Surface Properties of “Jellyfish”: Langmuir Monolayer and Langmuir

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Surface Properties of “Jellyfish”: Langmuir Monolayer and Langmuir-Blodgett Film Studies of Recombinant Aequorin Chengshan Wang,† Miodrag Micic,‡ Mark Ensor,§ Sylvia Daunert,§ and Roger M. Leblanc*,† Department of Chemistry, UniVersity of Miami, Coral Gables, Florida 33146, MP Biomedicals LLC, 15 Morgan, IrVine, California 92618, and Department of Chemistry and Department of Pharmaceutical Sciences, UniVersity of Kentucky, Lexington, Kentucky 40506-0055 ReceiVed March 14, 2007. In Final Form: May 4, 2007 In this paper, we studied the surface properties of recombinant aequorin at the air-water interface. Using the Langmuir monolayer technique, the surface properties of aequorin were studied, including the surface pressure and surface potential-area isotherms, compression-decompression cycles, and stability on Trizma Base (Tris/HCl) buffer at pH 7.6. The results showed that aequorin formed a stable Langmuir monolayer and the surface pressure-area isotherms were dependent on both pH and ionic strength. At a pH higher or lower than 7.6, the limiting molecular area decreased. The circular dichroism (CD) spectra of aequorin in aqueous solutions explained this result: when the pH was higher than 7.6, the R-helix conformation changed to unordered structures, whereas at a pH lower than 7.6, the R-helix conformation changed to β-sheet. The addition of calcium chloride to the Tris/HCl buffer subphase (pH 7.6) caused an increase of the limiting molecular area of the aequorin Langmuir monolayer. The fluorescence spectra of a Langmuir-Blodgett (LB) film of aequorin in the presence of calcium chloride indicated that the aequorin transformed to the apoaequorin.

Introduction The bioluminescent photoprotein aequorin, originally isolated from the jellyfish Aequorea Victoria, consists of apoaequorin, coelenterazine, and molecular oxygen.1-3 This photoprotein emits blue light (λmax at 469 nm) during the conformational change induced by Ca2+ in the presence of oxygen, resulting in oxidation of coelenterazine to coelenteramide with emission of blue light and CO2.4 The amphiphilic aequorin is an ideal luminophore for Ca2+ monitoring. Aequorin expresses many benefits compared with standard fluorophores, such as low toxicity, low leakage rate, and virtually no background noise.5-7 However, for many practical biosensing applications, the luminophore needs to be immobilized on surfaces, so studying the surface properties of the luminophore is of crucial importance for developing biosensors. Due to its low abundance in jellyfish, practical applications of aequorin started being of interest only after the first recombinant proteins were synthesized.8,9 Since then, many different variants of aequorin and aequorin-like systems were synthesized in a variety of expression systems. Recently, a novel recombinant aequorin, a cysteine-free mutant,10 was synthesized with increased pH stability and which incorporates the coelenterazine substrate.11 This interesting pro* Fax: +1-305-284-6367; Tel: +1-305-284-2194; E-mail [email protected]. † University of Miami. ‡ MP Biomedicals LLC. Fax: +1-949-859-5095; Tel: +1-949-833-2500 [email protected]. § University of Kentucky. [email protected]. (1) Johnson, F. H.; Shimomura, O. Methods Enzymol. 1978, 57, 271-291. (2) Vysotski, E. S.; Markova, S. V.; Frank, L. A. Mol. Biol. 2006, 40, 355367. (3) Vysotski, E. S.; Lee, J. Acc. Chem. Res. 2004, 37, 405-415. (4) Hirano, T.; Ohashi, M. J. Synth. Org. Chem. Japan 1996, 54, 596-606. (5) Nomura, M.; Inouye, S.; Ohmiya, Y.; Tsuji, F. I. FEBS Lett. 1991, 295, 63-66. (6) Creton, R.; Kreiling, J. A.; Jaffe, L. F. Microsc. Res. Technol. 1999, 46, 390-397. (7) Mattheakis, L. C.; Ohler, L. D. Drug DiscoVery Today S 2000, 15-19. (8) Inouye, S.; Sakaki, Y.; Goto, T.; Tsuji, F. I. Biochemistry 1986, 25, 84258429. (9) Inouye, S.; Aoyama, S.; Miyata, T.; Tsuji, F. I.; Sakaki, Y. J. Biochem. 1989, 105, 473-477.

