Anal. Chem. 1995,67,149-159
Dynamics Surrounding Cys=34in Native, Chemically Denatured, and SilicamAdsorbed Bovine Serum Albumin Run Wang, Shiying Sun,t Evan J. Bekos, and Frank V. Bright*#*
Department of Chemistty, Natural Sciences and Mathematics Complex, State University of New York at Buffalo, Buffalo, New York 74260-3000
We report the steady-stateand time-resolvedfluorescence of 6-acryloyl(dimethylamino)naphthalene (acrylodan) covalently attached to Cys-34 in bovine serum albumin (BSA). For this conceptually simple system, complicated fluorescence intensity and anisotropy decay kinetics are observed. The steady-state and time-resolved results demonstrate the presence of an excited-statereaction for the BSA-acrylodan system. Additional analysis shows that dipolar relaxation of the environment surrounding acrylodan within BSA is responsible for most of the observed time-dependentevolution of the emission spectrum. The effects of temperature, chemical denaturation, and protein adsorption to a bare silica substrate are also investigated. These results demonstrate the complexity of the changes within a proteidbiorecognition element that affect the signal from a single fluorescent reporter grOUP. Protein molecules are dynamic in nature,1,2and experimental results have shown that there is considerable local motion within a protein under ambient c0nditions.3-~The characteristic times of these motions extend over a broad range from picoseconds to seconds. Additional structural data have shown that residue or subunit displacements play a key role in the activity and affinity of proteins (e.g., enzyme catalysis,6hemoglobin cooperativity? and immunoglobulin action*). During the past decade, the functional significance of protein structural dynamics and internal or segmental motions on their overall biological function has been recognized.l-12 As a result, considerable effort has centered on characterizing the dynamical features of various protein systems. ' Permanent address: Chemistry Department at the Branch Campus of Peking University, 59Tu Cheng Bei Lu Rd., Beijing, 100083,People's Republic of China. Present address: School of Chemical Engineering, Georgia Institute of Technology, 778 Atlantic Dr, Atlanta, GA 30332-0100. (1)(a) Metzler, D. E. Biochemistry, the Chemical Reactions of Living Celki; Academic Press: New York, 1977. (b) McCammon, J. A; Harvey, S. C. Dynamics of Proteins and Nucleic Acid Cambridge University Press: New York, 1987. (c) Welch, G.R Fluctuating Enzyme; Wiley: New York, 1986. (2) (a) McCammon, J. A; Gelin, B. R; Karplus, M. Nature 1977,267,58595. (b) Karplus, M.; McCammon, J. A CRC Chi. Rev. Biochem. 1981,9, 293-349. (3)Eftink, M. R;Ghiron, C. A Biochemistry 1976, 15,672-8. (4)Frauenfelder, H.; Sliger, S . G.; Wolynes, P. G. Science 1991,254, 1598603. (5)Genberg, L.;Richard, L.; McLendon, G. L.Science 1991,251,1051-4. (6)Lipscomb, W. N. Acc. Chem. Res. 1970,3,81-9. (7) Karplus, M.; Weaver, D. L. Nature 1976,260,404-6. (8)Huber, R;Deisenhofer, J.; Colman, P. M.; Matsushima, M.; Palm, W. Nature 1976,264,415-20. (9)Daragan, V. A;Mayo, K. H. J. Am. Chem. SOC.1992, 114,4326-31. (10)Ross,J. B. A; Rousslang, K W.; Brand, L.Biochemistry 1981,20,4361-9. 0003-2700/95/0367-0149$9.00/0 0 1994 American Chemical Society
Toward this end, temperature-dependent X-ray d&action, NMR relaxation, molecular dynamics simulations, and fluorescence depolarization techniques have been used to address many aspects of protein dynamics.2~9-12 Advances in instrumentation and theory have allowed timeresolved fluorescence techniques to directly probe biomolecule dynamic~.'~"J~ Modem developments in laser technology and electronics now permit subnanosecond measurements on a routine basis with considerable reliability. As an example, our group recently used steady-state and time-resolved fluorescence anisotropy measurements to study the rotational reorientation kinetics of a fluorescent probe covalently attached to bovine serum albumin @SA).14 This system served as a convenient benchmark for the type of probe-protein interactionsand processes occurring in our self-contained, fiber-optic-based analytical immunosensors.15 The activity, afhity, and/or conformation of proteins at interfaces is clearly of great importance to the development of biosensors, understanding and controlling bioadhesion, and addressing biocompatability.16-n In the case of chemical biosensors, (11) Marzola, P.;Gratton, E.]. Phys. Chem. 1991,95,9488-95. (12) (a) Topics in Fluorescence Spectrosco& Lakowicz, J. R Ed.; Plenum Press: New York, 1991,Vols. 1-3. @) Idpati, G.;Szabo, A Biophys. /. 1980,30, 489-96. (13)Lakowicz, J. R;Szmacinski, H.; Gryczynski, I. Photochem. Photobiol. 1988, 47,31-41. (14) (a) Wang, R;Bright, F. V.J Phys. Chem. 1993,97,4231-8.(b) Wang, R; Bright, F. V.J. Phys. Chem. 1993,97,10872-8.(c) Wang, R;Bright, F. V. AppL Spectrosc. 1993,47,800-6.(d) Wang, R;Bright, F. V. Appl. Spectrosc. 1993,47,792-9. (15) (a) Bright, F. V.; Litwiler, K S.;Vargo, T.G.; Gardella, J. A, Jr. Anal. Chim. Acta 1992,262,323-30.(b) Beth, T. A; Catena, G. C.; Huang, J.; Litwiler, K. S.; Zhang, J.; Zagrobelny, J.; Bright, F. V. Anal. Chim. Acta 1991,246, 55-63. (c) Bright, F. V.; Betts, T.A;Litwiler, K S. Anal. Chem. 1990,62, 1065-9. (16)Taylor, R F. Immobilized Antibody- and Receptor-Based Biosensors. In Protein Immobilization: Fundamentals and Applications; Taylor, R F., Ed.; Marcel Dekker, Inc.: New York, 1991;pp 263-303. (17) Horbett, T.A;Brash, J. L. Proteins at Intetfaces: Current Issues and Future Prospects in Proteins at Interfaces, Physicochemical and Biochemical Studies; ACS Symposium Series 343;American Chemical Society: Washington, DC, 1987;Chapter 1, pp 1-33. (18)Velez, M.; Barald, K F.; Axelrod, D.J. Cell Biol. 1990,110,2049-59. (19)Scalettar, B. A; Selvin, P. R; Axelrod, D.; Klein, M. P.; Hearst,J. E. Biochemistry 1990,29,4790-8. (20)Scalettar, B. A;Selvin, P. R; Axelrod, D.; Hearst, J. E.; Klein, M. P.Biophys. J. 1988,53,215-26. (21)Hlady, V.; Andrade, J. D. Colloid Su?f 1988,32, 359-69. (22)Hlady, V.; Andrade, J. D. Colloid Su?f 1989,42, 85-96. (23)Cheng, Y.L.;Darst, S.A; Robertson, C. R J. Colloid Interface Sci. 1987, 118,212-3. (24)Oroszlan, P.; Lu, X. M.; Yarmush, D.; Karger, B. L.J Chromatogr. 1990, 500,481-502. (25)Rainbow, M. R;Atherton, S.; Eberhart, R C. J Biomed. Mater. Res. 1987, 21,539-55.
