J . Phys. Chem. 1989, 93,7635-7640 more correct in assessing this aspect of the PES under study. On the other hand, its description of the damping function, which controls the general shape of the potential well, still needs some refinement. As a final remark, one should try to verify the reliability of IOSA cross sections in generating first moments like those of eq 8. In general, one knows, in fact, that the approximation tends to overestimate inelasticity especially in the region of large Aj. Thus, its use as a parameter for comparing different PES for the same system is ceAainly justified, while the analysis of the results for different systems and at different collision energies should at least remove the ES simplification before being carried out.
7635
Results along these lines will be reported e l ~ e w h e r e . ~ ~
Acknowledgment. We are grateful to Dr. A. Palma for his generous help with the plotting routines and with the VAX 8600 operating system. We also thank Dr. M. Venanzi for several useful discussions and for help with the IOSA cross sections. The financial support of the Italian Ministery of Education (MPI) is also gratefully acknowledged. Registry No. Ne, 7440-01-9; N1, 7727-37-9. (31) Bernardi, M.; Gianturco, F. A.; Palma, A.; Venanzi, M. Manuscript in preparation.
Raman Spectra and Water Absorption of Bovine Serum Albumin Barbara A. Boltont and James R. Scherer*.t Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Albany, California 94710 (Received: March 30, 1989)
Integrated optics and Raman spectroscopy are used in a study of the stepwise hydration of bovine serum albumin (BSA) films. Integrated Raman intensities of the amide I11 spectral region indicate an increase in a-helical content with increasing water concentration. Carboxylate site hydration is observed as low as 2% RH, half the sites are hydrated at 20% RH, and all the sites are hydrated by 90% RH. Hysteresis observed in the refractive index and calculated water concentration for BSA during an absorption/desorption cycle is attributed to changes in secondary structure and a small loss of void volume.
Introduction The importance of water in biological systems has prompted many studies of its interaction with macromolecules and macromolecular assemblies.14 Water activity is known to affect the folding, stability, and biological activity of globular proteins and a variety of experimental techniques have been used to form a coherent molecular picture of water-protein interactions.l-’ The hydration shell of a protein has been pictured as a layer of water interacting with the macromolecule (often referred to as “bound water”) which has distinctly different thermodynamic, kinetic, and spectroscopic properties from those observed for “bulk” water. Since dissimilar techniques often measure different properties, it is not surprising to find disparity in the detection of bound water. Few vibrational investigations of the nature of water-protein interactions at intermediate levels of water activity can be found in the literature. Proteins, usually in the form of films, were exposed to known levels of relative humidity and spectral measurements made.8-12 One method combined gravimetric and infrared techniques to generate spectra and sorption isotherms.12 Changes in the spectral characteristics of protein bands sensitive to conformation or water content, or the OH (OD) stretching bands of water, were used to monitor the protein/water interaction. It is generally accepted that proteins absorb water vapor at low humidity by binding water molecules to specific hydrophilic sites which, for water soluble globular proteins, are primarily a t the surface of the p r ~ t e i n . ’ ~As the humidity is raised water-water interactions increase. Some of the strongest hydrogen-bonding sites are polar groups, acidic side chains, and/or the backbone amide carbonyls. Bovine serum albumin (BSA) is a globular water-soluble protein whose primary structure (66 267 Da) is known.14 Many vibrational bands of BSA in the solid state and solution have been *Send correspondence to this author at 1309 Arch St., Berkeley, CA 94708. ‘Present address: Varian Instrument Group, 2700 Mitchell Drive, Walnut Creek, CA 94598. !Present address: University of California at San Francisco, School of Dentistry, Bldg. 158A, 1301 So. 46th St., Richmond, CA 94804.