tein has been utilized in creating fused proteins for a variety of biosensing applications in solution.12-20 However, in order to construct a practical bioluminescence-based biosensor, it is preferred that the detecting ligand and luminophore are immobilized on the solid surface. Although aequorin has been a subject of intensive research studies in the last two decades, its surface properties are still largely unknown. In this paper, we are presenting first results of surface properties of aequorin, as derived from our studies of the aequorin Langmuir monolayer at the air-water interface. The effect of ionic strength and pH on the surface pressure-area isotherm of the aequorin Langmuir monolayer was studied, which was helpful to optimize the conditions for aequorin to construct a Langmuir monolayer. The stability of the monolayer was also studied, because it is a key factor for construction of reliable biosensors, with a solid-phase supported sensing layer. Besides surface pressure and surface potential-area isotherms, we have explored the spectroscopic behavior of aequorin and apoaequorin as dependent on surface pressure, and corresponding states of 2D condensed matter, ranging from gaseous through liquid expanded and liquid condensed phases. Materials and Methods 1. Materials. All the chemicals, namely, disodium ethylenediaminetetraacetate dihydrate (EDTA), Trizma base (Tris), Bis-Trispropane (Bis-Tris), and potassium dihydrogen phosphate, H2O2, and (10) Kurose, K.; Inouye, S.; Sakaki, Y.; Tsuji, F. I. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 80-84. (11) Lewis, J. C.; Lo´pez-Moya, J. J.; Daunert, S. Bioconjugate Chem. 2000, 11, 65-70. (12) Deo, S. K.; Daunert, S. Anal. Chem. 2001, 73, 1903-1908. (13) Deo, S. K.; Lewis, J. C.; Daunert, S. Anal. Biochem. 2000, 281, 87-94. (14) Deo, S. K.; Lewi, J. C.; Daunert, S. Bioconjugate Chem. 2001, 12, 378384. (15) Desai, U. A.; Wininger, J. A.; Lewis, J. C.; Ramanathan, S.; Daunert, S. Anal. Biochem. 2001, 294, 132-140. (16) Roos, W. Planta 2000, 210, 347-370. (17) Chiesa, A.; Rapizzi, E.; Tosello, V.; Pinton, P.; de Virgilio, M.; Fogarty, K. E.; Rizzuto, R. Biochem. J. 2001, 355, 1-12. (18) Deo, S. K.; Daunert, S. Fresenius J. Anal. Chem. 2001, 369, 258-266. (19) Monteith, G. R. Immunol. Cell Biol. 2000, 78, 403-407. (20) Zerefos, P. G.; Ioannou, P. C.; Traeger-Synodinos, J.; Dimissianos, G.; Kanavakis, E.; Christopoulos, T. K. Hum. Mutat. 2006, 27, 279-285.

10.1021/la700756e CCC: $37.00 © 2007 American Chemical Society Published on Web 06/08/2007