Analytical Chemistry, Vol. 67, No. 1, January 1, 1995 149
the act of surface “immobilization”,to produce the actual biosensor because the precise location of the reporter group (Ac) within interface, generally decreases protein activity or aftinity due to the the BSA is known and emission from acrylodan reports on the conformationalchanges that result from the interactions between dynamics from a single domain (Le.,the microenvironment around the surface and the chemical recognition element (Le., protein Cys-34). Second, the acrylodan photophysics have been studmolecule).’6J7 In addition, the biointerface produced in any ied.32*33 Third, the acrylodan fluorescence spectrum is extremely practical biosensor is not static and will change from day to day. sensitive to the physicochemical properties of the solvent and Thus, if one wishes to develop viable biosensors, it becomes dipolar perturbation from the local environment around the imperative to understand the dynamics of proteins that make up pr0be.3~-~~ Therefore, acrylodan can, in principle, be used to study the interface of a biosensor. This type of information can local physicochemical properties, conformational changes, and biological activity information on more specific domains within potentially provide valuable insights into the operation, perforp r o t e i n ~ . 2 ~ However, , ~ ~ - ~ ~ previous reports27,32-39 on acrylodanmance, and inevitable failure of modern biosensors.15 labeled proteins used primarily steady-state fluorescence techBSA has been used extensively as a model protein to study niques or did not focus directly on the dynamical aspects of the surface-induced conformational changes in proteins.17~21~22J5-27 For probe attached to the proteins. Specifically, previous work27s32-39 example, Hlady and Andrade used total internal reflection fluodid not aim to determine the origin of spectral shifts, probe the rescence (TIRF) to probe the conformational changes of BSA adsorbed at a silica interface.21122These experiments used the dynamics surrounding the acrylodan residue withii the labeled intrinsic fluorescence from tryptophan residues in BSA and the protein systems (e.g., native and surface adsorbed), or determine fluorescenceof an extrinsic probe, 1-anilinonaphthalen&sulfonate how surface adsorption affected the aforementioned dynamics. (l,&ANS). The authors found that the most strongly adsorbed In this paper we use steady-state and time-resolved fluoresBSA exhibited a tryptophan emission that was blue shifted relative cence techniques to investigate acrylodan-labeled BSA @SA-Ac) to native BSA in solution. In contrast, the emission spectrum of in the bulk and adsorbed to a bare silica surface. Toward this 1,gANS bound to surface-adsorbed BSA red shifted relative to end, multifrequency phase and modulation fluorescence native BSA/l,&ANS in solution. From these results, the authors (MPMF‘) ,40-42 multifrequencyphase and modulation total internal concluded that conformational changes were induced by BSA reflection fluorescence (MPM-TIFF) and phaseresolved evaadsorption such that the tryptophan environments became more nescent wave-induced fluorescence (pREwIF‘)45are used to hydrophobic and the l,&ANS binding sites became more hydroinvestigate how the acrylodan reporter group within BSA-Ac philic.21,22 behaves and is influenced by temperature, chemical denaturation, and adsorption to a silica surface. BSA has two tryptophan residues that are located in grossly different microenvironments,a and the excited-statedecay kinetics from even tryptophan alone in water is inherently c ~ m p l e x . ~ ~THEORY ~~ In addition, a single BSA molecule can bind several l,M” The theory of frequency-domain fluorescence for recovering fluorescence intensity and anisotropy decays and the time evolumolecules.3l Therefore, the observed fluorescence in the aforetion of an emission process (e.g., dipolar relaxation) have been mentioned experiments arises from the collective response of multiple emissive centers or what amounts to an inherently described e l s e ~ h e r e . 4 ~ - ~ 2We ~ ~review ~ - * ~ briefly the key exprescomplex emission process. Hence, it is difficult at best for one sions describing the timedependent spectral relaxation and the to attribute the observed steady-state emission shift from tryp anisotropy decay kinetics because they are the most germane to tophan residues or extrinsic probes with high binding stoichiomthe remaining discussion. etry (i.e., not 1:l probe to protein) to any change withii a specific Tie-Dependent Spectral R e h t i o n . Absorption of light microenvironment. by a fluorophore generally increases the fluorophore’s dipole Reichert and co-workersn have studied BSA adsorption to bare m ~ m e n t . ’ ~The ~ , ~change ~ in dipole moment disrupts the equiand alkyl silane-treated glass cover slips using steady-state (34)Sommer, A; Gorges, R; Kosher, G. M.; Paltauf, F.; Hermetter, A. fluorescence. In an elegant approach, these authors overcame Biochemistry 1991,30, 11245-9. many of the aforementioned difficulties by using a site-selective (35)Clark, I. D.;Burtnick, L. D. Arch. Biochem. Biophys. 1988,260,595-600. fluorescent probe, &acryloyl-2-(dimethylamino)naphthalene (36)Yem, A W.; Epps, D. E.; Mathews, W. R; Guido, D. M.; Richard, K A; Staite, N. D.; Deibel, M. R, Jr. I. Biol. Chem. 1992,267,3122-8. (acrylodan,Ac) .32 The advantages of using acrylodan are several. (37)Epps, D. E.; Yem, A W.; Fisher, J. F.; McGee, J. E.; Paslay, J. W.; Deibel, First, acrylodan reacts only with free thiol gr0ups,3~-~~ and BSA M. R,Jr. J. Biol. Chem. 1992,267,3129-35. has one free thiol at C ~ s - 3 4 .Thus, ~ ~ this system is more simple (38)Lehrer, S. S.; Ishii, Y. Biochemistry 1988,27, 5899-906. (26) (a) Crystal, B.; Rumbles, G.; Smith, T. A; Phillips, D. J. Colloid Intetface Sci. 1993, 155, 247-50. (b) Phillips, D. Analyst 1994,119, 543-50. (27) (a) Garrison, M. D.; Iuliano, D. J.; Saavedra, S. S.; Truskey, G. A; Reichert, W. M. /. Colloid Intetface Sci. 1992, 148, 415-24. (b) Iuliano, D.J.; Saavedra, S. S.; Thlskey, G. A J. Biomed. Mater. Res. 1993,27,1103-13. (28)Brown, J. R;ShocMey, P.Serum Albumins: Structure and Characterization of Its Ligand Binding Sites in Lipid Protein Interactions;Jost, P. C., Griffith, 0. H., Eds., John Wiley & Sons: New York, 1982;Chapter 2,pp 25-68. (29)(a) Szabo, A. G.; Tayner, D. M. J. Am. Chem. SOC.1980,102,554-63.(b) Jameson, D. M.; Weber, G. J. Phys. Chem. 1980,85,953-8.(c) Bivin, D. B.; Khoroshev, M. I. J. Photochem. Photobiol. A: Ckem. 1994,78,209-17. (30)Demchenko, A P. In Topics in Fluorescence Spectroscopy; Lakowicz, J. R, Ed.; Plenum Press: New York, 1991;Vol. 3,Chapter 2,pp 65-111. (31)Kolb, D. A;Weber, G. Biochemistry 1975, 14,4476-81. (32)Prendergast, F. G.; Meyer, M.; Carlson, G. L.; Iida, S.; Potter, J. D. J. Biol. Chem. 1983,258,7541-4. (33)Ilich, P.; Prendergast, F. G. J. Phys. Chem. 1989,93, 4441-7. 150 Analytical Chemistty, Vol. 67,No. 7, January 1, 1995
