Langmuir 1991, 7, 727-737
Preparation of Active Langmuir-Blod Oxidase
727
!tt Films of Glucose
Songcheng Sun, Phuoc-Hoa Ho-Si, and D. Jed Harrison* Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2 Received December 12, 1989. I n Final Form: August 24, 1990 Active Langmuir-Blodgett (LB)films of glucose oxidase (GO,Aspergillus niger)can be prepared directly by spreading the enzyme at the air-water interface and transferring to a substrate; no interaction with lipid films is required. Native enzyme LB film activity is poor, however, as determined by electrolysis of H202 produced by reaction with glucose and 02. Modified enzyme can be prepared by reaction with glutaraldehyde before spreading on the subphase, and this results in LB films on Pt substrates that are 5 to 15 times more active, depending on reaction conditions. These films exhibit an activity similar to conventional bovine serum albumin immobilized enzyme electrodes and a response time of less than 3 s. Nominally monomolecular layers are formed by using GO modified by reaction with glutaraldehyde for 24 h at 5 "C, followed by ultrafiltration to remove oligomers before spreading on the subphase. Deposition at a surface pressure of 30 mN/m onto Si gives a film thickness of 48 A/layer, compared to 30 A/layer for the native enzyme. Films of ultrafiltered, modified GO appear smooth at 70 000 times magnification using scanning electron microscopy (SEM). LB films of modified GO that is not ultrafiltered show even greater activity and are apparently thicker; however, SEM shows these films have an island structure so that the mass deposited is greater than for a true monolayer. Homogeneous assays of the glutaraldehyde treated enzyme show about 89 90 of the activity of the native enzyme, with minimal change in selectivity. Gel electrophoresis of the native enzyme using denaturing conditions gives only the 80 000 dalton subunit, while the modified enzyme shows the presence of 11%subunit, 60% holoenzyme, and 29% oligomers of the enzyme when first reacted 24 h a t room temperature in 2.5 9; glutaraldehyde. The product distribution can be controlled by reaction conditions. The results indicate an increased resistance to denaturing following inter- and intramolecular cross-linking with glutaraldehyde.
Introduction The preparation and nature of protein and enzyme films a t the air-water interface have been the subject of continued in~estigationl-~ since the original studies of Langmuir and Schaeffer,4 Groter,5 and others.617 It is believed that proteins that are spread a t low surface concentration (1mg/m2) is thought to explain their retention of activity. However, the ability to retain some level of activity in concentrated films is dependent on the specific e n ~ y m e . l - ~ , ~ - ~ The Langmuir-Blodgett (LB) technique for transfer of films a t the air-water interface to a solid substrate is a powerful means of obtaining organized, densely packed assemblies for a variety of applications.'3J4 Through control of the subphase or surface composition the method may provide a means of preparing oriented enzyme films with an increased accessibility of active sites compared to other immobilization techniques. Further, many enzymes are known to self-aggregate in vivo and are most stable in
* Author t o whom correspondence should be addressed. (1)Miller, I. R.; Bach, D. Surf. Colloid Sci. 1977,7,185. (2)James, L. K.;Augenstein, L. G. Adu. Enzymol. Relat. Subj. Biochem. 1966,28,1. (3) Adamson, A. W. Physical Chemistry of Surfaces, 4th ed.; J. Wiley: New York, 1982;Chapter 3 and references cited. (4)Langmuir, I.; Schaefer, V. J. Science 1937,85,76;J. Am. Chem. SOC.1938,60,1351 and 2803. ( 5 ) Gorter, E. Trans. Faraday SOC.1937,33,1125. (6)Cheesman, D. F.; Davies, J. T. Adu. Protein Chem. 1954,9,439. (7)Bull, H.B. Adu. Protein Chem. 1947,3,95. (8)Cheesman, D. F.; Schuller, H. J . Colloid Sci. 1954,9,113. (9)Loeb, G.I.; Baier, R. E. J. Colloid Surf. Sci. 1968,27,38. (10)Loeb, G.I. J. Polym. Sci., Part C: Polym. Symp. 1971,34,63. (11)Winter, C. S.; Tredgold, R. H. Thin Solid Films 1985,123,L1. (12)Jacobsen, R. J.; Cornell, D. G. Appl. Spectrosc. 1986,40,318. (13)Roberts, G. G. Adu. Phys. 1985,34,475,and 1983,4,131. (14)Kuhn, H. Thin Solid Films 1983,99,1.