Surface Properties of “Jellyfish” concentrated sulfuric acid were purchased from VWR Co. (Westchester, PA). The water utilized as the subphase (pH 5.8) for a surface chemistry study was obtained from a Modulab 2020 water purification system (Continental Water System Corp., San Antonio, TX) with a surface tension of 72.6 mN‚m-1 and a resistivity of 18 MΩ·cm at 20.0 ( 0.5 °C. Quartz slides used for Langmuir-Blodgett (LB) film deposition of aequorin were purchased from Hellma Cells Inc. (Plainview, NY). The quartz slides were cleaned by a mixture of H2O2 and concentrated sulfuric acid (1:5, v/v). The preparation of the aequorin sample is described below. 2. Recombinant Aequorin Production. The Tris/HCl, Bis-Tris, phosphate, and potassium phthalate buffers were made according to the CRC Handbook of Chemistry and Physics.21 The coding sequence for the cysteine-free5 apoaequorin was ligated into pIN422 plasmid vector, which contains an lpp promoter and an OmpA leader sequence. This plasmid vector was used to transform E. coli Top10 Chemically Competent E. coli cells (Invitrogen, Carlsbad, CA). The apoaequorin produced by these cells was released into the culture medium. The cells were grown in 1 L of Lennox LB medium (MP Biomedicals, Irvine, CA) preseeded with ampicillin at a concentration of 100 µg‚mL-1, incubated overnight at 37 °C on a 250 rpm shaker. The cells were pelleted by centrifugation, and the apoaequorin in the supernatant was precipitated by the dropwise addition of glacial acetic acid while stirring until the pH reached 4.3. The precipitated protein was then pelleted by centrifugation at 12 000 g for 30 min. The pellet was resuspended in 35 mL of 30 mM Tris/HCl, 2 mM EDTA buffer, and the pH was adjusted to 7.6 using 1 M NaOH. The protein solution was then filtered through a 0.2 µm filter. The apoaequorin was purified by chromatography on Poros HQ (Applied Biosystems, Foster City, CA) and Butyl Sepharose Fast Flow (Amersham Biosciences, Uppsala, Sweden) columns using a BioCad Sprint system (Applied Biosystems, Foster City, CA). The protein solution was applied to a Poros HQ column (20 × 65 mm). This column was equilibrated with a 30 mM Tris/HCl, pH 7.6, 2 mM EDTA buffer eluted with a 0-50% gradient of 30 mM Tris/HCl, pH 7.6, 2 mM EDTA, 1 M NaCl, over 10× column volumes. The most active fractions were pooled together and ammonium sulfate was added to a 1 M final concentration. This protein solution was then applied to a Butyl Sepharose Fast Flow column (16 × 90 mm) equilibrated with 20 mM Bis-Tris, pH 7.6, 2 mM EDTA, 1 M ammonium sulfate. The column was washed with 20 mM Bis-Tris, pH 7.6, 2 mM EDTA, 0.2 M ammonium sulfate. The apoaequorin was then eluted using a gradient from 0.2 M ammonium sulfate to zero ammonium sulfate over two column volumes. Fractions were analyzed for purity using SDS-polyacrylamide gels, and by assaying for activity. This recombinant aequorin, synthesized as per procedure described above, is now commercially available through MP Biomedicals. 3. Generation of Aequorin and Assay for Activity. Active aequorin was generated by incubating the purified apoprotein with a 3× molar excess of coelenterazine in the dark at 4 °C overnight. The luminescence activity of the aequorin was measured by using an Optocomp I Luminometer (GEM Biomedical, Carrboro, NC). The luminescence was triggered by injecting 50 µL of 100 mM Tris/HCl, pH 7.6, 100 mM CaCl2. The luminescence signal was collected over a 6 s time period at 0.1 s intervals. 4. General Methods for Surface Chemistry Study. All the isotherm measurements and LB film preparation were conducted in a clean room (class 1000) where temperature (20.0 ( 0.5 °C) and humidity (50 ( 1%) were kept constant. A Kibron µ-trough (Kibron Inc., Helsinki, Finland) with an area of 124.50 cm2 (5.9 cm × 21.1 cm) was utilized for the surface pressure-area (π-A) isotherm, compression-decompression cycle, and stability studies. Surface potential-area (∆V-A) isotherms were obtained on the Kibron trough using a Kelvin probe consisting of a capacitor-like system. The vibrating plate was set at approximately 1 mm above the surface of (21) Covington, A. K.; Davison, W. In CRC Handbook of Chemistry and Physics, 75th ed.; Lide, D. R., Frederikse, H. P. R., Eds.; CRC Press: Boca Raton, 1994; pp 8-42. (22) Porter, T. D.; Chang, S. Y. Drug Metab. ReV. 1999, 31, 159-174.

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Figure 1. Surface pressure and surface potential-area isotherms of the aequorin Langmuir monolayer on Tris/HCl buffer (pH 7.6). the monolayer, and a gold-plated trough was used as a counter electrode. The clean subphase was taken as the zero potential. The stock solution of aequorin was 1 mg‚mL-1 in Bis-Tris buffer at pH 7.6. The stock solution was diluted to 0.25 mg‚mL-1 with pure water. This diluted solution was used for surface chemistry study. 15-30 µL of the 0.25 mg‚mL-1 aequorin solution was spread at the air-water interface, followed by a period of 15 min waiting for the Langmuir monolayer formation. The compression rate was set at 600 Å2‚molecule-1 min-1. The circular dichroism (CD) spectra were measured by a JASCO J-810 spectropolarimeter fitted with a 150 W xenon lamp. Quartz cells of 1 mm path length were used for all CD measurements of aequorin solutions, and the spectra were recorded with a response time of 8 s and scan speed of 20 nm‚min-1. The concentration of aequorin was 0.1 mg‚mL-1 under all pH conditions for the CD measurements. The in situ UV-vis absorption spectra of the Langmuir monolayer were obtained with an HP Spectrophotometer model 8452 A, settled on a rail close to the KSV trough (KSV Instrument Ltd., Helsinki, Finland) which had a dimension of 7.5 cm × 30 cm, suitable for approach toward the quartz window which is located at the center of the KSV trough. The fluorescence of LB film on the quartz slide was measured on a Fluorolog 3 fluorospectrometer (Horiba Jobin Yvon, Edison, NJ).