(39)Reid, S. W.; Koepf, E. K; Burtnick, L. D. Arch. Biochem. Biophys. 1993, 302,31-6. (40)Bright, F.V.; Betts, T.A; Litwiler, K. S. CRC Crit. Rev. Anal. Chem. 1990, 21,389-405. (41)Gratton, E.; Jameson, D. M.; Hall, R D. Annu. Rev. Biophys. Bioeng. 1984, 13,105-24. (42) Lakowicz, J. R Principle ofFIuorescence Spectroscopy, Plenum F’ress: New York, 1983; Chapter 6,pp 155-185. (43)Bright, F. V.Appl. Spectrosc. 1993,47,1152-60. (44)Bright, F. V.; Wang, R; Li, M.; Dunbar, R A. Immunomethods 1993,3, 104-11. (45)Lundgren, J. S.; Bekos, E. J.; Wang, R; Bright, F. V. Anal. Chem. 1994, 66,2433-40. (46)Lakowicz, J. R;Cherek, H. Chem. Phys. Lett. 1985,122,380-4. (47)Maroncelli, M.; Fleming, G. R]. Chem. Phys. 1987,86,6221-9. (48)Maroncelli, M.; Castner, E. W., Jr.; Bagchi, B.; Fleming, G. R Faraday Discuss. Chem. SOC.1988,85,199-210. (49)Maroncelli, M.; MacInnis, J.; Fleming, G. R Science 1989,243, 1674-81.
librium between the fluorophore and its surrounding microenvironment. As a result, photoexcitation results in a change in the total system energy. In order to lower the overall system energy, the solvent molecules and/or local groups within the environment surrounding the fluorophore "reorganize" to accommodate and minimize the unfavorable interactions produced by the sudden change in the dipole moment of the fluorophore. If this reorganization or dipolar relaxation process occurs on the same time scale as the excited-state fluorescence lifetime, it results in an easily quantjfled, timedependent shifting of the emission spectrum of the fl~orophore.'~~4~,~~-~9 The time-dependent spectral evolution of the probe emission spectrum provides insight into the temporal changes in the local environment around the probe (i.e., the dynamics within the cybotactic region).42,46 The local environment could represent solvent molecules if the fluorophore is dissolved in a neat liquid. Alternatively, the cybotactic region could represent the immediate amino acid residues surrounding the fluorescent reporter group located within the protein matrix (e.g., around Cys-34 in BSA). If the fluorophore is located at the exterior of a protein, the cybotactic region may involve amino acid residues surrounding the probe and solvent molecules (e.g., water) that can access the probe. However, fluorescent reporter groups sequestered deep within the protein are probably less likely to encounter water and the probe thus exclusively senses the cybotactic region offered by the neighboring amino acid residues. The timeresolved emission spectra are recovered from the timeresolved fluorescence intensity decay profiles at several emission wavelengths (,le,,) and the normalized steady-state emission The wavelength dependence of the timeresolved fluorescence intensity decay can thus be written as&
where a&) and t i are the recovered apparent fluorescence intensity decay parameters obtained from analysis of the frequencydomain data.4°-42 The corresponding timeresolved emission spectra are subsequently formed from the normalized impulse response functions:46
where N(,l) represents the wavelength-dependent normalization factor. The time-dependent emission center of gravity ( ~ ( t )is) in turn given by46
(after the probe and its surrounding environment have reached equilibrium), and t (during the spectral relaxation process), respectively. S(t) serves to normalize the observed spectral shift to the total shift and allows one to compare systems in which the absolute spectral shifts differ. For the BSA-Ac system studied here, it appears inappropriate to use S(t) as it may lead to some confusion as to what is actually being measured or reported. To make it clear that we are following the evolution of a heterogeneous microdomain about a fluorescent probe within a protein (which may or may not include solvent molecules), we use D(t) to denote that we are observing relaxation of the domain around the acrylodan residue. Additional reasons for this choice of terminology are as follows: (1) the spectral relaxation of a fluorescent probe within a protein results from reorganization of the dipoles or residuesg-" within the cybotactic region; and (2) it is known that the three dimensional arrangement of residues within a protein is capable of shielding the fluorophore from solvents (e.g., water)? To extract the rate information about the observed dipolar relaxation process, we model the recovered D(t) data as a sum of exponentials?7-49 n
D(t) =
Biexp(-k,t)
where n denotes the number of independent rate processes describing the total spectral relaxation and ki and gi are the dipolar relaxation rate and its fractional contribution, respectively. The ki and gi terms are recovered using nonlinear least squares The form of D(t) depends on the physicochemical properties of the environment around the acrylodan residue. Thus, the recovered relaxation rates provide information on the cybotactic region surrounding the acrylodan residue within BSA-Ac. In effect, ki and gi provide one with information on the origin of the steady-state spectral shifts seen as a result of surface adsorption, temperature variations, chemical denaturation, and protein-ligand binding. Decay of Fluorescence Anisotropy. The rotational reorientation of a biomolecule is often monitored by use of an extrinsic fluorophore covalently attached to the p r ~ t e i n . ' ~ However, ,~~ analysis of such data is complicated if any form of internal (local) rotational reorientation from the probe is present. In these cases, the rotational motion from the probe becomes superimposed upon that of the entire biomolecule. For the case of a fluorophore free to rotate through a limited angular range that is attached to a larger particle that in turn undergoes isotropic rotational diffusion, the anisotropy decay kinetics are well approximated by a double exponential decay of the form42 = ro[PlexP(-t/dJ,)
In previous studies on normal liquids, time-dependent spectral shifts are often followed by using a solvent correlation function (S (t)):47-49 (4)
where v(O), Y(-), and ~ ( trepresent ) the spectral centers of gravity at time zero (immediately following optical excitation), infinity
(5)
i=l
+ Bz ex~(-t/dJJl
(6)
In this expression, Y, is the limiting anisotropy, & depends solely on the local rotational reorientation of the probe, and /31 and / 3 ~ are the fractional contributions of total anisotropy decay from the local and the global motions, respectively Q 3 i = 1). In the (50) Bevington, P. R Data Reduction and ErrorAnalysisfor the Physical Sciences; McGraw-Hill Book Co.: New York, 1969; pp 204-42. (51) The curve fitting capabilities of SigmaF'lot (version 5.1, Jandel Scientific, Inc., Corte Madera, CA) are used.
