spacers (HD-Sn -Gal, n = 1, 2, 3, 6, 9, 13, and 20, Scheme 1a) were prepared as previously reported (21). L-α-Dimyristoyl phosphatidylcholine (DMPC) and galactose oxidase (from Dactylium dendroides, 240 unit/mg) were from Sigma. Other reagents were commercially available. Deionized water was distilled before use.
261
GALACTOSE OXIDASE
Since sufficient amounts of HRP, 4-AA, and phenol were added to the reaction solution, the reaction of galactose oxidase to the galactose residue is the rate-determining step. The initial increase in optical density per minute was calculated from the initial linear portion of the curve (ε = 12,000). One unit of galactose oxidase causes the formation of one micromole of hydrogen peroxide per minute at 25◦ C. The stock solution of galactose oxidase was diluted to the appropriate concentration, and the catalytic activity of the enzyme was checked just prior to use. 4. Kinetic Analyses The Vmax and K m values of the enzyme reaction were determined by the Hanes–Woolf plot (24), [S]/v = (1/Vmax )[S] + K m /Vmax
SCHEME 1. Chemical structure of galactolipid, (a) HD-Sn -Gal (n = 1, 2, 3, 6, 9, 13, and 20), (b) DHDG-Gal, and (c) DODA-PMEGal.
2. Determination of CMC The critical micellar concentration (CMC) of galactolipids was determined by both surface tension (γ ) and fluorimetric methods. For the surface tension method, a du No¨uy apparatus (Taihei Rika Co., Osaka, Japan) was used to determine the CMC value from the bending point in the plot of γ vs the concentration of HD-Sn -Gal. In the fluorescence measurements (F-777 spectrofluorometer, Japan Spectroscopic Co., Tokyo, Japan), 8anilino-1-naphthalenesulfonic acid ammonium salt (8,1-ANS, 1 µM in the sample solution) was used as a hydrophobicity probe. The wavelengths of excitation and emission were 350 and 450 nm, respectively. The CMC value was estimated from a bending point in the plot of fluorescence intensity vs the concentration of HD-Sn -Gal. 3. Enzyme Activity Measurements The oxidation reaction of galactose residues catalyzed by galactose oxidase (0.14 units/mL HEPES buffer (10 mM, pH 7.0, total volume, 1.26 mL)) was followed using a spectrophotometric method (22, 23). In liposome systems, HD-S9 -Gal (10 mg) was ultrasonicated with a suspension of L-α-dimyristoyl phosphatidylcholine (DMPC) liposome (30 mg in the HEPES buffer), and after passing a disposable filter (Millipore Millex GV, 0.22 µm), applied to the enzymatic reaction. Hydrogen peroxide produced by the oxidation of galactose was reduced by reaction with 4-aminoantipyrine (4-AA, 49.2 mM) and phenol (531 mM) catalyzed by horseradish peroxidase (HRP, 1.5 mg/mL), and the increase in optical density at 500 nm due to the production of quinoneimine dye was measured for 5 min by a spectrophotometer (Ubest-35, Japan Spectroscopic Co.) thermostated at 25◦ C.
[1]
where [S] is the concentration of substrate (total concentration of galactose residues in the reacting solution), v is the initial reaction velocity, Vmax is the limiting value of initial reaction velocity (= kcat [E]o ; kcat = turnover number of the enzyme; [E]o = initial concentration of enzyme), and K m is the Michaelis constant. Graphing [S]/v versus [S] yields a straight line where the slope is 1/Vmax , the y-intercept is K m /Vmax , and the x-intercept is −K m . The data in the figures and table represent the mean values of two measurements. The uncertainties of the kinetic parameters were within 10%. RESULTS AND DISCUSSION
1. Critical Micellar Concentration of Galactolipids At first, we determined the critical micellar concentration (CMC) of the galactolipids, HD-Sn -Gal. The CMC values for HD-Sn -Gal with a short spacer (n = 1, 2, and 3) could be determined by both surface tension and fluorimetric methods (Table 1). The CMC values obtained by these methods were quite similar. With the increase in the spacer length (n = 6, 9, 13 and 20), however, the bending point could not be definitely determined by the fluorescence measurements probably due to a loose structure of the micelle as discussed in Section 2. Therefore, the CMC values for HD-Sn -Gal (n = 6, 9, 13, and 20) TABLE 1 The CMC Values for HD-Sn -Gala Number of Ethylene glycol units in the spacer CMC (µM)
1
2
3
6
9
13
20
42 (54)b 94 (107)b 150 (166)b 490 1030 1580 2280
a In the HEPES buffer (10 mM, pH 7.0) at 25◦ C. By the surface tension method. b By the fluorometric method. [8,1-ANS] = 1 µM. Ex = 350 nm, and Em = 450 nm.