Results and Discussion 1. Surface Pressure and Surface Potential-Area Isotherms of the Aequorin Langmuir Monolayer on Tris/HCl Buffer at pH 7.6. The π-A and ∆V-A isotherms on Tris/HCl buffer at pH 7.6 are shown in Figure 1. Tris/HCl buffer (pH 7.6) was chosen because this medium is usually used to observe the bioluminescence of aequorin in aqueous solution. The liftoff point of the π-A isotherm was situated at 5470 Å2‚molecule-1, followed by a steady increase of the surface pressure up to a kink point at 2940 Å2‚molecule-1. Decreasing the surface area further caused the surface pressure to increase quickly, and the collapse was observed at 2100 Å2‚molecule-1. The limiting molecular area was obtained at 3920 Å2‚molecule-1 by extrapolating the higher surface pressures of the isotherm to zero surface pressure. This value corresponds to the close-packed arrangement of the protein molecules. The diameter of the aequorin molecule in this arrangement was calculated from the limiting molecular area value, and a diameter of 70.6 Å was found. A much lower value of the diameter of the aequorin molecule was determined by single-crystal X-ray diffraction,23 i.e., 47.2 Å. Three reasons might explain this discrepancy: (i) In the single crystal of aequorin, the molecules are in a 3-D arrangement compared with the aequorin molecules in a 2-D arrangement at the air-water interface. (ii) The water content in a crystal of aequorin is different compared with the aequorin Langmuir monolayer. (iii) The irregular sphere shape of aequorin at the air-water interface (23) James, F. H.; Satoshi, I.; Katsunori, T.; Osamu, S. Nature (London) 2000, 405, 372-376.

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Wang et al.

Figure 2. Compression-decompression cycles (a) and stability studies (b) at 15 mN‚m-1 of the aequorin Langmuir monolayer on Tris/HCl buffer (pH 7.6).

presents its short axis perpendicular to the interface. The surface potential of the aequorin Langmuir monolayer increased from 12 500 Å2‚molecule-1 up to a kink point observed at 6100 Å2‚molecule-1, followed by a sharp increase up to 190 mV at molecular area of 2550 Å2‚molecule-1. Further compression of the Langmuir monolayer increased the intensity of the surface potential slowly. By extrapolating the slopes before and after the kink point at 6100 Å2‚molecule-1, there was an intercrossing point at 5510 Å2‚molecule-1, which was within the experimental error to the liftoff point of the π-A isotherm, i.e., 5470 Å2‚molecule-1. This indicated that there is a correlation between the π-A and ∆V-A isotherms of the aequorin Langmuir monolayer. On the basis of the π-A and ∆V-A isotherms, three regions of the isotherms could be characterized, namely, the gaseous, liquid expanded, and liquid condensed phases at 150006100, 6100-2940, and 2940-2100 Å2‚molecule-1, respectively. 2. The Compression-Decompression Cycles and Stability of the Aequorin Langmuir Monolayer. From the π-A isotherm, the aequorin Langmuir monolayer was in a liquid condensed phase at surface pressures above 11 mN‚m-1. Consequently, 15 mN‚m-1 was chosen to run the compression-decompression cycles and the long-term stability of the Langmuir monolayer as shown in Figure 2. For the compression-decompression cycles, the cycles overlapped within 50 Å2‚molecule-1, which indicated that the aequorin Langmuir monolayer was stable after 4 cycles (Figure 2a). When the surface pressure was held at 15 mN‚m-1 for 100 min, the molecular area changed only by 120 Å2‚molecule-1, which was less than 5% of the limiting molecular area (Figure 2b). On the basis of the evidence of these two experiments, we concluded that aequorin formed a stable Langmuir monolayer. 3. pH Effect on the Surface Pressure-Area Isotherm of the Aequorin Langmuir Monolayer. The π-A isotherms of the aequorin Langmuir monolayer on Tris/HCl, phosphate, and potassium phthalate buffers are shown in Figure 3. For Tris/HCl buffer (Figure 3a), the pH ranged from 9.0 to 7.0. For pH 9.0,

