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Langmuir 2002, 18, 3390-3391
Transmission Electron Microscopy at Cryogenic Temperatures and Dynamic Light Scattering Studies of Glucose Oxidase Molecules and Self-Aggregated Nanoparticles Alexander Kamyshny,*,† Dganit Danino,‡ Shlomo Magdassi,† and Yeshayahu Talmon‡ Casali Institute of Applied Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel, and Department of Chemical Engineering, TechnionsIsrael Institute of Technology, Haifa 32000, Israel Received November 2, 2001. In Final Form: January 24, 2002
Introduction Glucose oxidase (GOx) is the most widely used enzyme in the field of biosensors.1 It catalyzes the oxidation of β-D-glucose by molecular oxygen to D-glucono-1,4-lactone and hydrogen peroxide and has many important applications for quantitative determination of glucose in body fluids, foodstuffs, beverages, and fermentation liquor.1,2 The structure of bacterial GOx from Aspergillus niger is well characterized. It is a rigid dimeric glycoprotein of molecular weight 150-160 kDa (583 amino acid residues and high mannose-type carbohydrate up to 16% of the total molecular weight) containing one tightly bound flavin adenin dinucleotide (FAD) per monomer as cofactor.3-7 According to the crystal structure data at 2.3 Å resolution, the monomeric partially deglycosylated molecule is a compact spheroid with approximate dimensions 60 × 52 × 37 Å. The corresponding dimensions of the dimer are 60 × 52 × 77 Å.8,9 Dissociation of the subunits is possible only under denaturing conditions and is accompanied by the loss of the cofactor.10 The carbohydrate moiety does not contribute significantly to the structure, stability, and activity of GOx.11 The efficiency of the GOx-based sensors is limited, mainly, due to the heterogeneity of the enzyme distribution in the biosensor membrane, which makes difficult the creation of the effectively functioning compact analytical devices.12 To improve the molecular architecture of the biosensor films composed of the organized layers of membrane lipids and enzyme, the attachment of hydrophobic anchors to the latter may be fruitful. * To whom correspondence should be addressed. Tel.: 972-26584965. Fax: 972-2-6528250. E-mail:
[email protected]. † The Hebrew University. ‡ Technion. (1) Wilson, R.; Turner A. P. F. Biosens. Bioelectron. 1992, 7, 165. (2) Schmid, R. D.; Karube, I. Biotechnology 1988, 6b, 317. (3) Pasur, J. H.; Kleppe, K. Biochemistry 1964, 3, 578. (4) Swoboda, B. E. P.; Massey, V. J. Biol. Chem. 1965, 240, 2209. (5) Hayashi, S.; Nakamura, S. Biochim. Biophys. Acta 1981, 657, 40. (6) Kriechbaum, M.; Heilmann, H. J.; Wientjes, F. J.; Hahn, M.; Jany, K.-D.; Gassen, H. G.; Sharif, F.; Alaeddinoglu, G. FEBS Lett. 1989, 255, 63. (7) Frederick, K. R.; Tung, J.; Emerick, R. S.; Masiarz, F. R.; Chamberlain, S. H.; Vasavada, A.; Rosenberg, S.; Chakraborty, S.; Schopfer, L. M.; Massey, V. J. Biol. Chem. 1990, 265, 3793. (8) Hecht, H. J.; Kalisz, H. M., Hendle, J.; Schmid, R. D.; Schomburg, D. J. Mol. Biol. 1993, 229, 153. (9) Hecht, H. J.; Schomburg, D.; Kalisz, H.; Schmid, R. D. Biosens. Bioelectron. 1993, 8, 197. (10) Jones, M. N.; Manley, P.; Wilkinson, A. Biochem. J. 1982, 203, 285. (11) Kalisz, H. M.; Hecht, H. J.; Schomburg, D.; Schmid, R. D. Biochim. Biophys. Acta 1991, 1080, 138. (12) Pickup, J. C.; Thevenot, D. R. Advances in Biosensors; JAI Press: London, 1993; p 201.
We have reported on hydrophobization of antibodies and enzymes by covalent attachment of alkyl chains to the protein lysine amino groups with retention of biological activity after modification.13-15 It was found that modification of GOx was not accompanied by dissociation of subunits and loss of FAD, and the enzymatic activity measurements have indicated that GOx containing five covalently attached palmitic chains has retained 70% of its specific activity.15 Hydrophobized GOx displays increased surface activity at the air/water interface15 as well as increased ability to penetrate into lipid monolayers spread at the same interface as compared with the native enzyme.16 It was found that attachment of hydrophobic chains to various proteins by such chemical modification resulted in formation of typical surfactant-like molecules, which spontaneously form colloidal clusters in aqueous solutions and display enhanced surface activity at the air/water interface.13,15,17,18 In the present study we used two complementary techniques, dynamic light scattering and transmission electron microscopy at cryogenic temperatures (cryo-TEM) to characterize the microstructure of the native and hydrophobized GOx assemblies in aqueous solution. Experimental Section Materials. GOx from Aspergillus niger (type VII-S) and palmitic acid hydroxysuccinimide ester were purchased from Sigma Chemical Co. The modification reaction was performed as described previously15 at a protein-to-ester molar ratio 1:56. The number of covalently modified lysine residues was determined by the TNBS method,19,20 which is based on the reaction of trinitrobenzenesulfonic acid (TNBS) with free amino groups of proteins. This number was found to be eight palmitic chains per dimeric GOx molecule (8C16-GOx). Protein concentrations were determined spectrophotometrically (Hitachi double-beam spectrophotometer U-2000) by the improved Lowry method21 using Bio-Rad protein assay reagents or by absorbance at 453 nm. Particles Size Measurements. The average particle size of the protein samples was measured by dynamic light scattering method with the use of a Zetasizer 3000, Malvern Instruments, U.K. (Ar-laser, wavelength 488 nm, power 70 