Dynamics of Acrylodan-Labeled Bovine and Human Serum Albumin

Taylor A. McCarty, Phillip M. Page, Gary A. Baker, and Frank V. Bright ... as a Means for Water-Regulated Release/Delivery of Actives in Fabric Care A...
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Anal. Chem. 1995, 67, 2436-2443

Dynamics of Acrylodan-Labeled Bovine and Human Serum Albumin Entrapped in a Sol-Gel-Derived Biogel Jeffrey D. Jordan, Richard A. Dunbar, and Frank V. Bright* Department of Chemistry, Natural Sciences and Mathematics Complex, State Universiv of New York at Buffalo, Buffalo, New York 14260-3000

We investigate aaylodan-labeled bovine and human serum albumin (BSA-Ac and HSA-Ac) entrapped within a tetramethylorthosilane-derived biogel composite. The effects of biogel aging and drying were studied by following the acrylodansteady-stateand time-resolvedemission, the decay of anisotropy, and the dipolar relamtion kinetics as a function of ambient storage time. The results indicate that there is a substantial amount of nanosecond and subnanosecond dipolar relaxation within the local environment surrounding cysteine-34 in both proteins, even when they are fully encapsulated in a dry biogel. Timeresolved anisotropy experiments show that the aaylodan residue and the protein are able to undergo nanosecond motion within the biogel. The semiangle through which the aaylodan can precess is the same for a freshly formed biogel and the native protein in buffer. However, once the biogel begins to dry, the semiangle increases (-20" and 10" for BSA-Ac and HSA-Ac, respectively)). This suggests that the "pocket"hosting the aaylodan reporter group opens as the biogel dries. Much work in the analytical sciences has centered on the development of so-called chemical sensors and biosensors. In these schemes, a chemical recognition element is usually immobilized at a transducer interface and the interactions of the recognition element with the target analyte are measured as a means to quantify the target analyte. Over the years many attractive sensors have been reported, but the dirsculty associated with actually producing a stable chemical recognition elementtransducer interface impedes routine use of many laboratory-based schemes. Sol-gel processing provides a lowtemperature means to incorporate organic species within an inorganic-organic c~mposite.l-~To date, sol-gel-processed materials have been used mainly for the development of optical coatings, mechanical devices, and electrooptic application~.l-~Additional effort has focused on encapsulating molecular recognition elements within the porous sol-gel matrix and using these composite materials (1) Chemical Processing ofAdoanced Materials; Hench, L. L,West J. IC, Eds.; Wiley: New York, 1992. (2) Hench L. L.; West, J. K. Chem. Rev, 1992, 90, 53-72. (3) Paul, A. Chemistry ofClasses, 2nd ed.; Chapman and Hall: New York, 1990; p 51. (4) Brinker, C. J.; Scherer, G. W. Sol-Gel Science; Academic Press: New York, 1989.

2436 Analytical Chemistry, Vol. 67,No. 14, July 15, 1995

as platforms for chemical sensors.j-1° More recent work has demonstrated sol-gel processing as a vehicle to encapsulate biomolecules.'0-2aFurther, because of the inherently porous nature of the room-temperature, sol-gel-processed a sub stantial subpopulation of the entrapped biorecognition elements ~~~~