0743-7463/91/2407-0727$02.50/0
this form or in the crystalline f ~ r m . ' ~ Densely J~ packed protein films prepared by using the LB technique may resemble these forms,17 although it is not known whether the stability will be significantly different than for other immobilization methods. However, the problem of retaining enzyme activity in the LB film is a difficult one that dates from the original work of Langmuir and Schaeffer, and developing a method to address this problem is the principal thrust of this report. Besides the early work of Langmuir and others1-*J8J9 on proteins spread on the air-water interface, relatively little has been reported regarding formation of enzymatically active LB films of pure enzyme.20y21 Much more work has focused on protein-lipid films formed by adsorption of protein from the subphase onto lipid films spread on the s u r f a ~ e . ' ~ t ~LB ~ - films ~ ~ consisting of alternate lipid and enzyme layers show activity in the case of some enzymes but not other^.^^-^^ Adsorption of penicillinase onto LB films of stearic acid on a pH-sensitive electrode has been used as a means of preparing penicillin sensors.28 Recently, films of glucose oxidase (GO) have been prepared by covalent bonding onto an LB film of (15)Osumi, M.; Nagano, M.; Yamada, N.; Hosoi, J.; Yanagida, M. J. Bacteriol. 1982,151, 376. (16)Vainshtein, B. K.;Kiselev, N. A.; Kaftanova, A. S.; Orlova, E. V.; Boedanova. V. P.: Morozkin. A. D.:. Deetvar.. R. G. Dokl. Akad. Nauk SS3R 1973;213,217. (17)Fromherz, P. Nature 1971,231,267. (18) Kaplin, J. G. J. Colloid. Sci. 1952,7,382. (19)Hayashi, T.;Eidson, G. R. J . Colloid. Sci. 1950,5,437. (20)Benjamins, J.; DeFeister, J. A.; Evans, M. T. A.; Graham, D. E.; Phillips, M. C. Faraday Discuss. Chem. SOC.1975,59,218. (21)Adams, D. J.; Evans, M. T. A,; Mitchell, J. R.; Phillips, M. C.; Rees, P. M. J . Polym. Sci., Part C: Polym. Symp. 1971,34,167. (22)Quin, P. J.; Dawson, R. M. C. Biochem. J. 1969,113,791,1969, 115,65,and 1970,116,671. (23)Fromherz, P.FEES Lett. 1970,11, 205. (24)Steinemann, A,; Lauger, P. J. Membr. Biol. 1971,4,74. (25)Fromherz, P.Eiochim. Biophys. Acta 1971,225,382. (26)Peters, J.; Fromherz, P. Biochim. Biophys. Acta 1975,394,111. (27)Fromherz, P.;Marcheva, D. FEES Lett. 1975,49,329.
0 1991 American Chemical Society
728 Langmuir, Vol. 7, No. 4, 1991
surfactant predeposited on Sn02,29as well as by adsorption of GO to a lipid layer at the air-water interface prior to deposition on a s0lid.~0*31 These methods form active films that generate HzO2 in the presence of glucose and 02, but even monolayer coatings exhibit relatively low sensitivity and response times of several minutes, which makes them poor candidates for glucose sensing. Hydrophobic lipid or surfactant layers are known to be densely packed and poorly permeable to many ions and molecules,32 so that the presence of the lipid layer probably accounts for the poor sensing characteristics. Improved response times have been reported for films prepared by first forming a water-insoluble enzyme-lipid complex before spreading the compound on the ~ u b p h a s e .Unfortunately, ~~ this method dilutes the enzyme density in the film, and reports of deposits thicker than two monolayers have not appeared. When the current is limited by the amount of enzyme, as it is for the monolayer films presented here, increasing the amount of enzyme will increase sensitivity. Also, to increase a sensor's longevity it is desirable to have a significant reservoir of enzyme in the film.34 We report here on the preparation of active LB films of glucose oxidase via transfer of a Langmuir film of enzyme adsorbed at the surface of a clean subphase without the use of additional surfactant. Active films with no added protein or surfactant diluent can be obtained by using spreading conditions that denature the native enzyme. This is accomplished by intramolecular cross-linking of the enzyme with glutaraldehyde to increase its durability before spreading it on the subphase. Up to at least ten monomolecular layers can be deposited, providing rapid response times and increased electrochemical sensitivity to glucose relative to lipid/GO films prepared by the LB technique.