assigned to vibrations of secondary structure and functional g r o ~ p s . l ~ These - ~ ~ studies have concluded that the amount of a-helix conformation increases in going from the solid state to solution. In addition, a new band, appearing in the solution spectrum at 1402 cm-l, was assigned to hydrated ionized side chain carboxyl groups.I8 Qualitative spectroscopic measurements of water associated with BSA have shown that the OH stretching bands occur in the same spectral region as those of liquid water.I5 Quantitative spectroscopic studies at levels of water activity between low humidity and saturation have not been done. Scherer et al. using the simultaneous application of integrated optics and Raman spectroscopy have devised a novel approach for investigating the absorption characteristics and state of hydrogen bonding of water (or ethanol) in thin (2 pm) cellulose (1) Membranes, Metabolism, and Dry Organisms: Leopold, A. C., Ed.; Comstock, Cornell University Press: Ithaca, NY, 1986. (2) Methods in Enzymology, Biomembranes Part 0,Protons and Water: Structure and Translocation;Packer, L., Ed.; Academic Press: New York, 1986, Vol. 127. (3) Finney, J. L. In Water, a Comprehensive Treatise; Franks, F., Ed.; Plenum: New York, 1979; Vol. 6, Chapter 2, p 47. (4) Ling, G N. In Water in Aqueous Solutions-Structure, Thermodynamics and Transport Processes; Home, R. A., Ed.; Wiley Interscience: New York, 1972; Chapter 16, p 663. ( 5 ) Rupley, J. A.; Gratton, E.; Careri, G. Trends Biochem. Sci. 1983, 18. ( 6 ) Cooke, R.; Kuntz, I. D. Annu. Rev. Biophys Bioeng. 1974, 95. (7) Kuntz, I. D.; Kauzmann, W. Adv. Protein Chem. 1974, 28, 239. (8) Riregg, M.; HLni, H. Biochim. Biophys. Acta 1975, 400, 17. (9) Susi, H.; Ard, J. S.;Carroll, R. J. Biopolymers 1971, I O , 1597. (IO) Poole, P. L.; Finney, J. L. Biopolymers 1984, 23, 1647. (1 1) Bendit, E. G. Biopolymers 1966, 4 , 539. (12) Careri, G.; Giansanti, A,; Gratton, D. Biopolymers 1979, 18, 1187. (13) Saenger, W. Annu. Rev. Biophys. Chem. 1987, 16,93. (14) Peters, T, Jr. Adu. Protein Chem. 1985, 37, 161. (1 5) Buontempo, U.; Careri, G.; Fasella, P. Biopolymers 1972, 11, 5 19. (16) Bellocq, A. M.; Lord, R. C.; Mendelsohn, R. Biochim. Biophys. Acta 1972, 257, 280. (17) Chen, M. C.; Lord, R. C. J . Am. Chem. Soc. 1976, 98, 990. (18) Lin, V. J. C.; Koenig, J. L. Biopolymers 1976, 15, 203. (19) Jakobsen, R. J.; Cornell, D. G. Appl. Spectrosc. 1986,40, 318.
This article not subject to US.Copyright. Published 1989 by the American Chemical Society
7636 The Journal of Physical Chemistry, Vol. 93, No. 22, 1989
Bolton and Scherer
i-
~M(E..E,)
'1
,
1.56
,
,
,
,
,
,
,
I
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n,= A i r n 2 = BSA F i l m
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IC
tFigure 1. Schematic diagram for controlled humidity waveguide cell. vi is the refractive index (for light polarized the TE or TM directions),@ is the incoupling angle, a is the prism angle, and t is the film thickness.