Figure 3. The effect of pH on surface pressure-area isotherms of the aequorin Langmuir monolayer. Buffer: (a) Tris/HCl, (b) phosphate, (c) potassium phthalate.

the liftoff point of the π-A isotherm was at 3230 Å2‚molecule-1, and the limiting molecular area was at 1880 Å2‚molecule-1, which was much smaller than the value of pH 7.6. When the pH was 8.0, the π-A isotherm was almost identical to the one at pH 7.6, and the only difference was that the limiting molecular area at pH 8.0 was at 4010 Å2‚molecule-1. As for the pH at 7.0, the liftoff point was at 5300 Å2‚molecule-1, and the limiting molecular area was at 3550 Å2‚molecule-1. When the subphase was phosphate buffer (Figure 3b), the liftoff point of the isotherm at pH 8.0 was observed at 5500 Å2‚molecule-1 and the limiting molecular area at 3490 Å2‚molecule-1. As for the pH at 7.6, the liftoff point was at 5180 Å2‚molecule-1 and the limiting molecular area at 3780 Å2‚molecule-1. For pH 7.0, the liftoff point was at 5230 Å2‚molecule-1 and the limiting molecular area at 3540 Å2‚molecule-1. As for pH 5.9, the liftoff point was at 4200 Å2‚molecule-1; however, the surface pressure increased quickly after the liftoff point. The limiting molecular area was situated at 3640 Å2‚molecule-1. As for the potassium phthalate buffer (Figure 3c), the shape of the isotherm at pH 5.9 was identical to the one on the phosphate buffer at pH 5.9 with a liftoff point at 4580 Å2‚molecule-1 and a limiting molecular area situated at 3800 Å2‚molecule-1. The

Surface Properties of “Jellyfish”

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Table 1. The Values of the Liftoff Point and the Limiting Molecular Area from the Surface Pressure-Area Isotherms of the Aequorin Langmuir Monolayer under Different Buffers at Different pHa pH 9.0