Experimental Section Glucose oxidase was used as received from Sigma (types VI1 and X). The product data sheet specified a content of 63.8", protein for the batch of type X we used for most of this work. The activity of other enzymatic impurities varied from batch to batch based on the specifications, but only galactose oxidase (1 to 4c, ), maltase ( l o c )and , catalase (2"O were present in significant quantities. Type VI1 GO caused a shift of 10 m N / m in the baseline of a surface pressure-surface area isotherm when spread on the subphase. Since type X GO did not show this effect, it was used exclusively. Glutaraldehyde (25";) in HzO) was obtained from Sigma and Aldrich and either was used as received or was purified, either by distillation and redissolution in boiling water ( l o o c in HzO) or by treating with activated charcoal.35 The ultraviolet (UV) spectrum of the glutaraldehyde preparations showed peak absorbances at 236 nm due to polymeric impurities and a t 282 nm due to the monomer.35 The ratio of oligomer to monomer in the glutaraldehyde was found to affect the activity of GO treated with it. Reagent grade glutaraldehyde (Sigma, grade 11) was stored a t 5 "C, while purified electron microscope grade glutaraldehyde (Sigma, grade I) was shipped and stored on dry ice. Methanol (reagent grade) was doubly distilled, and distilled, deionized H20 was redistilled from (28) Anzai, J.; Furuya, K.; Chen, C.; Osa, T.; Matsuo, T. Anal. Sci. 1987, 3, 271. (29) Tsuzuki, H.; Watanabe,T.; Okawa, Y.; Yoshida, S.; Yano, S.; Koumoto, K.; Komiyama, M.; Nihei, Y. Chem. Lett. 1988, 8, 1265. (30) Sriyudthsak, M.; Yamagishi, H.; Moriizumi, T. Thin Solid Films 1988, 160, 463. (31) Moriizumi, T. Thin Solids Films 1988, 160, 413.
(32) Porter, M. D.; Bright, T. B.;Allara, D. L.; Chidsey, C. E. D. J.Am. Chem. SOC.1987, 109, 3559. (33) Okahata, Y.; Tsuruta, T.; Kuniharu, I.; Ariga, K. Langmuir 1988, 4, 1373. (34) Gough, D. A.; Lucisano, J. Y.; Tse, P. H. S . Anal. Chem. 1985,57, 2351. (35) Gillet, R.; Gull, K. Histochemie 1972, 30, 162.
Sun et al. alkaline permanganate, discarding the first 20 7% fraction, and retaining the next 50%. Other reagents and bufferswere reagent grade. Silicon slides (1 cm X 4 cm) were prepared by scribing from 3 in. diameter n-type Si wafers (S.E.H., Malaysia), 100orientation, with a resistivity of 1 to 2 R cm. Platinum foil (0.004 in. thick, Johnson-Matthey) was cut into 1cm X 4 cm slides. All substrates were cleaned before deposition of LB films in the following manner. Slides were sonicated for 15 min in 5 % aqueous Sparkleen (Fisher), replacing the solution every 5 min, then sonicated in distilled methanol for 15 min, replacing the methanol every 5 min, and then sonicated again in distilled water for 25 min, changing the HzO every 5 min. This gave uniformly hydrophilic substrates. The Si was then dried in an oven (100 "C) for 5 min and used. The Pt was immersed in 0.5 M HzS04, anodized a t +1.9 V vs SCE for 5 min, cycled between +1.1and -0.24 V vs SCE for 10 min, and held a t +1.1 V for 5 min before removing, rinsing with distilled H20, and drying in the oven. GO Reaction Conditions. GO cross-linked with glutaraldehyde was prepared by using a variety of conditions. The most common procedure involved dissolution of 40 mg of GO in 2 mL of water and addition of 1 mL of 25% glutaraldehyde (100001 mole ratio of glutaraldehyde to GO), and then 1mL of Hz0. This solution was allowed to stand for about 5 min and then diluted with HzO or a methanol/HzO mixture to 10 mL, giving a solution of 4 mg/mL of GO, 2.5OL glutaraldehyde, and, in the latter case, 20% methanol. After dilution, solutions were allowed to react for lOmin to 24 h, either a t room temperature or in the refrigerator a t 5 "C. Reaction for 20 to 24 h a t either temperature was most common. When reacted a t room temperature with methanol present, a precipitate often formed. However, the extent of precipitation depended on the batch of glutaraldehyde, with purified glutaraldehyde producing no precipitate. Bovine serum albumin (BSA) immobilized films of GO were prepared according to the method of Ya0.~6 BSA (Sigma 73.2 mg) and GO (19.5 mg) were dissolved in 1mL of aqueous acetate buffer, pH 5.5, to which 5 WLof 25% glutaraldehyde was then added. A measured weight of this mixture was spread on a Pt electrode and allowed to dry 2 h before rinsing in p H 5.5 buffer, and then HzO. The amount of enzyme on the electrode was determined both from the weight of solution deposited and by weighing a coated electrode (microbalance), removing the film by gentle wiping after 3 h in 20% KOH in ethanol, and reweighing the electrode. Activity Measurements. The activity of a homogeneous solution of the enzyme was determined by using the assay outlined in the literature shipped by Sigma with every batch of GO. I t is based on the colorimetric determination of oxidized o-dianisidine (Aldrich) in the presence of peroxidase (Sigma), GO, and