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KITANO, ISHINO, AND AL-ARIFI
obtained by the surface tension method are compiled in Table 1. By the increase in the spacer length, the CMC value monotonously increased, which is intuitively understandable. 2. Catalytic Behavior of Galactose Oxidase for Galactose-Carrying Amphiphiles
TABLE 2 Kinetic Constants of Galactose Oxidase for Various Galactose Residues at Different Reaction Fieldsa
Sacccharides
Km (mM)
kcat 102 kcat / (mM · mL/ K m (mL/ min · units) min · units)
Notice
After the incubation of galactose-carrying amphiphile (HDSn -Gal, n = 6, 9, 13, and 20) with a solution of galactose oxidase, the production of H2 O2 was definitely confirmed by the spectrophotometric method. As for the galactolipids with a shorter spacer (HD-Sn -Gal, n = 1, 2, and 3), on the contrary, the production of H2 O2 could be only very vaguely detected, probably because of the very low solubility of the lipids. Though a steric hindrance for the enzyme to approach the galactose residue in the amphiphile seemed to be effective, the poor solubility made the estimation of catalytic activity to the galactolipids with a short spacer below and above the CMC impossible. Figure 1 shows a typical example of Hanes–Woolf plot for the catalytic reaction of galactose oxidase to a galactose-carrying amphiphile (HD-S9 -Gal). From the x-intercept, the Michaelis constant, K m , could be determined, and compiled in Table 2. The affinity (estimated by the inverse of K m value) of galactose oxidase for the amphiphiles with a long spacer (HD-Sn -Gal, n = 6, 9, 13, and 20) was much larger than those for free Dgalactose and β-D-galactopyranosides with a small molecular weight (lactose, p-nitrophenyl β-D-galactopyranoside (PNPG) and methyl β-D-galactopyranoside (MG)). Furthermore, by varying the spacer length of the amphiphiles, we could examine the effect of the distance between the galactose residue and the aliphatic group on K m (Table 2 and Fig. 2). The larger the spacer length of galactolipid was, the smaller the affinity of galactose oxidase was. This tendency could be observed only when the substrate had a sufficient solubility. In a separate experiment, it was also found that an ethyleneglycol oligomer (degree of polymerization, 8.5) did not show any effect on the catalytic property of galactose oxidase. These results suggest that galactose oxidase can effectively recognize and oxidize
Galactose Lactose PNPG MG HD-S6 -Gal HD-S9 -Gal HD-S9 -Gal HD-S9 -Gal
FIG. 1. Hanes–Woolf plot for the catalytic reaction of galactose oxidase with a galactose-carrying amphiphile (HD-S9 -Gal) in a micellar state in HEPES buffer (10 mM, pH 7.0). [Enzyme]=0.14 U/mL. CMC for HD-S9 -Gal, 1.03 mM.
FIG. 2. Effect of the spacer length on K m and kcat /K m values at 25◦ C in HEPES buffer (10 mM, pH 7.0). [Enzyme] = 0.14 U/mL. , : Bellow CMC. , : Above CMC.
HD-S13 -Gal HD-S13 -Gal HD-S20 -Gal HD-S20 -Gal DODA-PMEGal (DP = 8.5)
a b
58 528 22 52 0.12 0.30 0.19 0.20 0.41 0.40 2.00 2.00 0.0030
1.0 0.39 0.40 1.6 0.032 0.032 0.029 0.21 0.074 0.065 0.091 0.088 0.012
1.7 0.081 1.8 3.0 25 11 15 99 18 16 4.6 4.3 400
Ref. (20) Ref. (20) Ref. (20) Ref. (20) Below the CMC Below the CMC Above the CMC Incorporated in DMPC liposomeb Below the CMC Above the CMC Below the CMC Above the CMC Incorporated in Polymerized DDPC liposome Ref. (20)
At 25◦ C unless mentioned. At 23◦ C.
galactose residues in the vicinity of nonpolar group, if the substrate can be dissolved in the reacting solution sufficiently. We at first expected that the enzyme would catalyze the oxidation of galactose residue more effectively above the CMC than below that. This is because it was previously reported that the galactose oxidase from Polyporus circinatus had a high affinity for galactomannan guaran (polysaccharide constituted from galactose and mannose) (22). Furthermore, the K m value for galactose residue in poly(2-methacryloyloxyethyl βD-galactopyranoside) (PMEGal) (0.35 and 0.13 mM (degree of polymerization (DP) = 16 and 53), respectively) was much smaller than that for D-galactose (58 mM) (19, 20).