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(5) Shahriari, M. R; Ding J. Y. Doped Sol-Gel Films for Fiber Optic Chemical Sensors. In Sol-Gel Optics: Processing and Applications; Klein, L. C., Ed.; Kluwer Academic Publishers: New York, 1994; Chapter 13. (6) Carraway, E. R.: Demas, J. N.: DeGraff, B. A. Langmuir 1991, 7, 2 9 9 2998. (7) Avnir, D.; Braun, S.; Lev, 0.;Ottolenghi, M. SPIE Sol-Gel Optics II 1992, 1758,456-463. (8) Narang, U.; Dunbar, R. A.; Bright, F. V.; Prasad, P. N. Appl. Spectrosc. 1993, 47, 1700-1703. (9) Lee, J. E.; Saavedra, S. S. Anal. Chim. Acta 1994,285, 265-269. (10) Aharonson, K.;Alstein, M.; Avidan, G.; Avnir, D.; Bronshtein, A,; Lewis, A; Lieberman, R; Ottolenghi, M.; Polevaya, Y.; Rottman, C.; Samuel, J.; Shyalom, S.: Strinkovski, A.; Turniansky, A In Better Ceramics Through Chemisty Vt Sanchez, C., Mecartney, M. L., Brinker, C. J., Cheetham, A,, Eds.; Mater. Res. SOC.Symp. Proc. 1994,346, 1-12. (11) Shtelzer, S.; Braun, S. Biotechnol. Appl. Biochem., in press. (12) Narang. U.; Prasad, P. N.; Bright, F. V.; Kumar.A.; Kumar, N. D.; Malhotra, B. D.; Kamalasanan, M. N.; Chandra, S. Chem. Mater. 1994,6,1596-1598. (13) Narang, U.; Prasad, P. N.; Bright, F. V.; Kumar, A.; Kumar, N. D.; Malhotra. B. D.; Kamalasanan, M. N.; Chandra, S. Anal. Chem. 1994,66,3139-3144. (14) Carturan, G.; Campostrini, R.; Dire, S.; Scardi, V.; De Alteriis, E. J. Mol. Catal. 1989,57, L13-Ll6. (15) Braun, S.; Rappoport, S.; Zusman, R.; Avnir, D.; Ottolenghi, M. Mater. Lett. 1990, 10,1-5. (16) Ellerby, L.; Nishida, C. R.; Nishida, F.;Yamanaka, S. A.; Dunn, B.; Valentine, J. S.; Zink, 1. I. Science 1992,255, 1113-1115. (17) Shtelzer, S.; Rappoport, S.; Avnir, D.; Ottolenghi. M.; Braun, S. Biotechnol. Appl. Biochem. 1992, 15, 227-235. (18) Tatsu, Y.; Yamashita, K.; Yamaguchi, M.; Yamamura, S.; Yamamoto, H.; Yoskikawa, S. Chem. Lett. 1992, 1615-1618. (19) Avnir, D.; Braun, S.; Lev, 0.;Ottolenghi. M. Sol-Gel Optics II 1992, 1758, 1-8. (20) Avnir, D.; Braun, S.; Ottolenghi, M. In Supramolecular Architecture; Bein, T.. Ed.; ACS Symposium Series 499 American Chemical Society: Washington, DC, 1992. (21) Inama, L.; Dire, S.; Carturan, G.; Cavazza, A.J. Biotechnol. 1993,30,197210. (22) Wang, R ; Narang, U.: Prasad, P. N.; Bright, F. V. Anal. Chem. 1993.65. 2671-2675. (23) Wu, S.; Ellerby, L. M.; Cohan, J. S.; Dunn, B.; El-Sayed, M. A,;Valentine, J. S.; Zink, J. I. Chem. Mater. 1993,5, 115-120. (24) Edmiston, P. L.; Wambolt, C. L.; Smith, M. K.; Saavedra, S. S. J. Colloid Inte@ce Sci. 1994, 163, 395-406. (25) (a) Zink, J. I.; Dunn, B. In Proceedings of the First European Workshop on Hybrid Organic-Inorganic Materials (Synthesis, Properties, Applications); Sanchez, C., Ribot, Eds. 1993; pp 143-152. (b) Dave, B. C.; Dunn. B.; Valentine, J. S.; Zink, J. I. Anal. Chem. 1994,66, 112OA-1127A. (26) Kurokawa, Y.; Sano, T.; Ohta, H. Nakagawa, Y. Biotechnol. Bioeng. 1993, 42, 394-397. (27) Kurokawa, Y.; Ohta, H.: Okubo, M.: Takahashi, M. Carbohydr. Polym. 1994, 23, 1-4. (28) Cmmbliss, A. L.; Stonehuemer, J.; Henkens, R. W.; O'Daly, J. P.; Zhao, J. New J. Chem. 1994, 18,327-339. 0003-2700/95/0367-2436$9.00/0 0 1995 American Chemical Society