glucose. Solutions were prepared as described, but the assays were performed at room temperature (-22 "C) so the Sigma units/g of protein reported here are lower than those given in the catalogue. Both native and glutaraldehyde treated GO were tested in this way, the latter with glutaraldehyde still present. The activity toward the interfering sugars galactose, fructose, and sucrose was determined with this assay by preparing solutions of equal molarity of the appropriate sugar. A qualitative test of the activity of LB films on Si and Pt substrates was performed by using a modification of the peroxytitanic acid colorimetric H202assay.37 A solution of Ti(IV) was prepared by dissolution of 10 g of Ti(SO4)2in 50 mL of HzO with 20 g of concentrated HzS04. This was centrifuged to clarify it after 24 h. LB film coated substrates were soaked for 24 h in 0.1 M glucose and 0.1 M acetic acid (adjusted to p H 5.5 with NaOH). To 3 mL of this solution was added 3 drops (Pasteur pipet) of the Ti(1V) solution and the absorbance determined a t 405 nm. A calibration curve was prepared by adding H202 to the acetate buffered 0.1 M glucose solution and determining the absorbance when Ti(1V) was added. The test is viewed as qualitative because the solutions were not stirred or agitated to increase (36) Yao, T. Anal. Chim. Acta 1983, 148, 27. (37) Schumk, W. C.; Satterfield, C. N.; Wentworth, R. L. Hydrogen Peroxide; Reinhold: New York, 1955; p 561.
P r e p a r a t i o n of Active LB Films of Glucose Oxidase glucose transport to the immobilized enzyme, and H202 added to the buffered 0.1 M glucose solution was found to partially decompose (10-20 Yo decrease) over 24 h. T h e activity of L B films was determined quantitatively by electrochemical methods. Films coated on Pt electrodes were immersed in 50 mL of 0.1 M phosphate buffer a t p H 7.4. The solution was magnetically stirred with a stir bar and an 89055A air-driven cell stirring module (Hewlett-Packard). The electrode was potentiostated a t +0.7 V versus a saturated calomel electrode (SCE) with a Pine RDE-4 bipotentiostat, and the current was recorded with a Kipp and Zonen Model BD90 x-y recorder equipped with a time base. The current was allowed to stabilize in a glucose-free solution, a t which point aliquots of 0.1 M glucose (pH 7.4, phosphate) were added and the change in signal recorded. At the highest stir rates used, the current density was independent of exact electrode placement within k 3 % in the presence of glucose. Gel Electrophoresis. The molecular weight distribution of glucose oxidase before and after glutaraldehyde treatment was determined by sodium dodecyl sulfate poly(acry1amide) gel electrophoresis. Electrophoresis was performed according to the method of Laemmli,38 using a Mini-Protean I1 apparatus (BioRad Laboratories) and a Brinkman power supply. Both gradient (7.5-20'( ) and 3.5L acrylamide gels were used. The former provided reasonably accurate estimates of molecular weight but did not resolve the higher oligomers. The 3.570 gelgavemolecular weights that tended to be high, as is well recogni~ed,3~3~~ but resolved the higher Dalton fractions. Sigma molecular weight standards kit MW-SDS-200 was used for the gradient gel, and a series of cross-linked phosphorylase b (rabbit muscle) standards (Sigma P8906) was used for the 3.5% gel. The gels were stained with Coomassie Blue R-250 to visualize the protein bands, and quantitative data were obtained from densitometer tracings of the gels. Langmuir Trough. A Joyce-Loebel Langmuir Trough (Model 6-11-4025) with a subphase volume of 4 L was controlled with an IBM PC-XT and interface electronics and software provided by Joyce-Loebel. Some minor modifications to the software were made to facilitate film deposition. A Wilhelmy plate (Whatman 1 CHR chromatography paper) was used to measure changes in surface tension. It was necessary to support the trough, mounted inside the arborite box supplied by Joyce-Loebel, on a Technical Manufacturers Micro-g air-table with a steel laminate top plate to reduce the effect of vibration. The trough components were cleaned by rinsing with Sparkleen (Fisher) detergent and distilled HzO. Before use the surface of the subphase was cleaned by repeatedly removing the top layer with a Pasteur pipet attached to a vacuum aspirator and replacing the lost volume, until the surface tension remained constant as the barrier was compressed from 1000 to 100 cm2 surface area. Methanol free enzyme spreading solution was introduced by using a Pipetman (Gilman) 20-200 pL variable pipet and allowing the solution to flow along a glass rod contacting the subphase. This gave a larger apparent surface area/molecule than obtained by delivering the solution dropwise. Spreading solution containing methanol could be delivered in either fashion with no difference in area/molecule. Initial formal surface concentrations before compression were varied from 1 to -20 mg/m2. Typical