GALACTOSE OXIDASE
The table shows that the affinity and effectiveness of the enzyme for HD-Sn -Gal (n = 9, 13, and 20) below and above the CMC were not largely different each other. This is probably because the structure of the HD-Sn -Gal micelle is not so rigid that the accessibility of the enzyme to the galactose residue is not significantly affected by the formation of micelle. This result is consistent with the very gradual increase in fluorescence intensity of ANS above the CMC as discussed in Section 1. Furthermore, the kcat /K m value, which represents the catalytic efficiency of galactose oxidase for the galactose residue, decreased with the spacer length (Fig. 2). Since the spacer provides accessibility for the enzyme to the galactose residue, the results in the figure strongly supports that the catalytic behavior of galactose oxidase represents an increased efficiency for the HDSn -Gal due to the nonpolar environment around the substrates with the appropriate accessibility. It should be mentioned here that the binding of sugar residues by lectins are also promoted by the introduction of nonpolar group near the sugar residues. For example, a lectin from Canavalia ensiformis (Concanavalin A) much more strongly binds to a α-mannose residue linked to a 4methylumbelliferryl group than methyl α-D-mannopyranoside (25). 3. Catalytic Behavior of Galactose Oxidase for Galactose-Carrying Liposomes Next, we examined the catalytic behavior of galactose oxidase to the galactolipid, HD-S9 -Gal, incorporated in a Lα-dimyristoyl phosphatidylcholine (DMPC) liposome. The production of H2 O2 was definitely confirmed spectrophotometrically, which shows that the galactose-carrying head group functions as a substrate for galactose oxidase. Since the lipid can be dissolved in a buffer, the reaction occurs both in a bulk solution and the lipid bilayer–liquid interface. The turnover number, kcat , and the effectiveness (kcat /K m ) of the enzyme in the presence of DMPC liposome were, however, much larger (ca. 7–9 times) than those for the monomolecular and micellar systems (Table 2), which definitely shows the advantage of the liposome surface for the enzymatic oxidation of galactose. When the galactose-carrying liposome consisted of photopolymerized bis(trans,trans-2,4-dioctadecadidienoyl) phosphatidylcholine (DDPC) and 1,3-di-O-hexadecyl-2-O-(β-Dgalactopyranosyl)glycerol (DHDG-Gal, Scheme 1b) (20, 26) nor HD-S3 -Gal was incubated with the solution of galactose oxidase, no production of H2 O2 was detected by the spectrophotometric method, which is in contrast to the results for HD-S9 Gal. Previously, it was shown that galactose oxidase could not oxidize the galactosyl residue of ceramide dihexoside in liposomes, probably because its carbohydrate moiety is too close to the membrane surface (17). Since the galactose residue of both DHDG-Gal and HD-S3 -Gal is quite near the anchoring group, galactose oxidase would not easily approach the substrate. Therefore, the present result is understandable. Previously, we prepared the galactose-carrying amphiphiles (poly(2-methacryloyloxyethyl β-D-galactopyranoside)–
263
dioctadecylamine conjugate, DODA-PMEGal, Scheme 1c, which have a galactose-carrying polymer chain as a hydrophilic group, from the lipophilic radical initiator and MEGal. The K m value for the galactose-carrying lipid with many pendant galactose residues (DODA-PMEGal, DP = 8.5, 20 wt%) incorporated into the polymerized DDPC liposome (0.0030 mM) (20) was much lower than that for the lipid with a single galactose residue (HD-S9 -Gal, 0.20 mM) (Table 2), probably because of the microscopically very high substrate concentration on the DODAPMEGal/polymerized DDPC liposome. In spite of the steric disadvantage that the reaction field is above the lipid bilayer surface, the affinity of galactose oxidase for HD-S9 -Gal on the liposome surface was comparable to those for PMEGal in bulk solution (K m , 0.35 and 0.13 mM (DP = 16 and 53), respectively) (20). The kcat value, which is defined as the number of substrate molecules converted into product per enzyme molecule per unit time, of galactose oxidase for the HD-S9 -Gal/DMPC liposome (0.21 mM · mL/min · units) was lower than those of free Dgalactose and small β-D-galactopyranosides and comparable to those of PMEGals (0.091 and 0.14 mM · mL/min · units (DP = 16 and 53), respectively), whereas the kcat /K m value, which represents the catalytic efficiency of an enzyme, of galactose oxidase for the HD-S9 -Gal/DMPC liposome system (0.99 mL/ min · units) was much larger than those for free D-galactose and small sugars (Table 1), and comparable to those for PMEGals (0.26 and 1.10 mL/min · units (DP = 16 and 53), respectively). Since the galactose-carrying chains are fixed to the liposome surface, the mobility of the enzyme molecule attaching the galactose residue above the liposome surface might be restricted, and therefore, it is understandable that the turnover number (kcat ) of galactose oxidase for HD-S9 -Gal was smaller than those for galactose, and other small sugars. However, the mobility of the galactose residue is still afforded by the oligo-ethylene glycol spacer. Consequently, the fixation of galactose-carrying chain to the membrane surface at a large surface density might enhance the reactivity of the enzyme-substrate complex (kcat ) to be much larger value than those at the monomolecular and micellar phases. In addition, since the K m value for galactolipid is much smaller than those of small galactose-derivatives, the sufficiently large turnover number (kcat ) resulted in a larger catalytic efficiency (kcat /K m ) of galactose oxidase for the HD-Sn -Gal-carrying liposome than for the small galactose derivatives. In conclusion, the affinity of galactose oxidase for sugar residues in the galactolipid was dependent on the length of the spacer between the galactose residue and aliphatic anchor group. The incorporation of galactolipids into the liposome largely enhanced the catalytic effectiveness of the enzyme, due to the increase in the turnover number of the catalysis above the liposome surface. The present study indicates that the spatial distribution of substrates significantly affects the catalytic behavior of the enzyme. Furthermore, the nonpolar group in the vicinity of sugar residues enhances the affinity of sugar-recognizing
264
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proteins (not only lectins but also enzymes), which would be valuable information in carbohydrate chemistry. ACKNOWLEDGMENTS This work was supported by Grants-in-Aid for Scientific Research 12450381, 13022225, and 13555260 from the Ministry of Education, Science, Sports and Culture.