remain accessible and can selectively interactheact with chemical species (analytes) within the sample milieu. One of the first examples of sol-gel encapsulationof biological materials was by Carturan et al.,2I who demonstrated that invertase-active whole yeast cells could be entrapped within a solgel-derived Si02 thin film. Avnir, Braun, and co-~orkers'~ used sol-gel processing to entrap alkaline phosphatase (AP)in a tetramethylorthosilicate (TMOS) sol-gel monolith. The encap sulated AP exhibited 30%activity for up to 2 months and showed improved thermal stability compared to native AP. The Dunn, Valentine, and Zink g r o ~ p s demonstrated ~~J~ that copper-zinc superoxide dismutase, cytochrome c, and myoglobin functioned within a sol-gel-derived monolith. Shtelzer et al.17 entrapped trypsin and AP within a sol-gel composite composed of tetraethylorthosilicate UEOS) and poly(ethy1ene glycol). These particular enzymes were stable, and trypsin did not undergo autodigestion while encapsulated within the sol-gel composite. Additional work, most notably, by Avnir, Braun, and co-workers and the Dunn, Valentine, and Zink has shown that other enzymes such as glucose aspartase,2Operoxidase,2O and u r e a ~ e land ~ . ~even ~ non-enzymes such as bacteriorh~dopsin~~ (the photosynthetic membrane protein) can be successfully entrapped in active forms within a sol-gel glass composite. In an effort to understand how the evolving sol-gel composite influenced an entrapped protein, Saavedra and c o - ~ o r k e r s ~ ~ recently reported fluorescence experiments on myoglobin and acrylodan-labeled bovine serum albumin (BSA-Ac) within aTMOS derived sol-gel monolith. These biogel composites were immersed in solutions containing ionic quenchers or chemical denaturants, and the protein response and conformation were probed by following shifts in the acrylodan emission maximum. This work served to illustrate that analytes (i.e., quenchers and denaturant) can diffuse into the sol-gel matrix and interact with the entrapped protein. The work also showed that the sol-gel matrix affected the entrapped protein. Our group recently reported29 on the steady-state and timeresolved fluorescence of native, chemically denatured, and silicasorbed BSA-Ac. These results demonstrated that the emission is affected by nanosecond and subnanosecond dipolar relaxation of the local protein environment surrounding the acryl0dan3~ residue. They also showed that motion of the acrylodan residue within the native BSA-Ac was restricted to a semiangle of 23 f 1". Thus, there is a substantial level of dynamics within the domain surrounding the acrylodan residue in BSA-Ac. In this paper we aim to determine how the aging sol-gel matrix influences the internal dynamics of an entrapped protein. Toward this end, we report on the static and time-resolved fluorescence of BSA-Ac and acrylodan-labeled human serum albumin (HSA-Ac) entrapped within a TMOSbased sol-gelderived bi0gel.2~This particular system was chosen for several reasons. First, TMOS is the alkoxide precursor best suited for use with biological dopants."Q5 Second, the dynamics of the acrylodan residue within native, chemically denatured, and surfaceadsorbed BSA-Ac has been reported previously29 and serves as a benchmark for the current experiment. Third, the fluorescence in the current experiments arises from a single, covalently attached residue (Ac) at a known location (cysteine-34) within the proteins. (29) Wang, R; Sun, S.; Bekos, E. J.; Bright, F. V. Anal. Chem. 1994,67, 14959. (30)Prendergast, F. G.; Meyer, M.; Carlson, G. L.; Iida, S.; Potter, J. D.]. Biol. Chem. 1983,258, 7541-7544.

Therefore,there is no confusion over multiple probe binding sites or conformation-induced probe migration. Fourth, although BSA and HSA are quite different proteins, much of their amino acid sequence is conserved.31 Thus, we can determine whether subtle changes in amino acid sequencing affect the observed dynamics and determine how different proteins are affected by an evolving biogel. THEORY The theory of frequency domain fluorescence for recovering intensity and anisotropy decays and determining the time evolution of the fluorescence emission spectrum (e.g., following dipolar relaxation within the cybotactic region) has been described e l s e ~ h e r e . 2 ~We ~ ~review ~ - ~ ~ only the key expressions here; the interested reader is referred to the aforementioned references for a more full description of the acquisition techniques, data analysis schemes, and basic interpretation. Time-Dependent Spectral Relaxation. Photoexcitation of a fluorophore generally leads to an instantaneous change in the fluorophore's dipole and production of an excited-state species that is no longer in equilibrium with its surrounding environment. To lower the overall system energy, dipolar species (solvent, residues, or both) surrounding the fluorophore reorganize to minimize the unfavorable interactions between themselves and the excited-state dipole. If this relaxation process is on the same time scale as the excited-statefluorescence lifetime, it results in a quantifiable,timedependent shift of the fluorophore emission spe~trUm.~9~3~-~* The time dependence of this spectral evolution, in turn, provides information on the kinetics of dipolar reorganization within the environment surrounding the fluorophore (Le., the cybotactic region).29J4,35 In the current work we follow this process using the time-resolved emission center of gravity ( ~ ( t ) ) , the time-dependent full width at half-maximum of the emission profile, and the time-resolved normalized domain relaxation function (D(t)):29