values were -7 mg/m2 for 20% methanol spreading solutions and 14 mg/m* for aqueous solutions. Substrate coatings were deposited by using a subphase containing 0.5-2 g/L BaC12 with no noticeable difference in behavior. The p H was adjusted to 4 or 5 by using HC1 to be near the enzyme isoelectric point of p H 4.2.30 A t a p H of 9 film deposition was not observed. A constant surface pressure of either 5 or 30 mN/m was maintained during deposition of the film. Only two to three layers of enzyme could be deposited at surface pressures of both 5 or 30 m N / m with a dipping speed of 20 mm/ min, since the film stripped off again as the slide reentered the subphase, as evidenced by an increase in surface area at constant surface pressure. To alleviate this a two-stage process was used. While the substrate was submerged in the subphase, a surface
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(38) Laemmli, U. K. Nature 1970, 227, 680. (39)Segrest, J. P.; Jackson, R. L. Methods Enzymol. 1972, 28, 54. (40) Tsuge, H.; Natsuaki, 0.; Ohashi, K. J. Biochem. 1975, 78, 835.
Langmuir, Vol. 7, No. 4, 1991 729 pressure of 5 or 30 m N / m was established and then maintained as the substrate was withdrawn a t 10 mm/min (2.5 min total). It was then allowed to air-dry about 8 min, and before reentering the subphase a t 48 mm/min the surface area was expanded to give a pressure of 0 mN/m. After the substrate was submerged, the barrier was recompressed to give 5 or 30 m N / m for the next withdrawal. This allowed films of up to 10 layers to be deposited and was used since enzyme deposited only on the upward stroke under all deposition conditions tried. The film transfer coefficients were measured on large surface areas to minimize error (3 in. diameter Si wafer, four separate 4 cm2Pt foils dipped together). Since the surface area decreased slowly a t constant surface pressure, the change in surface area on dipping a substrate was corrected for the drift in determining the transfer coefficient. Film Characterization. The infrared spectra of deposited films were measured by using a Nicolet 7199 FTIR in transmission mode. The films were deposited on Si wafers that were first thinned to about 0.5 mm by reaction in a Si etchant composed of 8% (v/v) concentrated HF, 17 7; (v/v) glacial acetic acid, and 75% (v/v) concentrated nitric acid for about 30 min. Thinning was done to increase transmission through the Si slide. Typically 128 interferograms were acquired per spectrum. Ellipsometry was performed with a Gaertner L125B twowavelength ellipsometer equipped with a rotating analyzer. Data acquisition and reduction and ellipsometer control were done with an IBM PS-2 system and software provided by Gaertner. Microspot optics gave a sampled area of 0.002 mm2 a t a 70' incident angle and 0.001 mm2a t a 50" incident angle. The 632.8nm source was employed and an angle of 70' was most commonly used. The uncoated regions of a sample Si slide were measured to determine the native oxide thickness, which was typically 19 A. Data for LB film coated regions were then analyzed assuming a two-layer coating of enzyme/oxide/Si substrate with the following parameters: Si substrateNs = 3.850,Ks = -0.020, oxide layer N = 1.46, t = measured thickness from uncoated region. For LB film thicknesses greater than 150 A, both thickness and refractive index could be determined from the data. An average value of NLB= 1.50 was obtained for the enzyme and this value was used as a fixed parameter for calculating the thickness of films less than 150 A. The ellipsometric effect was found to be in the first order by measurement on the same spot at both 50" and 70°, with the calculations performed with all parameters fixed except for the LB film thickness. A 9-MHz quartz crystal microbalance (ICM, Oklahoma City) was used to evaluate the mass of enzyme transferred to the crystal in a single dip coating following the method of McCaffrey et al.41 The crystal was driven with a circuit based on several inverters, and the frequency was measured with an H P Model 5335 univera1 counter. The crystals were repeatedly dipped into pure water followed by baking a t 60 "C for 10 min and then allowed to cool until a reproducible oscillation frequency was obtained after each dip. Baking was found to be a necessary step in this procedure. The crystals were then dipped into the Langmuir trough, using the same conditionsas for Pt electrodes. On removal the crystals were again baked, the frequency change recorded, and the mass change calculated according to the Saubrey equation using the same crystal parameters as Bruckenstein and S h a ~ . ~ ~ An area per molecule was calculated based on the geometric electrode area, the mass change, and the enzyme's molecular weight. Scanning electron microscopy was performed with a Cambridge 250 Stereoscan instrument. Samples were shadow coated with P d / P t (10/90) alloy a t about a 12" angle under high vacuum (10-6 Torr).