REFERENCES 1. 2. 3. 4. 5. 6. 7.
8.
9.
Wassarman, P. M., Science 235, 553 (1987). Imai, Y., Lasky, L. A., and Rosen, S. D., Nature 361, 555 (1993). Bliss, T. V. P., and Collingridge, G. L., Nature 361, 31 (1993). Mizuochi, T., Mattews, T. J., Kato, M., Hamako, J., Titani, K., Solomon, J., and Feizi, T., J. Biol. Chem. 265, 8519 (1990). Lee, Y. C., and Lee, R. T., Eds., “Methods in Enzymology,” Vol. 242. Academic Press, New York, 1994. Lee, Y. C., and Lee, R. T., Eds., “Methods in Enzymology,” Vol. 247. Academic Press, New York, 1994. Kim, S. W., Pai, C. M., Makino, K., Seminoff, L. A., Holmberg, D. L., Gleeson, J. M., Wilson, D. E., and Mack, E. J., J. Controlled Release 11, 193 (1990). Ouchi, T., and Ohya, Y., in “Neoglycoconjugates: Preparation and Applications” (Y. C. Lee and R. T. Lee, Eds.), p. 465. Academic Press, New York, 1994. Kitano, H., and Ohno, K., Langmuir 10, 4131 (1994).
10. Kitano, H., Sohda, K., and Kosaka, A., Bioconjugate Chem. 6, 131 (1995). 11. Suzuki, Y., and Suzuki, K., J. Lipid Res. 13, 687 (1972). 12. Eccleston, E. D., White, T. W., Howard, J. B., and Hamilton, D. W., Mol. Reprod. Dev. 37, 110 (1994). 13. Enrich, C., and Gahmberg, C. G., Biochem. J. 227, 565 (1985). 14. Kumarasamy, R., and Blough, H. A., Arch. Biochem. Biophys. 236, 593 (1985). 15. Stewart, R. J., and Boggs, J. M., Biochemistry 32, 5605 (1993). 16. Lampio, A., Rauvala, H., and Gahmberg, C. G., Eur. J. Biochem. 157, 611 (1986). 17. Lampio, A., Siissalo, I., and Gehmberg, C. G., Eur. J. Biochem. 178, 87 (1988). 18. Masserini, M., Sonnino, S., Ghidoni, R., and Chigormo, V., Biochim. Biophys. Acta 688, 333 (1982). 19. Ohno, K., Sohda, K., Kosaka, A., and Kitano, H., Bioconjugate Chem. 6, 361 (1995). 20. Ohno, K., and Kitano, H., Bioconjugate Chem. 9, 548 (1998). 21. Wilhelm, F., Chatterjee, S. K., Ratty, B., Nuhn, P., Benecke, R., and Ortwein, J., Liebigs Ann. 1673 (1995). 22. Cooper, J. A. D., Smith, W., Bacila, M., and Medina, H., J. Biol. Chem. 234, 445 (1959). 23. Avigad, G., Amaral, D., Asensio, C., and Horecker, B. L., J. Biol. Chem. 237, 2736 (1962). 24. Gerrett, R. H., and Grisham, C., “Biochemistry.” Saunders, Orlando, FL, 1995. 25. Frank, G. L., Robert, M. C., and Thomas, M. J., Biochemistry 16, 159 (1977). 26. Minamikawa, H., Murakami, T., and Hato, M., Chem. Phys. Lipids 72, 111 (1994).