8.0

7.6

7.0

5.9

buffer

LO

LMA

LO

LMA

LO

LMA

LO

LMA

Tris/HCl phosphate phtalate

3230

1880

5480 5500

4010 3490

5470 5180

3920 3780

5300 5230

3550 3540

a

4.8

4.0

LO

LMA

LO

LMA

LO

LMA

4200 4580

3640 3800

3100

2580

2210

1480

LO: liftoff point (Å ‚molecule ). LMA: limiting molecular area (Å ‚molecule ). 2

-1

isoelectric point of aequorin was at pH 4.8. Under this condition, the liftoff point was at 3100 Å2‚molecule-1 and the limiting molecular area at 2580 Å2‚molecule-1. As for the pH at 4.0, the liftoff point and the limiting molecular area further decreased to 2210 and 1480 Å2‚molecule-1, respectively. When the pH was lower than 4.0, no reproducible π-A isotherms were obtained. Table 1 gives a summary of the data (Å2‚molecule-1) of the liftoff point and the limiting molecular area from the π-A isotherms of the aequorin Langmuir monolayer. The effect of pH and the nature of the buffers on the aequorin Langmuir monolayer showed the following feature: (1) For Tris/HCl and phosphate buffers, the liftoff point at pH 7.0, 7.6, and 8.0 is at 5360 Å2‚molecule-1, on average, which is within (4% of each liftoff point value. This result means that in the range of pH 7.0-8.0, the liftoff point is dependent on neither the pH nor the nature of the buffers. A similar conclusion is reached for the limiting molecular area with an average value of 3715 Å2‚molecule-1, within (7% of each limiting molecular area value. (2) Higher than pH 8.0 or lower than pH 7.0, the value of liftoff point and limiting molecular area decreased, but the nature of the buffer at pH 5.9 has no effect on these values within the experimental error. How can we explain the fact that at high and low pH we observed a decrease in both the liftoff point and the limiting molecular area from the π-A isotherms? One hypothesis could be that, at these pH values, there is a conformational change of the protein at the air-water interface. The conformation change could make the protein aggregate24,25 and consequently decrease the limiting molecular area. In order to validate this hypothesis, we have examined the conformation of the protein in aqueous solutions of different pH values by CD spectroscopy. 4. Circular Dichroism Spectroscopy of Aequorin in Aqueous Solutions Under Different pH Values. The CD spectra of aequorin aqueous solutions at a concentration of 0.1 mg‚mL-1 under different pH values are shown in Figure 4. When the pH was 7.6, the spectrum showed two negative peaks at 208 and 221 nm and a positive peak at 192 nm. The crossing-zero point was at 201 nm. A typical CD spectrum of a protein with high R-helix content26 has the same peak positions as the ones shown in Figure 4b. Both higher and lower pH values than 7.6 reduced the contribution of the two peaks at 208 and 221 nm. This indicated that the content of R-helix in aequorin reached a maximum value at pH 7.6. To quantify the content in percentage of the secondary structures of aequorin aqueous solutions, the CD spectra were analyzed by the program CDPro, and the results are shown in Table 2. The reason to choose CDPro was that CDPro included the three popular methods for CD spectra analysis: SELCON3, CONTIN, and CDSSTR.24,27-29 The assignment of CD spectra (24) Zheng, J.; Constantine, C. A.; Rastogi, V. K.; Cheng, T.-C.; DeFrank, J. J.; Leblanc, R. M. J. Phys. Chem. B 2004, 108, 17238-17242. (25) Sopkova, J.; Vincent, M.; Takahashi, M.; Lewit-Bentley, A.; Gallay, J. Biochemistry 1998, 37, 11962-11970. (26) Homoelle, B. J.; Beck, W. F. Biochemistry 1997, 36, 12970-12975. (27) Sreerama, N.; Woody, R. W. Anal. Biochem. 2000, 287, 252-260. (28) Zheng, J.; Constantine, C. A.; Zhao, L.; Rastogi, V. K.; Cheng, T.-C.; DeFrank, J. J.; Leblanc, R. M. Biomacromolecules 2005, 6, 1555-1560.

-1

2

Table 2. Content in Percentage of Secondary Structures of Aequorin in Aqueous Solution under Different pHa pH

RR

RD

βR

βD

T

U

9.0 7.6 4.8 4.0

17.2 21.2 17.8 13.7

15.0 16.6 13.1 10.2

8.0 6.7 11.9 18.8

6.8 6.8 8.2 9.0

20.1 21.7 21.7 20.9

33.0 27.2 27.6 26.6

a RR: regular R-helix. RD: distorted R-helix. βR: regular β-sheet. βD: distorted β-sheet. T: turns. U: unordered structures.

from CDPro gave the content of six secondary structural classes: regular R-helix, RR; distorted R-helix, RD; regular β-sheet, βR; distorted β-sheet, βD; turns, T; and unordered, U. When the pH increased from 7.6 to 9.0, the content of regular R-helix decreased from 21.2% to 17.2% and the content of distorted R-helix decreased from 16.6% to 15.0%. On the contrary, the content of the unordered structures increased from 27.2% to 33.0%. The content in percentage of other secondary structures changed minimally. This indicated that the R-helix conformation changed to unordered structures when the pH increased up to 9.0. When the pH decreased from 7.6 to 4.8, the content of both regular and distorted R-helixes decreased from 21.2% and 16.6% to 17.8% and 13.1%. At pH 4.0, the content of the two types of R-helix further decreased to 13.7% and 10.2%. On the contrary, the content of both regular β-sheet and distorted β-sheet kept increasing when pH decreased down to 4.0. The contents of the two types of β-sheet were 11.9% and 8.2% at pH 4.8 and further increased to 18.8% and 9.0% at pH 4.0. This indicated that the R-helix conformation changed to β-sheet under acidic conditions. On the basis of these results, the R-helix has the highest content at pH 7.6. The conformation change of aequorin in acidic or basic aqueous solution supports the hypothesis to explain the lower limiting molecular area observed in the π-A isotherms at these pH values. 5. Salt Effect on the Surface Pressure-Area Isotherm of the Aequorin Langmuir Monolayer. The π-A isotherms of the aequorin Langmuir monolayer on KCl solution subphase at different concentrations are shown in Figure 5. When the concentration of KCl was 0.5 M, the liftoff point was at 4250 Å2‚molecule-1 and the limiting molecular area at 3850 Å2‚molecule-1. When the concentration of KCl decreased to 0.1 and 0.05 M, the π-A isotherms were almost identical. The liftoff point and limiting molecular area were at 4020 and 3610 Å2‚molecule-1, respectively. After further decreasing the concentration to 0.01 M, the liftoff point and limiting molecular area decreased to 3360 and 2290 Å2‚molecule-1, respectively. As for the concentration at 0.005 M, the liftoff point was at 1980 Å2‚molecule-1 and the limiting molecular area at 1520 Å2‚molecule-1. For the concentrations lower than 0.005 M, no reproducible π-A isotherms were obtained. When the subphase was pure water, surface pressure remained zero. The KCl was also added to Tris/HCl buffer at pH 7.6, and the π-A isotherms are shown in Figure 6. When the concentration of KCl was 0.1 (29) Ji, X.; Zheng, J.; Xu, J.; Rastogi, V. K.; Cheng, T.-C.; DeFrank, J. J.; Leblanc, R. M. J. Phys. Chem. B 2005, 109, 3793-3799.