Analytical Chemistry, Vol. 67, No. 1, January 1, 1995
151
situation where this local motion is much faster than the global motion of the entire protein, the rotational reorientation time of the local motion, h, is written as 1/(1/41 l/#d, where 41 reflects the global reorientation of the entire particle, (PG = 91. The semiangle (6) associated with the cone through which the probe rotationally reorients is related to the fractional contribution of local motion by12b
+
1 - p2 = cos2 e(i
+ COS e12/4
(7)
EXPERIMENTAL SECTION Reagents. The following chemicals were used Gacryloyl(dimethy1amino)naphthalene (acrylodan, Molecular Probes); essentially fatty acid-free bovine serum albumin (BSA),and 12 OOO MW cutoff dialysis tubing (cellulose membrane) ( S i a Chemical Co.); guanidine hydrochloride (Gd-HCl) (99%) and 1,4bis(4methyl-5phenyl-2-oxamlyl)benzene (Me2POPOP) (Aldrich Chemical Co.) ; Na2HP04, NaH2POc2H20, and NJV-dimethyl formamide (DMF) (Fisher Scientific Co.); and ethanol (200 proof, Quantum Chemical Corp.). All reagents were used as received without further purification, 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 Acrylodan-Labeled BSA (BSA-AC).~~~ A stock solution containing 50 pM BSA is prepared in 0.10 M phosphate buffer (PH = 7.0). A 4GmL aliquot of this solution is taken, and enough acrylodan (in the minimum amount of CH3CN) is added such that the molar ratio of BSA to acrylodan is 1:l. This mixture is stirred gently and maintained at room temperature for 10 h. The reaction mixture is then loaded into a 12000 MW cutoff cellulose dialysis bag and dialyzed at 4 "C against 250 mL of 1:20 (v/v) acetonitrile-phosphate buffer (0.1 M pH 7.0). M e r 12 h, the initial dialysate was replaced with 250 mL of 0.1 M pH 7.0 phosphate buffer. This solution was similarly replaced every 12 h for 4 days. Dialysis is complete when there is no detectable acrylodan fluorescence in the dialysate solution. In our hands, it was necessary to carry out this prolonged dialysis. Shorter term treatments lead to significant levels (>5%) of unreacted acrylodan in the "BSA-Ac" solution. The final BSAAc solution is stored at 4 "C and the molar ratio of acrylodan to BSA is 0.80 0.05.27a Adsorption of BSA-Ac to Silica.A l k m length of optical fiber (100@pm core diameter quartz multimode, General Fiber Optics) was treated with concentrated nitric acid overnight to remove the cladding from a l-cm segment. After the cladding was removed, the bare fiber was treated with 1 M NaOH for 30 min and washed with copious amounts of water and methanol. This process was repeated until a small drop of water spread evenly onto the bare silica and there were no detectable cladding components in the X-ray photoelectron spectrum of the optical fiber surface. In order to prepare a sample biosurface,we immerse the "clean end" of the optical fiber into a 50 pM BSA-Ac solution (T = 25 "C). After 30 min the optical fiber was removed from this solution, rinsed with buffer, and mounted within our f l ~ o r o m e t e r . ~A~ - ~ ~ similar type of configuration has been described previously by Andrade and co-workers.j2 Fluorescence Measurements. All steady-state fluorescence measurements were performed with a SLM 48000 MHF spectro-
+
152 Analytical Chemistry, Vol. 67, No. 1, January 1, 1995
fluorometer using a Xe arc lamp as the excitation source. All steady-state spectra were background subtracted. BSA-Ac in the Bulk. Timeresolved fluorescence intensity and anisotropy decay data of BSA-Ac in the bulk were acquired in the frequency domain using a SLM 48000 MHF multifrequency phasemodulation fluorometer. An argon ion laser (Coherent, Model Innova 90-6) operating at 351.1 nm was used as the excitation source. A 340 20 nm bandpass filter (Oriel) was placed in the excitation path to eliminate extraneous plasma discharge. Depending on particular experiments, emission was observed through a 385nm long-passfilter or a series of bandpass filters (Oriel). Magic angle polarization was used for all lifetime measurements3 MezPOPOP in ethanol was used as the reference liietime standard its lifetime was assigned a value of 1.45 ns.42 For all experiments, the Pockel's cell was operated at a repetition rate of 5 MHz. Typically, data were acquired for 60 s from 5 to 125 MHz (25 frequencies). All MPMF data were acquired and analyzed by a global analysis method as described elsewhere.14~" Phaseresolved emission spectras were acquired by converting the instrument from a multiharmonic Fourier scheme to a discrete frequency mode.4O BSA-Ac Adsorbed to Silica. A fused silica optical fiber, serving simultaneously as the light guide and the substrate, is optically aligned withiin the sample chamber. In this scheme, the excitation photons travel through the optical fiber and produce an evanescent field at the air-silica interfa~e.4~1~~ This evanescent wave in turn excites the surfaceadsorbed BSA-Ac. The evanescent wave induced fluorescence is collected by a lens and sent to our detection system. Timeresolved fluorescence decay data were acquired in the frequency domain by use of the MPM-TIRF techniq~e.4~944 Depending on the particular experiment, fluorescence was selected by a 385nm long-pass filter or a bandpass filter. Scattered radiation from the optical fiber surface was monitored selectively using a bandpass filter and used as the timing reference (z = 0 ns). The suitability of this scheme for fluorescence lifetime measurements at surfaces has been demonstrated previ0usly.4~ Under our experimental conditions, the intensity of the scattered light from the fiber surface is often substantially greater than the BSA-Ac fluorescence. In these situations, neutraldensity filters were selected and placed in the emission path with the bandpass filter in order to match the fluorescence and scatter intensities. It is well-known that the rotational reorientation of a fluorophore can bias the fluorescence intensity decay data.53 However, the simple magic angle conditions commonly used for isotropic liquid@ are somewhat different for surface measurements.@ This problem is exacerbated further because the optical fibers used here do not preserve the incident polarization of the excitation beam; therefore, such a bias could serious compromise our timeresolved interfacial fluorescence experiments. We tested for such a bias in a limited sense by acquiring MPM-TIRF data at several settings of the emission polarizer. The multifrequency traces at all polarizer settings tested were indistinguishable. This result
+
(52) Newby, IC;Andrade,J. D.;Benner, R E.; Reichert, W. M.J. Colloid Intetface Sci. 1986, 111,280-2. (53) Spencer, R D.;Weber, G. J. Chem. Phys. 1970, 52,1654-67. (54) (a) Beechem, J. M.; Gratton, E. PYOC.SPIE 1988,909,70-81. @) Beechem, 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. (55) McGown, L. B.; Bright, F. V. A n d . Chem. 1984, 56, 1400A-15A (56) Wuth, M. J. Appl. Spectrosc. 1993, 47, 651-3.