where v(O), Y(-), and ~ ( trepresent ) the spectral centers of gravity at time zero (immediately following optical excitation), infinity (after the probe and its surrounding environment have reached equilibrium), and t (during the spectral relaxation process), respectively. The terms in the exponential expression Cgi and ki) denote independent kinetic processes describing the total timedependent relaxation of the cybotactic region. The ki and gi terms (31) Brown, J. R; Shockley, P. In Serum Albumins: Structure and Characterization of Its Ligand Binding Sites in Lipid Protein Interactions; Jost, P. C., Griftith, 0. H.,Eds.; John Wiley & Sons: New York, 1982; Chapter 2, pp 25-68. (32) (a) Topics in Fluorescence Spectroscopy; Lakowicz, J. R, Ed.; Plenum Press: New York, 1991; Vols 1-3. (b) Bright, F. V.; Betts, T. A; Litwiler, K. S. CRC Crit. Rev. Anal. Chem. 1990,21, 389-405. (33) Gratton, E.; Jameson, D. M.; Hall, R D. Annu. Reu. Biophys. Bioeng. 1984, 13. 105-124. (34) Lakowicz, J. R Principle of Fluorescence Spectroscopy; Plenum Press: New York, 1983; Chapter 6, pp 155-185. (35) Lakowicz, J. R; Cherek, H. Chem. Phys. Lett. 1985,122,380-384. (36) Maroncelli, M.; Fleming, G. R J. Chem. Phys. 1987, 86, 6221-6229. (37) Maroncelli, M.; Castner, E. W., Jr.; Bagchi, B.; Fleming, G. R Faraday Discuss.Chem. SOC. 1988,85,199-210. (38) Maroncelli, M.; MacInnis, J.; Fleming, G. R Science 1989,243, 16741681.

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are recovered from the D(t) data using nonlinear least s q u a r e ~ . 3 ~ * ~ ~ Preparation of Sol-Gel-Derived Biogels. The sol-gel stock solution contained TMOS-water-HC1 in the molar ratio ki and g, provide one with information on the dynamics of the cybotactic region surrounding the acrylodan residue. 1:2:0.oooO16. The TMOS was placed within a beaker in a sonicator Decay of FluorescenceAnisotropy. Time-resolved fluoresand chilled to 4 "C, and the water-HC1 solution was added cence anisotropies provide insight into the mobility of the dropwise. Immediately after addition of the HC1-water solution, fluorescent reporter group within its cybotactic r e g i ~ n . How~ ~ . ~ ~ the reaction mixture was sonicated until a clear solution formed ever, in the current work, data interpretation becomes complicated (-30 min). (Note: In the absence of a cosolvent such as ethanol, because there is independent (local) rotational reorientation from hydrolysis must be carried out at reduced temperature or turbid, the probe itself. In this situation, the probe dynamics become heterogenous solutions result). The actual biogels for study were superimposed with those of the biomolecule. For the case of a prepared by adding the appropriate volume (typically 75-100 pL) fluorophore free to reorient over a limited angular range that is of the BSA-Ac or HSA-Ac stock solution (0.1 M, pH 7 phosphate attached to a larger particle that in turn undergoes isotropic buffer) to 1.5 mL of the sol-gel stock such that the final rotational diffusion, the time-resolved decay of anisotropy (r(t)) concentration of acrylodan-labeled albumin was 2.0 pM. These is well approximated by a double-exponentialdecay of the formz9 samples were then transferred to a fused silica cuvette and sealed with Teflon caps. After aging for 14 days in the dark at room 4)- ro[P1exp(-t/$J + Pz e x ~ ( - t / $ ~ ) l (2) temperature, the caps were removed and the biogels continued to age under ambient conditions (2' = 21 f 1 "C). FluorescenceMeasurements. All steady-statefluorescence where ro is the limiting anisotropy and #I depends solely on the measurements were performed with a SLM 48000 MHF spectroflocal rotational reorientation of the probe. In the situation where luorometer using a Xe arc lamp as the excitation source and all this local probe motion is much faster than the global motion of spectra were background subtracted. the entire protein, the rotational reorientation associated with the Tune-resolved fluorescence intensity and anisotropy decay data local motion, #L, is given as 1/(1/#1 1/42), where & reflects of the biogels were acquired in the frequency domain using a the global rotational reorientation of the entire protein; #G = 42. SLM 48000 MHF multifrequency phase-modulation fluorometer. The terms PI and PZrepresent the fractional contributions to the An argon ion laser (Coherent, Model Innova 90-6) operating at total anisotropy decay from the local and global motions, respec351.1 nm was used as the excitation source. A 340 f 20 nm tively (cpi = 1). bandpass filter (Oriel) was placed in the excitation path to A semiangle (8) can be associated with the cone within which eliminate extraneous plasma discharge. Depending on the the probe is able to precess during its excited-statefluorescence particular experiments, emission was observed through a 400 nm lifetime, and is given by32a long-passfilter or a series of bandpass filters (Oriel). Magic angle polarization was used for all excited-state intensity decay experim e n t ~ MezPOPOP .~~ in ethanol was used as the reference lifetime standard; its lifetime was assigned a value of 1.45 n ~ . ~For * all experiments, the Pockels cell was operated at a repetition rate of If the environment surrounding the probe were totally restrictive 5 MHz. Typically, data were acquired for 30 s from 5 to 175 MHz to local probe motion, 8 will approach 0". In contrast, complete (35 frequencies). All multifrequency phase and modulation data freedom to rotational reorient will result in 8 of 90". Intermediate were analyzed by the global analysis method as described 8 values reflect partial freedom of the probe to reorient within its else~here.29.~~-4~ There was no evidence of photodecomposition cybotactic region. under our experimental conditions.