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Figure 4. Circular dichroism spectra of aequorin at concentration of 0.1 mg‚mL-1 under different pH values: -b-, 9.0; -0-, 7.6; -O-, 4.8; -4-, 4.0. pHs of 9.0 and 7.6 were controlled by 0.02 M Tris solution and 0.02 M HCl solution. pHs of 4.8 and 4.0 were controlled by 0.1 M potassium dihydrogen phosphate solution and 0.1 M phosphoric acid solution.

Figure 5. The effect of potassium chloride on the surface pressurearea isotherms of the aequorin Langmuir monolayer (subphase pH 5.8): s, 0.5; -9-, 0.1; -b-, 0.05; -2-, 0.01; -0-, 0.005 M.

M in Tris/HCl buffer, the liftoff point was at 6100 Å2‚molecule-1 and the limiting molecular area at 4030 Å2‚molecule-1. When the concentration increased to 0.5 M in Tris/HCl buffer, the liftoff point and limiting molecular area increased to 6200 and 4570 Å2‚molecule-1, respectively. On the basis of these results, the higher the concentration of KCl was, the larger the limiting molecular area was. This experimental result was interpreted by the fact that the K+ and Cl- neutralized the charges in aequorin. The higher the concentration of KCl was, the more charges in aequorin were neutralized by K+ and Cl-. Consequently, the solubility of aequorin in water decreased, and the limiting molecular area increased. 6. Effect of Ca2+ on the Surface Pressure-Area Isotherms of the Aequorin Langmuir Monolayer at pH 7.6. The π-A isotherms of aequorin Langmuir monolayer on the Tris/HCl buffer subphase at pH 7.6 in the presence of calcium chloride (CaCl2) are shown in Figure 7. When the concentration of CaCl2 was 0.03 M in the Tris/HCl buffer subphase, the liftoff point increased to 6800 Å2‚molecule-1 and the limiting molecular area increased to 5250 Å2‚molecule-1 compared with the ones in the absence of CaCl2 in the subphase. Now, a question arises: Why did the limiting molecular area increase? Is it due to the transformation of aequorin to apoaequorin or due to the increment of ionic strength? In order to answer these questions, the concentration of CaCl2 in the subphase was increased to 0.05 and 0.1 M. The π-A isotherms at these concentrations were almost identical to the one when the concentration of CaCl2 was 0.03 M, which indicated the increment of concentration of CaCl2 does not increase the limiting molecular area. Consequently, the transformation of aequorin to apoaequorin may explain the increase

Wang et al.

Figure 6. Surface pressure-area isotherms of the aequorin Langmuir monolayer on Tris/HCl buffer (pH 7.6) at different concentrations of potassium chloride: s, 0; -9-, 0.1; -O-, 0.5 M.