1.0
. I
300
350
400
1.0 -a
0.8
--
0.2
--
Y
500
- Surface
1.2
.-
450
Excitation Wavelength (nm)
-
Bulk
+I
m c
0
G
1.2 1 .o
0.8 0.6
0.4 0.2 0.0
360
400
440
400
520
560
600
640
Emission Wavelength (nm)
Figure 1. Steady-state fluorescence spectra for BSA-Ac under various conditions. (upper panel) Steady-state fluorescence excitation spectra of native BSA-Ac at pH 7.5 with the emission monitored at two different wavelengths. (center panel) Steady-state emission spectra of native, chemically denatured (8 M Gd-HCI), and silicaadsorbed BSA-Ac; lex= 351.1 nm. (lower panel) Steady-state emission spectra of surface adsorbed and native BSA-Ac as a function of excitation wavelength.
suggests that the rotational reorientation of acrylodan in the adsorbed BSA is either substantially faster or slower compared to the average acrylodan excited-state fluorescence lifetime. The MPM-TIRF data were analyzed as previously For all experiments, the Pockel's cell was operated at a repetition rate of 5 MHz. Typically, data were acquired for 120 s from 5 to 125 MHz (25 frequencies). PREWIF45was used to acquire the phaseresolved emission spectra for BSA-Ac adsorbed to the silica substrate. RESULTS AND DISCUSSION
Steady-StateFluorescence. Only one acrylodan molecule can be attached covalently to a single BSA thus, the observed fluorescence under our experimentalconditions is from a single emissive center. Figure 1 depicts the static fluorescence spectra of native (PH 7.5), chemically denatured (8 M Gd-HCl), and silica-adsorbed BSA-Ac at 20 "C. Several aspects of these data merit special mention. Fist, the reported maximum emission wavelength for the acrylodan-mercaptoethanol adduct in water is 540 Second, a blue shift in the emission spectrum (central panel) was observed due to covalent attachment of acrylodan to
BSA, indicating the average environment surrounding acrylodan within BSA is less dipolar or less dynamical compared to water. Third, addition of 8 M Gd-HCI red shifts the emission spectrum, showing that the average local environment around acrylodan became more dipolar or more dynamical on protein denaturation. Fourth, the maximum emission wavelength of BSA-Ac with 8 M Gd-HCl (521 nm) is significantly shorter than 540 nm, showing that the dipolar nature of the average local environment around acrylodan in chemically denatured BSA is still less than in neat water. Fifth, there is an additional blue shift in the emission of BSA-Ac on adsorption to the silica surface. This result shows that the average local environment around acrylodan becomes less dipolar upon BSA adsorption. S i , different emission spectra (lower panel) are obtained at different excitation wavelengths for BSA-Ac. These results show that the emission from BSA-Ac is excitation wavelength dependent: the emission spectrum blue shifts and broadens when shorter wavelengths (Le., higher energy photons) are used to excite the sample. These results suggest heterogeneity of the silica-adsorbed BSA-Ac fluores~ence~~ and agrees with previous w0rk.2~~ Finally, the excitation spectra of native BSA-Ac that are obtained when one monitors the emission at the blue or red edge (upper panel) are similar. This indicates that the acrylodan ground state within BSA-Ac appears homogeneous. This demonstrates that the emission heterogeneity observed (lower panel) for acrylodan in BSA-Ac arises predominately from the excited state.3o Time-ResohedFluorescence of the Total Emission. Although there is only a single emissive center (i.e., acrylodan) in BSA-Ac, it is interesting that the steady-statefluorescence results demonstrate heterogeneity of the observed fluorescence. In an effort to determine the origin of this heterogeneity, MPMF and MPM-TIRF data were collected on BSA-Ac. In these particular experiments, a 385-nm long-pass filter was used to select the entire emission from BSA-Ac. Figures 2 and 3 illustrate the frequencydomain results for native and silica-adsorbed BSA-Ac, respectively, at 20 "C. In panel A, the experimental data (phase angle, 0; demodulation factor, 0 ) are compared with the best fits to a single-exponential (- - -) or a unimodal continuous Gaussian distribution14(-) model. For completeness, panels B and C show the corresponding residual plots (Gaussian, 0 and v; single, I and v). In both cases the Gaussian distribution apparently best models these experimental data. The results from the steady-state (Figure 1)and timeresolved fluorescence experiments (long-pass filter) (Figures 2 and 3) indicate heterogeneity of the BSA-Ac fluorescence. For this particular system, a continuous lifetime distribution could result from: (1) time-dependent spectral relaxation of the acrylodan residue and/or (2) multiple interconvertingconformations of BSA, resulting in a distribution of environments around acrylodan. It is well-known that the conformational structure of a protein is dynamic in nature.1-5J2J3~30 Thus, multiple conformations could certainly exist. However, the recovered excited-statefluorescence lifetimes for the acrylodan-mercaptoethanol adduct in water, dioxane, and acetonitrile are 1.28,2.47, and 3.38 ns, re~pectively.~~ For the native BSA-Ac, the apparent lifetime distribution is very broad; lifetime values range from 1 to 6 ns. Thus, although a distribution of static environments (i.e., BSA conformations) may indeed exist around acrylodan within BSA, it is difficult to assign such environments to real domains. That is, the excited-state lifetime of acrylodan in a neat liquid solvent does not exceed about Analytical Chemistry, Vol. 67, No. 1, January 1 , 1995
153
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4 ns nor is it less than about 1.3 ns. Therefore, the origin of acrylodan lifetimes beyond these extremes is difticult to explain in terms of acrylodan residues in domains with solventlike physicochemical properties. Phase-ResolvedEmission Spectra. Phaseresolved flub re~cence~5.55 is a convenient tool for resolving the steady-state emission spectrum into its individual components. Figure 4 presents typical phase-resolved emission spectra (broken lines) for native (upper panel) and silica-adsorbed (lower panel) BSAAc. These results clearly demonstrate that the static emission (-) is a result of emission from at least two different species. Additional analysis (vide infra) shows that the excited-state heterogeneity reflects a timedependent evolution of the acrylodan spectrum due to relaxation within the cybotactic region. Evidence for an Excited-State Reaction. In order to accurately determine the origin of the anomalous acrylodan emission, wavelength-dependent MPMF and MPM-TIRF data were acquired for native, chemically denatured, and silicaadsorbed BSA-Ac. In these particular experiments, 10 or more 154 Analytical Chemistry, Vol. 67, No. 1, January 1, 1995
3
-6l
'
'
I
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100
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Frequency (MHz)