+

EXPERIMENTAL SECTION

Reagents. The following chemicals were used &acryloyl(dimethy1amino)naphthalene (acrylodan, Molecular Probes); essentially fatty acid free bovine and human serum albumin and 12 000 MW cutoff dialysis tubing (cellulose membrane) ( S i a Chemical Co.) ; 1,4bis(4methyl-5-phenyl-2-oxazolyl)benzene (Mer POPOP) (Aldrich Chemical Co.); NazHP04, NaHzP04.2Hz0, N,I?dimethylformamide @MF), and HC1 Fisher Scientific Co.); ethanol (200 proof, Quantum Chemical Cow.); and tetramethylorthosilicate (TMOS) (Hiils). All reagents were used as received without further puritication, aqueous solutions were prepared in doubly distilled-deionized water, and stock solutions were refrigerated in the dark at 4 "C. Acrylodan was used immediately after it was dissolved in DMF. Preparation of BSA-Acand HSA-Ac. The protocol is similar to the scheme used previo~sly.2~~~~ (39) Bevington, P. R Data Reduction and ErrorAnalysis for the Physical Sciences; McGraw-Hill: New York, 1969; pp 204-242. (40) The curve fitting capabilities of Sigmaplot version 5.1 Uandel Scientific, Inc.,

Corte Madera, CA) are used. (41) Garrison, M. D.; Iuliano, D. J.; Saavedra, S. S.; Truskey, G. A; Reichert, W. M. J. Colloid Intetface Sci. 1 9 9 2 , 148, 415-424.

2438 Analytical Chemistty, Vol. 67,No. 14, July 15, 1995

RESULTS AND DISCUSSION

Steady-StateFluorescence. The site-selectivenature of the acrylodan labelling of the serum albumin at cysteine34 (loop 1, domain 1)2430,41defhes the cybotactic region from which the acrylodan residue reports. Thus, the information obtained is from a spec& site within the albumins that are entrapped within the sol-gel-derived biogel. Figure 1presents a series of normalized steady-state fluorescence spectra for a BSA-Ac-derived biogel as a function of storage time. Similar results were obtained with HSA-Ac. Several key points are readily apparent from these data. First, the emission spectra blue shift and broaden as the acrylodan residue is excited at higher energy. This demonstrates that the fluorescence process is inhomogeneous. Second, excitation (42) Spencer, R D.; Weber, G. J. Chem. Phys. 1 9 7 0 , 52, 1654-1667. (43) (a) Wang, R; Bright, F. V. J. Phys. Chem. 1993,97,4231-4238. @) Wang, R; Bright F. V. J. Phys. Chem. 1993,97,10872-10872. (c) Wang, R; Bright, F. V. Appl. Spectrosc. 1993, 47, 800-806. (d) Wang, R; Bright, F. V. Appl. Spectrosc. 1993, 47, 792-799. (44) Beecham. J. M.; Gratton, E. Proc. SPIE 1988, 909, 70-81. (45) Beecham, J. M.; Gratton, E.; Ameloot, M.; Knutson, J. R; Brand, L. In Topics in Fluorescence Spectroscopy; Lakowicz, J. R , Ed.: Plenum Press: New York, 1991; Vol. 2, Chapter 5, pp 241-305.

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Table 1. Recovered Kinetlc Parameters for the Spectral Relaxation for the Acrylodan Residue in BSAand HSAmAc Entrapped within a TYOS-Based Sol-Gel-Derlved Blogel

BSA-AC day 0

day 37

41 f 4 0.4 f 0.04 59 f 4 5.7 f 0.8

57 f 6 0.17 f 0.008 43 f 6 5.0 f 0.6

100 1.2 f 0.2 0 5.4 f 0.9

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c C

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c .n

0.5

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2,

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460

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0.0 0.0 400 450 500 550 600 400 450 500 550 600 1 0 . m 1 0 . m

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0.5

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8

8

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