Figure 7. Surface pressure-area isotherms of the aequorin Langmuir monolayer on Tris/HCl buffer (pH 7.6) at different concentrations of calcium chloride: CaCl2 ) 0, s; 0.03, -b-; 0.05, -O-; 0.1, -0-; 0.03 M, -2-; in the presence of KCl ) 0.5 M.

of the limiting molecular area. When KCl (0.5 M) was added to the Tris/HCl buffer subphase at pH 7.6 in the presence of 0.03 M CaCl2, the liftoff point and the limiting molecular area greatly increased to 9750 and 7180 Å2‚molecule-1, respectively. This observation indicated that increasing ionic strength in the subphase in the presence of Ca2+ increases the limiting molecular area of the apoaequorin Langmuir monolayer. In order to support this hypothesis, the photophysical properties of Langmuir monolayer and LB film of aequorin were studied. 7. In Situ UV-vis Spectra of the Aequorin Langmuir Monolayer and Fluorescence Spectra of the LB Film of Aequorin and Apoaequorin. The in situ UV-vis spectra of the aequorin Langmuir monolayer at different surface pressures on the Tris/HCl buffer subphase at pH 7.6 are shown in Figure 8a. The single peak in the spectra was situated at 208 nm, which was assigned to the amide group, and this peak increased linearly with the increase of surface pressure (as shown in the inset in Figure 8a). The peak positions of aequorin and coelenterazine in aqueous solution were observed at 208 and 427 nm, respectively. The peak at 427 nm was not observed in the UVvis spectrum of the Langmuir monolayer, because the value of the extinction coefficient was low, namely, 7400 M-1‚cm-1. When CaCl2 was added to the subphase, no change was detected in the in situ UV-vis spectra. The fluorescence spectrum of one monolayer of aequorin deposited on quartz slide by the LB technique was determined under two experimental conditions. First, the aequorin Langmuir monolayer was prepared in the absence of CaCl2 and compressed to a surface pressure of 10 mN‚m-1 before deposition on quartz slide with a deposition ratio of unity (LB film). Second, the aequorin Langmuir monolayer was deposited as LB film in the presence of CaCl2

Surface Properties of “Jellyfish”

Langmuir, Vol. 23, No. 14, 2007 7607

nm, respectively. The blue shift in the fluorescence peak positions for both LB films is interpreted as being due to the formation of aggregates at the air-water interface prior to the deposition of the monolayer on a quartz slide. It has to be noted that the same difference of 8 nm between the aequorin and apoaequorin peak positions was observed for aqueous solutions and LB films.

Conclusion

Figure 8. (a) In situ UV-vis spectra of the aequorin Langmuir monolayer on Tris/HCl buffer at pH 7.6 in the absence of Ca2+ at different surface pressures: s, 0; -9-, 5; -0-, 10; -O-, 15 mN‚m-1. (b) Fluorescence spectra of LB films (1 monolayer) of -0-, aequorin; and s, apoaequorin; on quartz slide deposited at surface pressure of 10 mN‚m-1.

in the subphase. For both LB films, the excitation wavelength was set at 340 nm. The fluorescence spectra are shown in Figure 8b. We observed a peak at 429 and 437 nm corresponding to aequorin (absence of CaCl2) and apoaequorin (presence of CaCl2), respectively. Satoshi30 has observed the fluorescence peak for aequorin and apoaequorin in aqueous solution at 459 and 467

In this paper, the surface pressure and surface potential-area isotherms of the aequorin Langmuir monolayer on Tris/HCl buffer at pH 7.6 were studied. Compression-decompression cycles and stability measurements showed that aequorin formed a stable Langmuir monolayer at the air-water interface. The effect of pH and ionic strength on the surface pressure-area isotherm of the aequorin Langmuir monolayer was also studied to optimize the experimental conditions. Variation of pH in the subphase from pH 7.6 decreased the limiting molecular area of the aequorin Langmuir monolayer because of the conformation change of aequorin. The limiting molecular area of aequorin increased with the increase of KCl concentration in the subphase. Addition of CaCl2 into the subphase also increased the limiting molecular area. The fluorescence spectra of LB films of aequorin in the presence and absence of CaCl2 indicated that aequorin transformed to apoaequorin, which caused the increase of limiting molecular area. This transformation indicated that aequorin was active at the air-water interface. Acknowledgment. This research was supported by the National Science Foundation (USA, CHE-0416095) and by the National Institute of Health (CHE-467917). We thank the Vice President of Research at the University of Kentucky for a University Research Professorship to S.D. Also, S.D. acknowledges support from a Gill Eminent Professorship. We appreciated the discussion with Jhony Orbulescu and Robert Triulzi on the content of our manuscript. LA700756E (30) Satoshi, I. FEBS Lett. 2004, 577, 105-110.