acetate films.2@26 Stepwise exposure of films to different levels of penetrant (water or ethanol vapor) and measurement of changes in the waveguiding characteristics of the film have provided information about the concentration of penetrant within the polymer film and the partial molar volume of penetrant as a function of penetrant concentration. Raman scattering obtained at different levels of penetrant concentration provided insight about the sites of penetrant/film interaction, hydrogen-bonding states, and molecular and/or morphological structure changes with penetrant absorption. Combining these two techniques provides useful information about mechanisms of penetrant absorption. The objective of the present investigation was to determine the waveguide characteristics of bovine serum albumin films over the full hydration range. In the following, we report measurements of the refractive index and film thickness as a function of water concentration and Raman measurements that monitor the incremental absorption of water by BSA films. We interpret the process of hydration with an analysis of four spectral regions: the amide I region (1500-1800 cm-I), the amide I11 region (1200-1350 cm-I), the OD (OH) stretching (2200-2800 cm-I), and the 1350-1500-cm-' vibrational region. We also present measurements of the integrated intensity of the amide 111 and carboxylate bands as a function of water concentration. The conclusions of the spectral analysis will be correlated with the results of our waveguide studies. Experimental Section Bovine serum albumin (BSA) having less than 0.005% fatty and used without acids was purchased from Sigma Chemical CO.~' further purification. BSA was dissolved in distilled water (27-29 wt%) and filtered ( 5 pm) to remove small particles. The resulting solution had a pH between 6.5 and 7.0. Films free of defects (dust, cracks, etc.) were fabricated by spinning the solutions on quartz or MgF, substrates at 2200-2600 rpm for 40 s. Films, typically 1.5-2.5 pm thickness, were dried for several hours in a stream of dry N, or under vacuum. The density of a dried BSA crystal, determined by the flotation method using benzene and bromobenzene, was found to be 1.278 g/cm3. The films were transferred to a humidifying cell and exposed to a continuous stream of dry nitrogen for 24 h. In the process of transfering, the films were exposed to ambient conditions (about 40-60% RH) for several minutes. Description of the humidifying cell, the assembly for holding the substrate, film and prisms, and a schematic diagram of a device which is capable of delivering 1.5 L/min of humidified N2gas have been reported.20 The relative humidity (RH),which was controlled to within 1% R H from 0 (20) Scherer, J . R.; Bailey, G. F. J . Membr. Sci. 1983, f3, 29. (21) Scherer, J . R.; Bailey, G. F. J . Membr. Sci. 1983, 13, 43. (22) Malladi, D. P.; Scherer, J. R.; Kint, S.; Bailey, G. F. J . Membr. Sci. 1984, 19, 209. (23) Scherer, J. R.; Bailey, G. F.; Kint, S.; Young, R.; Malladi, D. P.; Bolton, 8. A. J . Phys. Chem; 1985, 89, 312. (24) Scherer, J. R.; Bolton, B. A. J . Phys. Chem. 1985,89, 3535. (25) Bolton, B. A.; Kint, S.; Bailey, G. F.; Scherer, J. R. J . Phys. Chem. 1986, 90, 1207. (26) Bolton. B. A.; Scherer, J. R. J . Phys. Chem. 1986, 90, 1211. (27) Reference to a company and/or prbduct named by the Department is only for purposes of information and does not imply approval of the product to the exclusion of others which may also be suitable.
1.53-
I .52
~
r
1 soo
10
20
"
"
30
50
44
60
70
BO
Po
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% RH 1
"
"
'
I
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1.82 1.92
Figure 2. Average refractive index ((2nTE+ nTM)/2)and film thickness, t in km,as a function of increasing relative humidity. The dashed line
gives the results for subsequent dehydration. to 99% RH, was measured with a probe (Vaisala-HMP 310T) that was periodically calibrated against known humidities of saturated salt solutions.28 A detailed description of the waveguiding technique has been given previously.20 Briefly, the technique, illustrated in Figure 1, uses the protein film as an optical waveguide which propagates a focused laser beam within the film. The humidity chamber is placed over the protein film (index of refraction nz) which is supported on a substrate (index of refraction n3). A high-index prism couples a polarized (TE or TM) beam into the film at angles which satisfy the phase-matching constraints of a beam undergoing total internal reflection at the film/air and film/substrate interfaces. The refractive index n, and film thickness t are calculated from the measured incoupling angles, CP. The goniometer used to inject the laser beam into the BSA film has been describedS2' Light scattered from the m = 1 propagating