Results and Discussion Langmuir Films. A typical isotherm of surface pressure versus area is shown in Figure 1 for native glucose oxidase (GO) in 20% methanol spread on a subphase of 0.5 g/L BaClz adjusted to pH 5.6 with HC1. F o r the data (41) Bruckenstein, S.; Shay, M. Electrochim. Acta 1985, 30, 1295. (42) McCaffrey, R. R.; Bruckenstein, S.;Prasad, P. N. Langmuir 1986, 2, 228.
Sun et al.
730 Langmuir, Vol. 7, No. 4, 1991 50 I
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Figure 1. Surface pressure versus area curve for compression of 7.1 mg/m* of native glucose oxidase (GO) deposited from an aqueous 20°; methanol spreading solution. A 2.5 mM BaC12 subphase adjusted to pH 5.6 was used. in the figure the enzyme concentration was 7.1 mg/m2 before compression of the area, and the apparent surface area per molecule, extrapolated to 0 mN/m from the linear portion of the compression curve was 2025 A2/molecule. The observed surface area per molecule varied from 700 to 2200 A2 depending on the conditions used in spreading the enzyme on the subphase. The surface area/molecule increased as the initial surface concentration decreased, giving 2200 A2/molecule most consistently at 1 mg/m2 initial spreading concentration, and typically 1500 A2/ molecule for initial spreading concentrations of around 7 mg/m2. However, considerable variability (lt35% ) was found from run to run and this is likely due to variability in the efficiency of delivering the enzyme to the surface rather than to the subphase. The dimensions of glucose oxidase from Aspergillus niger are not well-known, but measurement of the hydrodynamic radius gives a value of 43 A.43 Using an electron micrograph technique, Vainshtein et al. have estimated the dimensions of crystalline GO from Penicillium vitale as 50 X 50 X 70 From the hydrodynamic radius an area per molecule of 5800 A2 would be predicted, while the area predicted by the dimensions of the Penicillium vitale strain, assuming an ellipsoidal shape, ranges from 2000 to 2750 A2 depending on the direction of the major and minor axis relative to the surface normal. The uncertainty in expected area per molecule is unfortunately large due to the lack of data on GO dimensions; however, the average experimental values are noticeably lower than the predicted range. This could result either from folding and stacking of molecules a t the air-water interface giving a multilayer ~ t r u c t u r eor ~ ,from ~ ~ loss of enzyme to the subphase. The data are consistent with increasing loss of enzyme to the subphase as the initial surface concentration is increased. This is suggested by the slow decrease in trough area when the surface pressure is held constant and is further supported by the fact that the walls and bottom of the Teflon trough become hydrophilic after spreading of the enzyme, indicating adsorption of GO from the subphase has occurred. A similar loss of lysozyme
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(43) Nakamura,S.;Hayashi,S.;Koga, K. Biochim. Biophys. Acta 1976, 445, 294. (44) Vainshtein, B. K.; Kiselev, N. A.; Kaftonova, A. S.; Orlova, E. A,; Lerner. F. Ya. Mol. Biol. (Kieu) 1974. 10. 19. (45)’Vainshtein,B. K.; Kiselev, N. A.; Kaftonova, A. S.; Orlova, E. V.; Bogdanov, V. P.; Morozkin, A. D.; Degtyar, R. G. Dokl. Akad. Nauk SSSR 1973, 223, 17.