Catalytic Effects of Galactose Oxidase on Micelle-Forming Galactolipids1 Hiromi Kitano,∗,2 Yukiko Ishino,∗ and Ali Hamed Al-Arifi† ∗ Department of Chemical and Biochemical Engineering, Toyama University, Toyama 930, Japan; and †Institute of Pharmaceutical Chemistry, University of Halle, D-06099, Germany Received January 25, 2002; accepted August 14, 2002
Catalytic effects of galactose oxidase on the oxidation of β-Dgalactose-carrying lipids with an oligo-ethylene glycol spacer (number of ethylene glycol units (n) = 1, 2, 3, 6, 9, 13, and 20) were examined. The affinity of galactose oxidase for the galactose residue in the amphiphile (estimated by the inverse of the Michaelis constant, Km ) was much higher than those for free D-galactose and small β-D-galactopyranosides, and dependent on the length of the ethylene glycol spacer. That is, both below and above the critical micellar concentration, the 1/Km values decreased with an increase in the n value. The effectiveness of the enzyme, which can be estimated by the kcat /Km value, showed the same tendency as the 1/Km value. These results could be attributed to the role of the nonpolar environment around the galactose residue in the binding by the enzyme. A significant enhancement of the enzymatic oxidation of galactose residue on the liposome surface was also observed. C 2002 Elsevier Science (USA) Key Words: galactolipid; galactose oxidase; CMC; liposome; spacer length; Michaelis constant.
INTRODUCTION
Recent studies on carbohydrate chains have been clarifying the importance of carbohydrate chains in vivo (e.g., immunological protection system, nervous system, virus infection, fertilization) (1–4). Based on these studies, glycoconjugates, which are carbohydrates linked to a variety of materials, including proteins, lipids, and synthetic polymers, have been practically applied in various fields (5, 6). For instance, techniques of medical treatment such as artificial organs or drug delivery systems (DDS) have been developed by taking advantage of the recognition of carbohydrates by cells in vivo and by investigating their therapeutic effects on diseases (7, 8). We have been studying the recognition between sugar-carrying liposomes and sugar receptors at liquid–lipid and solid–lipid interfaces (9, 10). Galactose oxidase catalyzes the oxidization of exposed primary hydroxyl groups in nonreducing, terminal galactose and
N -acetyl-D-galactosamine residues (11). A combination of galactose oxidase with NaB[3 H]4 is widely used as a technique to label cell-surface glycoconjugates (12–14). It was found that only a portion of glycolipids are available to galactose oxidase, and different glycolipids vary in their susceptibilities to the enzyme (15–18). The interaction of galactose oxidase with the major red-cell glycolipid (globoside) and GM1 ganglioside in liposome suspensions was also reported previously (17, 18). Recently, using a lipophilic radical initiator and galactosecarrying vinyl monomer, 2-methacryloyloxyethyl β-Dgalactopyranoside (MEGal), we prepared novel amphiphiles with many pendant galactose residues (galactolipids) (10). By incorporating the galactolipids into liposomes, we could examine the recognition of amino group-carrying liposomes by galactose-carrying liposomes, which had been treated with galactose oxidase beforehand, as a model system of induction of lymphocyte cytotoxicity (19). We also found that the affinity of galactose oxidase for the galactose residues in the polymer chains on the liposome surface was much stronger than that for free galactose (more than 104 times) (19, 20). In this report, to verify what happens in the catalytic reaction of enzyme with the carbohydrate chains on the colloid surfaces, galactose-carrying lipids with an oligo-ethylene glycol spacer, HD-Sn -Gal (Scheme 1), were prepared. Kinetic analyses of the catalytic behavior of galactose oxidase in the oxidation of galactose residues in lipids in various reaction fields including micelles and liposome surfaces were examined. Why galactose oxidase exhibits enhanced catalytic activity on the galactose residues on the colloid surfaces will be described below.
1 Presented at the 50th Annual Meeting of the Society of Polymer Science, Japan, at Osaka, in May 2001. 2 To whom correspondence should be addressed.
0021-9797/02 $35.00
C 2002 Elsevier Science (USA)
All rights reserved.
260
MATERIALS AND METHODS
1. Materials β-D-Galactolipids with hexadecyl groups and various oligoethyleneglycol spacers (HD-Sn -Gal, n = 1, 2, 3, 6, 9, 13, and 20, Scheme 1a) were prepared as previously reported (21). L-α-Dimyristoyl phosphatidylcholine (DMPC) and galactose oxidase (from Dactylium dendroides, 240 unit/mg) were from Sigma. Other reagents were commercially available. Deionized water was distilled before use.
261
GALACTOSE OXIDASE
Since sufficient amounts of HRP, 4-AA, and phenol were added to the reaction solution, the reaction of galactose oxidase to the galactose residue is the rate-determining step. The initial increase in optical density per minute was calculated from the initial linear portion of the curve (ε = 12,000). One unit of galactose oxidase causes the formation of one micromole of hydrogen peroxide per minute at 25◦ C. The stock solution of galactose oxidase was diluted to the appropriate concentration, and the catalytic activity of the enzyme was checked just prior to use. 4. Kinetic Analyses The Vmax and K m values of the enzyme reaction were determined by the Hanes–Woolf plot (24), [S]/v = (1/Vmax )[S] + K m /Vmax
SCHEME 1. Chemical structure of galactolipid, (a) HD-Sn -Gal (n = 1, 2, 3, 6, 9, 13, and 20), (b) DHDG-Gal, and (c) DODA-PMEGal.