mode was analyzed with a Spex 1401 double monochromator with a spatial filter and holographic gratings. The resolution at the 514.5-nm Ar' exciting line was =5 cm-I and the beam power a t the prism coupler was set between 20 and 50 mW. The data acquisition system has been described p r e v i o ~ s l y . ~ ~ Results and Discussion Refractive Index and Thickness Measurements. Films of BSA were exposed to relative humidity increasing from 0 to 100%RH. A plot of the average refractive index (ii = (2nm + nTM)/3)versus percent relative humidity (up to 92% R H ) for a 1.42-pm BSA film is shown in Figure 2. We observed an initial increased in R which reached a maximum by 10% RH. As the film was subjected to higher % RH, ii decreased with a more rapid drop observed above 50% RH. Also shown in Figure 2 is the refractive index for the stepwise dehydration (dashed line) of BSA from 92% RH. The dehydration curve lies approximatey 10%(below 70% RH) than the hydration curve. Furthermore, the film resulting from dehydraton (48 h) has a lower refractive index than that of the initially prepared film. The film thickness (Figure 2) increases rapidly up to 5% RH, then gradually increases (almost linearly) between 5 and 50% RH. Above 50% RH, the film expansion appears to be exponential, (28) Greenspan, L. J . Res. Nail. Bur. Stand. 1977, 81A, 89. Scherer, J. R. Appl. Spectrosc. 1976,30, 281. (29) Kint, S.; Elsken, R. H.;
Spectra of Bovine Serum Albumin
The Journal of Physical Chemistry, Vol. 93, No. 22, 1989 7637
reaching a value of 44% at 92% RH, a point beyond which it becomes tacky and loses its ability to propagate the laser beam without severe attenuation. The shape of the refractive index curve: : ’ ’ ’ ‘ 5 ’ can be explained by the following. The small increase in the value a of ii at low humidity is caused by a small increase in the effective v) a density of the film. At high concentration, the film expands and the refractive index decreases because the effective density of the film decreases. We also observe a hysteresis in film thickness with decreasing m humidity (dashed line). The dehydration curve lies approximately 5% above the hydration curve (midrange) and the dried film (48 h) is approximately 2% thicker than the initially prepared film. The hysteresis could be explained by the following processes: water 0 10 20 30 40 50 60 70 80 90 100 is more strongly bound in the hydrated BSA molecule than in the % RH dehydrated molecule (Le., water is harder to remove once absorbed); water has caused some structural change (i.e., new or Figure 3. Sorption isotherm derived from the smoothed curve in Figure stronger bonds between selective functional groups) altering the 2. Solid curve, hydration; dashed curve, dehydration. refractive index and thickness independent of the water content; and, water has altered the chemical composition of the BSA film. observe. Thus, we feel our calculated isotherm is reliable within This last mechanism has been shown to be in~a1id.I~ The value the range of 0-90% RH. of ii at maximum dehydration (1 S49.5) is the same as the value The isotherm in Figure 3 shows that, between 0 and 3% RH, of ii at 47% R H on the hydration curve, and the film thickness the water concentration increases sharply with a fairly distinct at 0% R H on the dehydration curve is the same as that at 7% R H “knee” occurring at 3% R H (1 13 HzO/BSA). It continues to on the hydration curve. After dehydration, the final state of the increase almost linearly between 7 and 65% R H (535 H20/BSA), BSA film at 0% R H has a lower refractive index but a larger and exponentially above 65% RH. The dehydration curve (dashed volume. Dilatometric and refractometric studies on poly-Lline) at 4 0 % R H is about 13% higher than the hydration curve. glutamic acid have shown an increased volume and a decreased The general shape of this sorption isotherm has been observed refractive index associated with a coil to helix t r a n s f ~ r m a t i o n . ~ ~ for many macromolecules (polymers and proteins). One explaThese results strongly suggest that a conformational change has nation suggests that the shape results from two separate sorption occurred during hydration which is not reversible on dehydraton. processes.34 One process is dominant at low % R H and involves We note that these films have already undergone partial hydration unimolecular sorption of water a t specific sites. The knee of the (to ~ 5 0 % RH) and dehydration before the hydration measurecurve has usually been associated with monolayer coverage. The ments shown in Figure 2 were made (see Experimental Section). other process occurs at higher R H and involves the formation of As we shall see in a later section, Raman spectra show small multilayers with extensive water-water interactions. conformational changes