mW; detector angle 90°; 25 °C, dispersant viscosity 0.89 cP; dispersant refractive index 1.33, sample refractive index 1.40, absorbance 0.30). The particle size (according to the particle number distribution) was taken as a mean value of four measurements. The measurements in the case of hydrophobized GOx were performed for samples, which were filtered through a 1.2 µm filter, as well as for samples, which were further filtered through 450 and 200 nm filters. Cryo-TEM. Vitrified specimens for electron microscopy were prapared in a controlled environment vitrification system (CEVS) at controlled temperature (25 °C) and 100% relative humidity, as previously described.22 In brief, a drop of about 3 µL was placed (13) Kamyshny, A.; Reuveni, T.; Magdassi, S. J. Colloid Interface Sci. 1996, 181, 470. (14) Kamyshny, A.; Magdassi, S. Colloids Surf., B 1997, 9, 147. (15) Baszkin, A.; Boissonnade, M. M.; Rosilio, V.; Kamyshny, A.; Magdassi, S. J. Colloid Interface Sci. 1997, 190, 313. (16) Baszkin, A.; Boissonnade, M. M.; Rosilio, V.; Kamyshny, A.; Magdassi, S. J. Colloid Interface Sci. 1999, 209, 302. (17) Toledano O.; Magdassi, S. J. Colloid Interface Sci. 1997, 193, 172. (18) Kamyshny, A.; Magdassi, S.; Relkin P. J. Colloid Interface Sci. 1999, 212, 74. (19) Habeeb, A. F. S. Anal. Chem. 1966, 14, 328. (20) Adler-Nissen, J. J. Agric. Food Chem. 1979, 27, 1256. (21) Peterson, G. L. Anal. Biochem. 1979, 100, 201. (22) Talmon, Y. In Modern Characterization Methods of Surfactant Systems; Binks, B. P., Ed.; Marcel Dekker: New York 1999; p 147.
10.1021/la011631p CCC: $22.00 © 2002 American Chemical Society Published on Web 03/09/2002
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Langmuir, Vol. 18, No. 8, 2002 3391
Figure 2. (A) Cryo-TEM image of the native GOx molecules showing individual globular and slightly elongated structures, about 6 to 8 nm. (B-D) Typical structures found in samples of the hydrophobized enzyme after filtration through a 1.2 µm filter. (B) Clusters of hydrophobized molecules (dark round images), 13.5-35 nm in diameter are the most abundant aggregates seen, although few larger clusters (insert) are also found. (C) Individual native molecules (black arrows) and organized regions of native molecules (white arrows). (D) Organized regions of hydrophobized and native molecules.
Figure 1. Particle size distribution according to dynamic light scattering: A, solution of the native GOx (curve 1) and 8C16GOx (curve 2) filtered through 450 nm filter; B, solution of 8C16-GOx filtered through 1.2 µm filter. on a TEM grid covered with a perforated carbon film (Ted Pella, USA) held by a tweezer connected to the plunger of the CEVS. The drop was blotted with filter paper to form a sample film, typically 100-250 nm thick, and the grid was then plunged into liquid ethane at its freezing temperature (-183 °C), producing a vitrified specimen. Specimens were examined in a Philips CM120 microscope operated at 120 kV, using an Oxford CT3500 cooling holder maintained below -178 °C. Images were recorded in the low-dose imaging mode to minimize electronbeam radiation damage by a Gatan MultiScan 791 CCD camera, using the Digital Micrograph 3.1 software package. Image processing was performed by the Adobe Photoshop 5.0 package.
Results and Discussion Results of particle size measurements by dynamic light scattering for the native GOx molecules and for 8C16-GOx are presented in Figure 1. The mean diameter of the native GOx molecule (Figure 1A, distribution curve 1) is 7 ( 1 nm. A cryo-TEM image of these molecules is shown in Figure 2A. Individual globular and slightly elongated particles of about 6-8 nm are seen, in good agreement with the light scattering findings and also with the size obtained from the crystal structure data8,9 (60 × 52 × 77 Å). Figure 1B shows the particle size distribution obtained by light scattering in a solution containing hydrophobically
modified GOx and filtered through 1.2 µm filter. It was found that there are large particles, having a mean size of 470 ( 15 nm (Figure 1B). It was found that after further filtration through 450 nm filter the mean particle size of modified protein was 13.5 ( 1.5 nm (Figure 1A). This size does not change after additional filtration through a 200 nm filter. These nanoparticles are, most probably, clusters of the hydrophobic 8C16-GOx molecules. Such clusters may contain approximately eight dimeric enzyme molecules (at cubic packing), which are tightly bound by the hydrophobic interactions due to the hydrophobic tails attached to the enzyme molecules. By cryo-TEM, several populations of particles are distinctly identified in samples of hydrophobized GOx. The most abundant structures are clusters of hydrophobized molecules, with a diameter of 13.5-35 nm (Figure 2B). In the spaces between these clusters, which correspond to the nanoparticles found by dynamic light scattering (Figure 1A, distribution curve 2), native molecules were also observed. Individual molecules of the native GOx, which did not undergo hydrophobic modification, are also seen in Figure 2C (black arrows). Aggregates, several hundred nanometers in size, are also formed by nanoclusters of the hydrophobized GOx molecules, as shown in Figure 2D. These regions correspond to the large particles found by dynamic light scattering (Figure 1B). In conclusion, it was shown that the two complementary methods used in the study of native and hydrophobized GOx molecules in solution are in good agreement and demonstrate unequivocally the existence of spontaneously formed nanoparticles composed of the hydrophobized enzyme molecules. LA011631P