adsorbed at the air-water interface to the subphase was elegantly demonstrated by Adams et al. using a radiotracer technique.21 For a constant initial surface concentration the area per molecule increased when methanol was added to the spreading solution (20% by volume). This effect is most likely due to the lower viscosity and density of the spreading solution compared to HzO. Methanolic droplets that submerge below the subphase surface can be seen to rise again and disperse when striking the surface. This leads to an increased efficiency of delivery of the enzyme to the surface rather than the subphase, and this method has been used previously to spread other proteins more effectively at the surface.46 Activity measurements of homogeneous GO in 20% methanol solutions, or after recovery from such solutions, show no difference in activity from the native enzyme. This is consistent with the increased apparent area/molecule arising from delivery of more mass to the surface rather than to the subphase. It is not possible to rule out that changes in the tertiary structure due to interactions with methanol lead to an increase in the enzyme’s size, but this interpretation is less likely since enzyme activity is often adversely affected by structural rearrangement. Langmuir-Blodgett Films. The Langmuir films on the subphase can be transferred to a number of different solid substrates such as Si, Pt, Au, and glass using the Langmuir-Blodgett technique. To facilitate analysis of the films Si and Pt substrates were used most commonly. Initially, confirmation that the protein was deposited was obtained from transmission FT-IR studies of Si slides. After a single dip cycle using any of a variety of film spreading conditions and surface pressures (vide infra), a narrow peak is introduced a t 1650 cm-l (4.2 X 10-3 absorbance unit) and a broad peak from 3200 to 3600 cm-l (5.4 X absorbance unit). Films of GO evaporatively deposited from aqueous solution on Si give much stronger absorbances at the same positions. Monitoring the change in surface area a t constant surface pressure while passing the substrate through the surface also indicates material is transferred. Under all conditions used the LB films deposited were “z-type”;that is, enzyme transferred to the substrate only on the upward stroke, as the substrate exited the subphase, and no deposition occurred on entering the subphase. For all the procedures used the transfer coefficients (i.e., the decrease in trough area per dip cycle at constant surface pressure ratioed to surface area of the substrate) were 1 f 0.05 for the first dip cycle. This value tended to decrease to about 0.8 or 0.9 (f0.1) by the fifth cycle, depending on the procedures used. Despite the relative ease of depositing LB films of GO, the thickness, uniformity, and enzymatic activity of the deposit depend strongly on spreading solution preparation and LB film transfer techniques (vide infra). Film thicknesses were determined by ellipsometry of films deposited on Si slides. For LB films of native GO prepared by transfer at a surface pressure of 5 mN/m, a thickness of only 10 to 15 A was measured following a single dip cycle. This thickness is consistent with denaturing of the protein through loss of the tertiary structure, which is known to result in a thickness of -10 A for protein^.^ Enzyme-coated substrates of Si and Pt were submerged in buffered glucose solutions for 24 h, and the H202 formed was determined by c0lorimetry3~based on peroxytitanic acid formation. Little or no activity was determined for all the films tested. Electrochemical determination of activity proved more sensitive and (46) Dervichian, D. Nature 1939, 144, 629.