Figure 3. MPM-TIRF data for the total emission from silicaadsorbed BSA-Ac. (A) Phase (0)and modulation (0)data and best fits to single exponential (- - -) and Gaussian distribution (-) models: (8) Residual phase angles. H and denote the residual for the single lifetime and Gaussian-distributedlifetime fits, respectively. (C) Residual demodulation factors. v and v denote the residual for the single lifetime and Gaussian-distributedlifetime fits, respectively. A,, = 351.1 nm; Aem > 385 nm.
bandpass filters spaced equally across the entire fluorescence contour were used and the frequencydomain data were acquired at each emission wavelength. Figure 5 presents several emission wavelengthdependent multifrequency phase (open symbols) and modulation (solid symbols) traces for native (upper panel) and silica-adsorbed BSA-Ac (lower panel). Clearly, the multifrequency phase and modulation traces at each wavelength are not superimposable,indicating that the fluorescence decay kinetics are strongly emission wavelength dependent. Global analysis" of the wavelengthdependent data (symbols) shows that a tripleexponential decay model gives a statistically adequate fit to the experimental data (solid curves). The recovered uppavent fluorescence intensity decay parameters for native, chemically denatured, and silica-adsorbed BSA-Ac, respectively, are available in the supplementary material. The single most important aspect of these data is that one of the preexponential factors (as)at the red edge of the emission become progressively more negative as the wavelength increases. This is unequivocal evidence for an excited-state reaction.46
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Figure 4. Phase-resolvedspectra for BSA-Ac. (upper panel) Static and phase-resolved spectra for native BSA-Ac. (lower panel) Static and PREWIF spectra for silica-adsorbed BSA-Ac. “Null” refers to electronic elimination of a fluorescent signal from a particular fluorescent A modulationfrequency of 50 MHz was used.
Figure 5. Emission wavelength-dependent multifrequency phase (open symbols) and demodulation (solid symbols) traces for native (upper panel) and silica-adsorbed(lower panel) BSA-Ac. The traces were obtained using the following bandpass filters: (native) 421 (0, O), 460 (v,v),and 500 nm (0, m); (silica adsorbed) 440 (0,O), 470 (v v),and 500 (0, m). le,= 351.1 nm. The solid curves denote the best fits to a three-component model.
The recovered wavelength-dependent fluorescence decay parameters (see supplementary material) are used to calculate the normalized timeresolved emission spectra. Figure 6 presents an abbreviated set of timedependent emission spectra for native (upper panel), chemically denatured (central panel), and silicaadsorbed (lower panel) BSA-Ac. Four features are readily apparent from these data. First, the timeresolved spectra for the acrylodan residue in BSA-Ac under these three sets of conditions are quite different from one another. Second, the time-resolved emission spectra of the acrylodan reporter group under all experimental conditions red shift following optical excitation. Third, the emission spectra of acrylodan withim native BSA-Ac is blue shifted compared to that of acrylodan within denatured BSA. Fourth, the emission spectra for BSA-Ac adsorbed to silica is blue shifted relative to native and chemically denatured BSAAc at all times following optical excitation. These results are in agreement with the steady-state result (vide supra); protein denaturation red shifts and silica adsorption blue shifts the emission spectrum of acrylodan within BSA-Ac (Figure 1). The kinetics associated with the acrylodan spectral shift are a measure of the rate of reorganization/dynamics within the cybotactic region. The actual timedependent shift (v(t))for native (-) , chemically denatured (. and silica-adsorbed BSA-Ac (- - -) are plotted in Figure 7 (upper panel) and reveal several interesting features. For example, there are dynamics surrounding the acrylodan reporter group within BSA-Ac regardless of the BSA environment (i.e., native, chemically denatured, or silica adsorbed). The center of gravity for the silica-adsorbed BSAAc is greater than that for native BSA-Ac. This result is consistent with our steady-state results (Figure l),showing that
BSA adsorption induces a significant blue shift in the emission spectrum of BSA-Ac in comparison to native BSA-Ac in buffer. The total timedependent shift of the emission for silica-adsorbed BSA-Ac is larger than that seen for native BSA-Ac. Thus, it appears that adsorption to bare silica actually increases the magnitude of the range of relaxation around the acrylodan reporter group in BSA-Ac. Compared to native BSA-Ac, the average center of gravity shifts more rapidly for BSA-Ac at the silica surface in the first few nanoseconds; however, the spectral shift is by far the fastest in chemically denatured BSA-Ac. Finally, all spectral relaxation is essentially over after about 4 ns. This result indicates that a new equilibrium between the surrounding environment and the acrylodan reporter group is reached within a 4ns time period. In order to provide a more quantitative comparison of the native, chemically denatured, and silica-adsorbed BSA-Ac, we calculated the D(t) function F i r e 7 , lower panel). These results more clearly illustrate two key aspects of the timeresolved spectral data. First, the overall rate of spectral evolution for silica-adsorbed BSA-Ac is clearly faster than native BSA-Ac, but slower in comparison to chemically denatured BSA-Ac. Second, a single exponential decay model (one rate process) is unable to accurately describe the experimental data well. A double-exponential decay model (two rate processes) fits the experimental data well and suffices to describe the spectral relaxation process. This indicates that at least two independent processes are responsible for the spectral relaxation of acrylodan within BSA-Ac. To determine the effects of temperature on the reorganization rates of the environment surrounding acrylodan within native BSA-Ac, wavelength-dependent MPMF data at 10 different
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Analytical Chemistry, Vol. 67, No. 1, January 1, 1995
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required to describe the spectral relaxation process of acrylodan withii BSA-Ac for all temperatures studied. Second, the recovered rates (kl and kz) are temperature independent (panels A and B) . It is known that Cys-34 is located in a crevice approximately 9 hi from the BSA surface and thus is somewhat protected from bulk waters5?Temperature independence of the recovered rates is consistent with the internal dynamics of the local dipoles and/ or amino acid residues within the cybotactic region surrounding acrylodan within BSA-Ac not coupling effectively to the bulk solvent. A similar type of result was reported by Pierce and Boxelss for the relaxation of 2'-(N,Ndimethylamino)-&naphthylCtrunscyclohexanoic acid bound to sperm whale apomyoglobin. Third, the relative contribution of these two rates to the total spectral relaxation is temperature dependent. The fractional contribution @I) associated with the faster relaxation process ( k l ) increases with increasing temperature.