associated with the hydration/dehydration In the following we estimate the amount of water necessary cycle. to give monolayer coverage of the exterior of the BSA molecule. Isotherm and Partial Molar Volume. In our studies on cellulose We model the “stubby cigar-shaped” albumin molecule as a we calculated the concentration of water (or ethanol) cylinder with a heights of 141 A and a diameter of 41.6 A.14The within the film from the observed average refractive index (ii) surface area is 2.1 X lo4 A2 and the volume is 1.9 X lo5 A’. With and thickness ( t ) of the film using the Clausius-Mosotti equation the dimensions of a water molecule from the ice lattice to approximate the height and area of a water molecule (3.6 A and 8.4 .&231), the surface of the BSA molecule could accommodate 2.5 X lo3 water molecules. (In this hypothetical icelike surface, half of the waters could be hydrogen bonded to sites above the where k is a constant and C is the concentration of water (w) surface and half could be hydrogen bonded below the surface. The and the BSA (f) in g/cm3. %e BSA concentration is related to c axis is taken perpendicular to the plane of the surface.) From the ratio of the thickness at dryness ( t o ) to the thickness at i% the isotherm in Figure 3, we calculate that between 4 and 10% R H and the density at dryness do (i.e., C,= (fod,,)/li),and the R H (the “knee”), there are 115-158 HzO molecules per BSA constant K is the specific refraction which is related to the momolecule. It is clear that there are insufficient water molecules lecular polari~ability.~~ We note that the specific refraction for to give “monolayer” coverage of the entire BSA molecule and that water is observed to be approximately constant for liquid, solid, monolayer coverage refers only to site hydration. There are 185 and gaseous states.31 Density data for BSA in different structural possible charged sites on the BSA molecule, 100 of which involve conformations is not available. However, assuming that the COO- groups.14 This suggests that there are more charged sites specific refraction of BSA is invariant, the observed decrease in than there are water molecules at monolayer coverage. (This does refractive index on dehydration (0.003) implies a density decrease not preclude that there may be certain sites that have much higher of 0.005 g/cm3. In Figure 3 we show the calculated water conhydration levels at very low RH.) At 70% R H (607 H,O/BSA) centration versus the % relative humidity. We have compared and 90% RH (1204 H20/BSA) the average water coverage is this calculated isotherm to isotherms obtained from gravimetric 3.3 and 6.5 molecules per charged site. Kuntz and Kauzmann’ measurements of samples exposed to different relative humidiindicate that ionic groups can bind 4-8 waters at relative huBoth the general shape of the sorption isotherm and the midities above 70% RH. In a later section we present spectroscopic degree of hysteresis are comparable to those observed in this study. evidence that 90% of the COO- sites are hydrated at 75% RH. The values of the water vapor pressure over BSA samples as a The partial volume of water may be determined from the defunction of water content reported by Fuller and Brey3*are within pendence of thickness with c ~ n c e n t r a t i o n ~ ~ +0.008 g of H20/g of BSA of our calculated values obtained from the smoothed curves shown in Figure 2. However, the sorptionK(A3/mo1) = (18 x 1 0 ~ ~ / ~ ) s ( t ~ / t , , ) / 6 (2) ~ ~ ~ desorption isotherms reported by Benson and Richardson33are where C,l = Cw(ti/to)gwater/cm3 dry BSA and N is Avogadro’s both approximately to 0.2 g H 2 0 / g of BSA lower than what we number. Figure 4 shows a plot of the partial molar volume of water versus concentration (Cw’) for hydrated and dehydrated (30) Noguchi, H.;Yang, J. T. Biopolymers 1963,I , 359. (dashed line) BSA films. We note that, a t zero concentration, (3 1) Oxford (32) (33)
Eisenberg, D.;Kaufmann, W. The Structure and Properties of Water; University Press: New York, 1969. Fuller, M. E., 11; Brey, W. S., Jr. J . Biol. Chem. 1968, 243, 274. Benson, S.W.; Richardson, R. L. J . Am. Chem. Soc. 1%5,77,2585.
(34) Rogers, C. E. In Engineering Design for Plastics; Baer, E., Ed.; Reinhold: New York, 1964; Chapter 9, p 609.
7638 The Journal of Physical Chemistry, Vol. 93, No. 22, 1989 H ~ O mol /SA
Bolton and Scherer
I
mol
BSA FILM OZRH
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1400
1300
1200
1100
C,(ti/t.)
c m-l
Figure 4. Partial molar volume of water as a function of water concentration in the film. Units of Cw(ti/t,-,)are g/cm3 dry BSA.
Figure 5. Raman spectra in the amide I11 region of a BSA film at 0% RH, BSA in D20solution, and difference spectra (100% RH or 0% RH - D20solution) showing the contribution from a-helix and random coil.