2. Determination of CMC The critical micellar concentration (CMC) of galactolipids was determined by both surface tension (γ ) and fluorimetric methods. For the surface tension method, a du No¨uy apparatus (Taihei Rika Co., Osaka, Japan) was used to determine the CMC value from the bending point in the plot of γ vs the concentration of HD-Sn -Gal. In the fluorescence measurements (F-777 spectrofluorometer, Japan Spectroscopic Co., Tokyo, Japan), 8anilino-1-naphthalenesulfonic acid ammonium salt (8,1-ANS, 1 µM in the sample solution) was used as a hydrophobicity probe. The wavelengths of excitation and emission were 350 and 450 nm, respectively. The CMC value was estimated from a bending point in the plot of fluorescence intensity vs the concentration of HD-Sn -Gal. 3. Enzyme Activity Measurements The oxidation reaction of galactose residues catalyzed by galactose oxidase (0.14 units/mL HEPES buffer (10 mM, pH 7.0, total volume, 1.26 mL)) was followed using a spectrophotometric method (22, 23). In liposome systems, HD-S9 -Gal (10 mg) was ultrasonicated with a suspension of L-α-dimyristoyl phosphatidylcholine (DMPC) liposome (30 mg in the HEPES buffer), and after passing a disposable filter (Millipore Millex GV, 0.22 µm), applied to the enzymatic reaction. Hydrogen peroxide produced by the oxidation of galactose was reduced by reaction with 4-aminoantipyrine (4-AA, 49.2 mM) and phenol (531 mM) catalyzed by horseradish peroxidase (HRP, 1.5 mg/mL), and the increase in optical density at 500 nm due to the production of quinoneimine dye was measured for 5 min by a spectrophotometer (Ubest-35, Japan Spectroscopic Co.) thermostated at 25◦ C.
[1]
where [S] is the concentration of substrate (total concentration of galactose residues in the reacting solution), v is the initial reaction velocity, Vmax is the limiting value of initial reaction velocity (= kcat [E]o ; kcat = turnover number of the enzyme; [E]o = initial concentration of enzyme), and K m is the Michaelis constant. Graphing [S]/v versus [S] yields a straight line where the slope is 1/Vmax , the y-intercept is K m /Vmax , and the x-intercept is −K m . The data in the figures and table represent the mean values of two measurements. The uncertainties of the kinetic parameters were within 10%. RESULTS AND DISCUSSION
1. Critical Micellar Concentration of Galactolipids At first, we determined the critical micellar concentration (CMC) of the galactolipids, HD-Sn -Gal. The CMC values for HD-Sn -Gal with a short spacer (n = 1, 2, and 3) could be determined by both surface tension and fluorimetric methods (Table 1). The CMC values obtained by these methods were quite similar. With the increase in the spacer length (n = 6, 9, 13 and 20), however, the bending point could not be definitely determined by the fluorescence measurements probably due to a loose structure of the micelle as discussed in Section 2. Therefore, the CMC values for HD-Sn -Gal (n = 6, 9, 13, and 20) TABLE 1 The CMC Values for HD-Sn -Gala Number of Ethylene glycol units in the spacer CMC (µM)
1
2
3
6
9
13
20
42 (54)b 94 (107)b 150 (166)b 490 1030 1580 2280
a In the HEPES buffer (10 mM, pH 7.0) at 25◦ C. By the surface tension method. b By the fluorometric method. [8,1-ANS] = 1 µM. Ex = 350 nm, and Em = 450 nm.