Rotational Reorientation Kinetics of Acrylodan in Native
0.2
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16
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Figure 7. Recovered time-resolved emission center of gravity (upper panel) and o(r) function (lower panel) for native, chemically denatured, and silica-adsorbed BSA- Ac.
temperatures, ranging from 10 to 55 "C, were collected and analyzed. The results of these experiments are illustrated in Figure 8 and can be summarized as follows. First, two rates are 156 Analytical Chemistry, Vol. 67, No. 7, January 1, 1995
BSA-Ac. The time evolution of the acrylodan emission (Figures 6 and 7) could result from rotational reorientation of the fluorescent probe within the "pocket" surrounding Cys-34 in BSA and/ or the reorganization of the dipoles/amino acid residues within the local protein environment around the acrylodan residue. In an effort to determine whether the local motion of acrylodan itself within the BSA molecule plays a signiticant role in the observed spectral relaxation, multifrequency differential polarized phase H.;Chang, R; Kaplan, L.J. Biochim. Biophys. Acta 1975,400, 132-6. (58) Pierce, D.W.; Boxer, S. G.J. Phys. Chem. 1992,96,5560-6. (57) Hull, H.
Table 1. Effects of Temperature on the Recovered Rotatlonai Reorientation Kinetics of 1 pM BSA-Ac (Native) in 0.1 M Phosphate Butfer (pH 7.5)
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Rotational correlation times. Limiting anisotropy is always linked over temperature. Global ,y2 for the linked analysis. The local ,y2 values are not shown for clarity. dAnisotropy at time inhity. Fractional contribution of the global protein motion. f Semiangle for the cone in which the acrylodan precesses.
10
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Frequency (MHr)
Figure 9. Rotational reorientation of Ac in native BSA-Ac at 20 "C: (A) Multifrequency polarized modulation ratio. (8) Differential polarized phase angle. The lines represent the best fits using an isotropic (- - -), hindered rotor (.e.), or anisotropic rotor (-) models. (C and D) Corresponding residual plots (isotropic rotor, v; hindered rotor, v; anisotropic rotor, 0 ) .A,, = 351.1 nm; Aem > 385 nm.
angle and polarized modulation ratio data were obtained for native BSA-Ac at six different temperatures. In these experiments, a 385-nm long-pass filter was used to select the entire fluorescence emission contour. Figure 9 illustrates a typical set of results for 1pM native BSAAc at 20 "C. We present the polarized modulation ratio (panel A) and d~erentialpolarized phase angle (panel B) data and compare our experimental results (points) to several test models. Specifically, we show fits% to an isotropic rotor (- - -1, a "hindered rotor (. .), and an anisotropic rotor (-) model. For completeness,panels C and D present the correspondingresidual plots (isotropic rotor, r;hindered rotor, v; and anisotropic rotor model, 0). Clearly, the anisotropicrotor model with two rotational correlation times best fits our experimental data. Table 1 summarizes the complete analysis for the BSA-Ac system, and several points are readily apparent. First, at least two rotational correlation times are required to accurately describe the total decay of anisotropy for native BSA-Ac. Second, the recovered rotational correlation times decrease with increasing temperature. Third, the longer of the two rotational correlation times is attributed to the global motion of the entire BSA m~lecule.'~Fourth, the short rotational correlation time is
assigned to the local motion of the acrylodan reporter group within BSA. Finally, the local motion of the acrylodan residue within BSA-Ac is limited to a small cone with a semiangle around 23", and this semiangle does not change significantly with temperature. This result should be contrasted with our previous work on dansylated BSA14 in which the semiangle associated with the reporter groups increased significantly with temperature. Additional inspection of Table 1also shows that the rotational correlation time associated with the local motion of the acrylodan residue within BSA-Ac clearly decreases (Le., reorients more rapidly) by a factor of 2 with temperature. Thus, one might speculate that the rotational reorientation of the probe could contribute to the observed temporal relaxation F i e 7) seen in the BSA-Ac system. However, the recovered rates associated with the spectral relaxation (Figure 8) are temperature independent. These results do not indicate any clear correlation between the local rotational reorientation of the acrylodan reporter group and the observed kinetics associated with the time-dependent spectral shift in the BSA-Ac system. Thus, it appears that the local motion of the probe does not contribute significantly to the observed time-dependent spectral shift of acrylodan within BSAAc. The timeresolved spectral and decay of anisotropy data for native BSA-Ac can be summarized as follows. First, the rate at which the acrylodan can rotationally reorient is affected by temperature and the semiangle through which the acrylodan residue precesses remains constant with temperature. Thus, the acrylodan reporter group can rotationally reorient more rapidly at increased temperatures, but it remains "restricted" in this movement to a 23" semiangle. Second, the rates associated with dipolar relaxation around the acrylodan residue are independent Analytical Chemistry, Vol. 67, No. 1, January 1, 1995
157
E-S-K-H-T-DNHz I/
\A-KR-F-K-DLGEEH-F-K-GLV-
IC
Figure 10. Sketch of the local amino acid residues that make up the cybotactic region around Cys-34 in BSA. Cys-34 resides in loop 1 of domain 1. The residues in the immediate vicinity of Cys-34 (local environment) are shown in the lower section of the figure. No particular orientation of the residues is implied. Table 2. Recovered Kinetic Parameters for Spectral Relaxation of Acryiodan in BSA-Ac.