Preparation of Active LB Films of Glucose Oxidase
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] 0.5
Relative mobility (Rf )
Figure 2. A plot of current density for Hz02 on a Pt electrode coated with five layers of enzyme, as a function of the molar ratio of glutaraldehyde to glucose oxidase. The first point at negative infinity corresponds to no added glutaraldehyde. Glucose oxidase was allowed to react with glutaraldehyde (batch5, see Table 11) for 24 h at 5 "C for all entries except one. At a molar ratio of lo4two data points are shown, the lower current corresponding to 10 min reaction time, the higher current to 24 h reaction time. indicated limited activity remains for these films, as discussed below. LB films of native GO were also prepared at 30 mN/m surface pressure, and this method gave films on Si with a 30-A thickness as determined by ellipsometry, whereas the expected thickness for a monolayer of native GO would be in the range of 50 to 80 A.43-45This suggests either that the enzyme is partially denatured or that the enzyme film is formed of islands on the surface with substantial regions of open space in the film that lead to a distorted ellipsometric measurement. Qualitative determination of the film activity showed that a modest glucose activity was retained by the deposited enzyme on Pt and Si substrates. Quantitative measurement of the film activity on a Pt substrate was done electrochemically, based on oxidation of the H202 produced enzymatically in the presence of glucose and oxygen.34*47,48 The current density for five layers of native enzyme was at most 0.05 (pA/cm2)/mM, as shown in Figure 2. However, the activity was lower than that reported for other methods of forming LB films of GO that involve additional lipid m a t e r i a l ~ . ~ ~ - ~ ~ , 3 3 To confirm the activity arises from an immobilized film, and not from GO which leaches into solution from the surface, a control experiment was performed that used the colorimetric peroxytitanic acid test. A coated slide when soaked 24 h in buffered glucose solution produced a solution 0.8 mM in H202. If a coated slide was instead soaked in buffer alone for the same period and removed, followed by the addition of glucose and incubation of the solution for 1 to 24 h, then the peroxytitanic acid test indicated no Hz02 was formed. This control demonstrates it is the modified surface that is active, not enzyme redissolved in solution. Since the film thickness measurements may be interpreted as indicating unfolding of GO, and both partial and complete unfolding of proteins at the air-water interface is known to occur, a method to prevent or reduce this effect via intramolecular cross-linking of the enzyme was evaluated. Glutaraldehyde treatment of GO was used for this study as the enzyme is well-known to retain activity when treated with this cross-linking reagent. (47) Updike, S. J.; Shults, M.; Ekman, B.Diabetes Care 1982,5, 207. (48) ThBvenot, D.R.Diabetes Care 1982,5, 184.
Figure 3. log (molecular weight) versus the relative mobility of molecular weight standards (O),native glucose oxidase, and glutaraldehyde cross-linked glucose oxidase prepared by reacting 24 h at 5 "C. Data were obtained by using 7.5-20% gradient acrylamide gel electrophoresis. A molecular weight of 80 000 for the subunit of the native enzyme produced by the denaturing conditions and a weight of 160 000 for the modified enzyme were observed, indicating resistance to the denaturing conditions.
Spreading solutions of modified GO were prepared by varying the quantity of glutaraldehyde present and the reaction time in 20% methanol. Figure 2 shows that the relative activity of a five monolayer LB film of the enzyme on a Pt substrate increases as the amount of glutaraldehyde present increases a t a reaction time of 24 h. Aging of the spreading solution for 10 min a t 5 "C instead of for 24 h resulted in a significant decrease in activity at a mole ratio of glutaraldehyde to glucose of 1OOOO:l. The control experiment described above proved the activity was due to a surface confined film in all cases. Both with and without the use of glutaraldehyde the shape of the surface pressure-area isotherms, and particularly the apparent area per molecule obtained from these data, was highly variable, as discussed earlier. Consequently, the isotherms provided little insight into the reasons for increased enzyme activity when glutaraldehyde is used, and so the nature of the reaction products of the glutaraldehyde treatment and the properties of the LB films formed by the products were examined in detail by using electrophoresis and ellipsometry. Enzyme Cross-Linking Products. Gel electrophoresis was used to characterize the product of the reaction of glucose oxidase and glutaraldehyde. A gradient gel of 7.5 to 20 %acrylamide ;I was used, and the enzyme was prepared for electrophoresis using denaturing condition^^^ by boiling in a sample buffer of glycerol, mercaptoethanol, and sodium dodecyl sulfate (SDS) a t pH 6.8 (Tris buffer). Figure 3 shows a plot of log (molecular weight) versus the retention factor, R f ,for a series of standards and both the native and cross-linked enzyme. The GO was cross-linked byreactingfor 24 h a t 5 "C in 2.5 7; glutaraldehyde solution and 20% methanol, which gave a high activity LB film on Pt as shown in Figure 2. The native enzyme gave a single band with a molecular weight of 80 000 daltons, in good agreement with previous electrophoretic studies of the enzyme using denaturing ~ o n d i t i o n s . ~ 9Reaction *~~ with SDS and either mercaptoethanol or urea is known to break the holoenzyme into identical subunits of approximately 80 000 daltons. The glutaraldehyde cross-linked enzyme is not completely decomposed into the subunits by the denaturing treatment and shows a predominant band a t 160 000 daltons and a much weaker band a t 80 000 dal-
732 Langmuir, Vol. 7, No. 4, 1991 A. standards
Sun et al. B. native GO
Table I. Relative Distribution of Molecular Weights for Glutaraldehyde-Treated Glucose Oxidase
GO oligomerb distribution, %, for modified GO preparationa glut, native glut, glut, R P glut! GO RTe RTd filter centrifuge 5 "C subunit >98 11 27