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KITANO, ISHINO, AND AL-ARIFI
obtained by the surface tension method are compiled in Table 1. By the increase in the spacer length, the CMC value monotonously increased, which is intuitively understandable. 2. Catalytic Behavior of Galactose Oxidase for Galactose-Carrying Amphiphiles
TABLE 2 Kinetic Constants of Galactose Oxidase for Various Galactose Residues at Different Reaction Fieldsa
Sacccharides
Km (mM)
kcat 102 kcat / (mM · mL/ K m (mL/ min · units) min · units)
Notice
After the incubation of galactose-carrying amphiphile (HDSn -Gal, n = 6, 9, 13, and 20) with a solution of galactose oxidase, the production of H2 O2 was definitely confirmed by the spectrophotometric method. As for the galactolipids with a shorter spacer (HD-Sn -Gal, n = 1, 2, and 3), on the contrary, the production of H2 O2 could be only very vaguely detected, probably because of the very low solubility of the lipids. Though a steric hindrance for the enzyme to approach the galactose residue in the amphiphile seemed to be effective, the poor solubility made the estimation of catalytic activity to the galactolipids with a short spacer below and above the CMC impossible. Figure 1 shows a typical example of Hanes–Woolf plot for the catalytic reaction of galactose oxidase to a galactose-carrying amphiphile (HD-S9 -Gal). From the x-intercept, the Michaelis constant, K m , could be determined, and compiled in Table 2. The affinity (estimated by the inverse of K m value) of galactose oxidase for the amphiphiles with a long spacer (HD-Sn -Gal, n = 6, 9, 13, and 20) was much larger than those for free Dgalactose and β-D-galactopyranosides with a small molecular weight (lactose, p-nitrophenyl β-D-galactopyranoside (PNPG) and methyl β-D-galactopyranoside (MG)). Furthermore, by varying the spacer length of the amphiphiles, we could examine the effect of the distance between the galactose residue and the aliphatic group on K m (Table 2 and Fig. 2). The larger the spacer length of galactolipid was, the smaller the affinity of galactose oxidase was. This tendency could be observed only when the substrate had a sufficient solubility. In a separate experiment, it was also found that an ethyleneglycol oligomer (degree of polymerization, 8.5) did not show any effect on the catalytic property of galactose oxidase. These results suggest that galactose oxidase can effectively recognize and oxidize
Galactose Lactose PNPG MG HD-S6 -Gal HD-S9 -Gal HD-S9 -Gal HD-S9 -Gal
FIG. 1. Hanes–Woolf plot for the catalytic reaction of galactose oxidase with a galactose-carrying amphiphile (HD-S9 -Gal) in a micellar state in HEPES buffer (10 mM, pH 7.0). [Enzyme]=0.14 U/mL. CMC for HD-S9 -Gal, 1.03 mM.
FIG. 2. Effect of the spacer length on K m and kcat /K m values at 25◦ C in HEPES buffer (10 mM, pH 7.0). [Enzyme] = 0.14 U/mL. , : Bellow CMC. , : Above CMC.
HD-S13 -Gal HD-S13 -Gal HD-S20 -Gal HD-S20 -Gal DODA-PMEGal (DP = 8.5)
a b
58 528 22 52 0.12 0.30 0.19 0.20 0.41 0.40 2.00 2.00 0.0030
1.0 0.39 0.40 1.6 0.032 0.032 0.029 0.21 0.074 0.065 0.091 0.088 0.012
1.7 0.081 1.8 3.0 25 11 15 99 18 16 4.6 4.3 400
Ref. (20) Ref. (20) Ref. (20) Ref. (20) Below the CMC Below the CMC Above the CMC Incorporated in DMPC liposomeb Below the CMC Above the CMC Below the CMC Above the CMC Incorporated in Polymerized DDPC liposome Ref. (20)
At 25◦ C unless mentioned. At 23◦ C.
galactose residues in the vicinity of nonpolar group, if the substrate can be dissolved in the reacting solution sufficiently. We at first expected that the enzyme would catalyze the oxidation of galactose residue more effectively above the CMC than below that. This is because it was previously reported that the galactose oxidase from Polyporus circinatus had a high affinity for galactomannan guaran (polysaccharide constituted from galactose and mannose) (22). Furthermore, the K m value for galactose residue in poly(2-methacryloyloxyethyl βD-galactopyranoside) (PMEGal) (0.35 and 0.13 mM (degree of polymerization (DP) = 16 and 53), respectively) was much smaller than that for D-galactose (58 mM) (19, 20).