kinetic parameters k l b (ns-l) glc (%I kzb (ns-')
gzc(%)
bulk
(native BSA)
conditions surface (adsorbed BSA)
1.2 f 0.3 57 f 6 0.12 f 0.03 43 f 6
2.6 f 0.5 63 f 7 0.16 f 0.05 37 f 7
bulk (denatured BSA) 4.7 f 0.7 80 f 8
0.50 f 0.09 20 f 8
T = 22 f 2 "C. Spectral rehation times. Fractional contributions.
of temperature, but the fractional contribution of the faster of these rates increases with temperature. Together these results paint the following picture for native BSA-Ac: (1)increasing temperature causes the local protein environment around the acrylodan to change such that acrylodan is influenced more by the faster of two relaxation pathways; (2) the actual kinetics associated with 158 Analytical Chemistry, Vol. 67, No. 1, January 1, 1995
the individual relaxation processes are not influenced by temperature; and (3) acrylodan is able to reorient within the binding pocket more rapidly at elevated temperature, but this increased mobility does not influence the rate with which the local protein residues can relax the emission spectrum. In effect, the protein backbone shifts with temperature around a "rapidly flailing" acrylodan residue and the proximity of the dipolar amino acid residues and/or solvent molecules within the Cys-34 pocket ultimately controls the spectral evolution. The increased mobility of the acrylodan does not apparently influence the spectral evolution per se since its motion, albeit faster, is restricted to a fixed semiangle. Comparison of BSA-Ac in Merent Environments. Table 2 summarizes the recovered relaxation rates and their relative contributions for acrylodan within native, chemically denatured, and silica-adsorbed BSA-Ac. These data show several interesting features. For example, the two rate processes describing D(t)
generally differ by about 1order of magnitude. Protein denaturation results in an increase in both rates (kland k2, and an increase in the relative contribution of the fast rate (k3 to the total spectral shift. The environment around acrylodan within chemically denatured BSA-Ac reorganizes most rapidly following optical excitation. Finally, the faster overall relaxation associated with the silica-adsorbed BSA-Ac (Figure 7, - - -), relative to that of native BSA-Ac (Figure 7, -), only arises from an increase in the faster rate (k3;the slow rate (kz) and its fractional contribution are essentially unaffected by adsorption to a bare silica surface. CONCLUSIONS
The BSA-Ac system consists of a single emissive center. However, an excited-state reaction leads to substantial heterogeneity in the observed static fluorescence, and the acrylodan emission spectrum within BSA-Ac red shifts with time following optical excitation. This spectral relaxation process is due to dipolar relaxation of the local environment around acrylodan within BSA-Ac. Detailed analysis of the time-dependent data shows that two rate terms are required to describe the overall spectral relaxation process. Adsorption of BSA-Ac to bare silica increases one of these rates significantly; however, chemical denaturation affects all recovered parameters (Table 2). Thus, only one of two acrylodan “relaxation channels” is affected when BSA adsorbs to bare silica, but both are affected on chemical denaturation. The observed time-dependent spectral shifts are attributed to the reorganization of the local environment around acrylodan within BSA-Ac; the mobility of the probe per se does not affect the spectral dynamics. Temperature-dependent experiments indicate that the bulk solvent does not couple well to the cybotactic region around Cys-34 but does affect the actual rotational reorientation rate of the probe. Surface adsorption to bare silica induces changes in the conformational structure of BSA in such a way that the cybotactic region around the lone acrylodan group at Cys-34 encounters a less dipolar environment that is simultaneously more dynamical (potentiallyless rigid) compared to native BSA-Ac. It is ideal to determine the actual correlation between structure and dynamics of the local environment around acrylodan and to address what specific dipoleshesidues are responsible for the observed spectral relaxation seen in BSA-Ac. BSA consists of 582 amino acid residues,28 and Cys-34 resides in a pocket approximately 9 A from the exterior surface of B S 5 ’ The adjacent amino acid residues are Pro-35, Gln-33, Leu-80, Arg-81, and Glu-82.2* Side chains such as Glu-82 also appear capable of segmental motions which could participate in the “relaxation”of (59) He, X. M.; Carter, D. C. Nature 1992,358,209-15. (60) Takeda, K; Harada, K; Yamaguchi, K.; Moriyama, Y. J Colloid Interface Sci. 1994, 164, 382-6.
the environment around acrylodan and contribute to the observed spectral shift (Figure 10). Unfortunately, the X-ray crystal structure of BSA is not known. Thus, correlation between the recovered spectral relaxation rates and the dipolar residues is difficult to determine. The X-ray crystallographic structure of human serum albumin (HSA)is known to 2.0A re~olution,~~ and it too has only one free cysteine at position 34.28 Therefore, in terms of realizing a detailed conclusion regarding the “responsible” amino acid residues, acrylodan-labeled HSA appears to have more potential when compared to BSA. Current work in our group is centering on HSA-Ac to determine what specific amino acid residues withii the Cys-34 pocket could/do participate in the observed dipolar relaxation process. Additional experiments are focusing on probing the effects of “hydration”around BSA-Ac and HSA-Ac dissolved within a reverse micellem that in turn contain different levels of water within the core region. The results of these efforts will be reported in due course. Finally, this work demonstrates that one must exercise caution when using static fluorescence spectra to infer issues about local “polarity”, “hydrophobicity”, “hydrophilicity”, and “rigidity” in proteins in the bulk and at surface. The photophysics for what appear to be simple systems (e.g., BSA and acrylodan) are indeed much more complicated than can be fully understood from a static fluorescence spectrum. Thus, one should be concerned with the fact that the cybotactic region about a fluorescent reporter group can evolve with time and that the static emission spectrum observed is time averaged and/or a result of internal dynamics around the residue and alone provides limited information about the origin of a particular spectral feature. Time-resolved techniques, long used to study proteins in the bulk,12330942 are now becoming more common in the study of bios~rfaces25~~6~~-~~ and can provide one with a more detailed picture of the dynamics of proteins at biointerfaces. ACKNOWLEWMENT
This work was supported by the National Science Foundation (CHE9300694). SUPPLEMENTARY MATERIAL AVAILABLE
Tables (A-1-A-3) containing recovered emission wavelengthdependent impulse response functions for native, chemically denatured, and silica sorbed BSA-Ac (3 pages). Ordering information is given on any current masthead page. Received for review July 8, 1994. Accepted October 19, 1994.a AC9406890 Abstract published in Advance ACS Abstracts, November 15, 1994.
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