GALACTOSE OXIDASE
The table shows that the affinity and effectiveness of the enzyme for HD-Sn -Gal (n = 9, 13, and 20) below and above the CMC were not largely different each other. This is probably because the structure of the HD-Sn -Gal micelle is not so rigid that the accessibility of the enzyme to the galactose residue is not significantly affected by the formation of micelle. This result is consistent with the very gradual increase in fluorescence intensity of ANS above the CMC as discussed in Section 1. Furthermore, the kcat /K m value, which represents the catalytic efficiency of galactose oxidase for the galactose residue, decreased with the spacer length (Fig. 2). Since the spacer provides accessibility for the enzyme to the galactose residue, the results in the figure strongly supports that the catalytic behavior of galactose oxidase represents an increased efficiency for the HDSn -Gal due to the nonpolar environment around the substrates with the appropriate accessibility. It should be mentioned here that the binding of sugar residues by lectins are also promoted by the introduction of nonpolar group near the sugar residues. For example, a lectin from Canavalia ensiformis (Concanavalin A) much more strongly binds to a α-mannose residue linked to a 4methylumbelliferryl group than methyl α-D-mannopyranoside (25). 3. Catalytic Behavior of Galactose Oxidase for Galactose-Carrying Liposomes Next, we examined the catalytic behavior of galactose oxidase to the galactolipid, HD-S9 -Gal, incorporated in a Lα-dimyristoyl phosphatidylcholine (DMPC) liposome. The production of H2 O2 was definitely confirmed spectrophotometrically, which shows that the galactose-carrying head group functions as a substrate for galactose oxidase. Since the lipid can be dissolved in a buffer, the reaction occurs both in a bulk solution and the lipid bilayer–liquid interface. The turnover number, kcat , and the effectiveness (kcat /K m ) of the enzyme in the presence of DMPC liposome were, however, much larger (ca. 7–9 times) than those for the monomolecular and micellar systems (Table 2), which definitely shows the advantage of the liposome surface for the enzymatic oxidation of galactose. When the galactose-carrying liposome consisted of photopolymerized bis(trans,trans-2,4-dioctadecadidienoyl) phosphatidylcholine (DDPC) and 1,3-di-O-hexadecyl-2-O-(β-Dgalactopyranosyl)glycerol (DHDG-Gal, Scheme 1b) (20, 26) nor HD-S3 -Gal was incubated with the solution of galactose oxidase, no production of H2 O2 was detected by the spectrophotometric method, which is in contrast to the results for HD-S9 Gal. Previously, it was shown that galactose oxidase could not oxidize the galactosyl residue of ceramide dihexoside in liposomes, probably because its carbohydrate moiety is too close to the membrane surface (17). Since the galactose residue of both DHDG-Gal and HD-S3 -Gal is quite near the anchoring group, galactose oxidase would not easily approach the substrate. Therefore, the present result is understandable. Previously, we prepared the galactose-carrying amphiphiles (poly(2-methacryloyloxyethyl β-D-galactopyranoside)–
263
dioctadecylamine conjugate, DODA-PMEGal, Scheme 1c, which have a galactose-carrying polymer chain as a hydrophilic group, from the lipophilic radical initiator and MEGal. The K m value for the galactose-carrying lipid with many pendant galactose residues (DODA-PMEGal, DP = 8.5, 20 wt%) incorporated into the polymerized DDPC liposome (0.0030 mM) (20) was much lower than that for the lipid with a single galactose residue (HD-S9 -Gal, 0.20 mM) (Table 2), probably because of the microscopically very high substrate concentration on the DODAPMEGal/polymerized DDPC liposome. In spite of the steric disadvantage that the reaction field is above the lipid bilayer surface, the affinity of galactose oxidase for HD-S9 -Gal on the liposome surface was comparable to those for PMEGal in bulk solution (K m , 0.35 and 0.13 mM (DP = 16 and 53), respectively) (20). The kcat value, which is defined as the number of substrate molecules converted into product per enzyme molecule per unit time, of galactose oxidase for the HD-S9 -Gal/DMPC liposome (0.21 mM · mL/min · units) was lower than those of free Dgalactose and small β-D-galactopyranosides and comparable to those of PMEGals (0.091 and 0.14 mM · mL/min · units (DP = 16 and 53), respectively), whereas the kcat /K m value, which represents the catalytic efficiency of an enzyme, of galactose oxidase for the HD-S9 -Gal/DMPC liposome system (0.99 mL/ min · units) was much larger than those for free D-galactose and small sugars (Table 1), and comparable to those for PMEGals (0.26 and 1.10 mL/min · units (DP = 16 and 53), respectively). Since the galactose-carrying chains are fixed to the liposome surface, the mobility of the enzyme molecule attaching the galactose residue above the liposome surface might be restricted, and therefore, it is understandable that the turnover number (kcat ) of galactose oxidase for HD-S9 -Gal was smaller than those for galactose, and other small sugars. However, the mobility of the galactose residue is still afforded by the oligo-ethylene glycol spacer. Consequently, the fixation of galactose-carrying chain to the membrane surface at a large surface density might enhance the reactivity of the enzyme-substrate complex (kcat ) to be much larger value than those at the monomolecular and micellar phases. In addition, since the K m value for galactolipid is much smaller than those of small galactose-derivatives, the sufficiently large turnover number (kcat ) resulted in a larger catalytic efficiency (kcat /K m ) of galactose oxidase for the HD-Sn -Gal-carrying liposome than for the small galactose derivatives. In conclusion, the affinity of galactose oxidase for sugar residues in the galactolipid was dependent on the length of the spacer between the galactose residue and aliphatic anchor group. The incorporation of galactolipids into the liposome largely enhanced the catalytic effectiveness of the enzyme, due to the increase in the turnover number of the catalysis above the liposome surface. The present study indicates that the spatial distribution of substrates significantly affects the catalytic behavior of the enzyme. Furthermore, the nonpolar group in the vicinity of sugar residues enhances the affinity of sugar-recognizing
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proteins (not only lectins but also enzymes), which would be valuable information in carbohydrate chemistry. ACKNOWLEDGMENTS This work was supported by Grants-in-Aid for Scientific Research 12450381, 13022225, and 13555260 from the Ministry of Education, Science, Sports and Culture.
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