Pi has a value of 15.2 A3/molecule in a dry BSA film hydration. Since the calculated van der Waals volume of a water molecule is 18.0A3(calculated from van der Waals radii of 1.20 A for H, 1.52 A for 0 of water, 1 .SO A for 0 of a carboxylate hydrogenbonding site; bond length of 0.96 A for 0-H;and a distance between Ow,,, and OCm- of 2.8 A calculated from a correlation of the uncoupled O D oscillator and 0-0distance by Falk35*36), we conclude that 7=3 A3/molecule of void volume is used by a water molecule initially entering the film. (We note that, if the molecular volume of water were larger than 18 A? an even larger void volume would be required to account for the ojserved q.) We interpret the increase in 6 up to the point here 6 = Vw(van der Waals volumes of 18.0 A3) as resultin from a depletion of void volume available to water. Above 18 the increase in can be attributed to an increase in the number of water molecules hydrogen bonding with each other, thereby taking additional space (larger 6 ) because of directed hydrogen bonding. At a water concentration of 0.14 g/cm3 and above, 8 reaches a limited value of 29.2 A3, indicating that water is binding to other water molecules instead of BSA (more like "bulk water"). With dehydration, the partial molar volume decreases more slowly with 8 = 18.6 A3/molecule for the dry film. These results suggest that the small void spaces present in the dry BSA film disappear with desorption. Rao and Das3' have suggested that the observed hysteresis in the first and successive sorption/desorption cycles of gelatin, egg albumen, and casein is a result of the gradual collapse of cavities. Secondary Structure. The secondary structure of polypeptides is controlled by hydrogen-bonding between peptide subunits. Information about the conformation structure of a protein can be obtained from observed frequencies and intensities of the amide I band ( C 4stretching vibration); the amide 111 band (mixture of C-N bond stretching and in-plane N-H bond bending); and the 900-1000-cm-' skeletal vibration regions. The amide I band of BSA (observed at 1663 cm-l with a full width at half-height, fwhh, of 57 cm-I at 0% RH) shifts to lower frequency and becomes narrower with increasing water content (90% RH, 1658 cm-l fwhh = 49.5 cm-I). The band is observed at 1656 cm-I in a 4 wt % solution. We used a concentrated solution of guanidine hydrochloride in the presence of 2-mercaptoethano13*to denature BSA and the Raman spectrum showed the amide I band at 1678 cm-I (fwhh = 64.5 cm-I). The shift of the amide I band to lower frequencies in the film could arise from an increase in a-helix content of the protein molecule with increasing hydration39and/or hydrogen bonding of the amide carbonyls with water.
i3,
vi
(35) Falk, M.In Chemistry and Physics of Aqueous Gas Solutions; Ada m , W. A., Ed.; Electrochemical Society: Pennington, NJ, 1975; p 19. (36) Scherer, J. R. In Advances in Infrared and Raman Spectroscopy; Clark, R.J. H., Hester, R. E., Eds.; Heyden: London, 1978; Vol 5, Chapter 3, p 149. (37) Rao, K. S.; Das, B. J. Phys. Chem. 1968, 72, 1223. (38) Tanford, C.; Kawahara, K.; Lapanje, S. J. Am. Chem. Soc. 1967, 89, 729. (39) Tu,A. T.Raman Spectroscopy in Biology; Wiley: New York, 1982.
b -
c,
E -
a -
c
c -
UQO
1300
1200
cm" Figure 6. Raman difference spectra (previous figure) of spectra at 100% RH, (c) and 0% RH (b), and a spectrum of denatured BSA solution (a) in the amide 111 region.
Protein conformation is more easily estimated from the analysis of the intensities in the amide 111 region (1320-1200 cm-I). A spectrum of the amide 111 region of a dry BSA film (0% RH) and a background of the amide 111 region without the amide 111 bands (28 wt %) BSA in DzO)are shown in Figure 5. The 1280and 1237-cm-I bands are associated with a-helix and random coil conformations and are shifted to lower frequencies on deuterat i ~ n .The ~ ~ Raman spectrum of the deuterated protein was subtracted from the BSA film spectra by using the integrated intensity of the breathing vibration of the phenyl ring (a1004 cm-') as an internal standard. The difference spectra (BSA film - BSA D20solution, smoothed with a 21-point cubic polynomial" is shown in Figure 5. This spectrum has a doublet which may be assigned to vibrations of the a-helical and random coil structures. An estimate of the a-helical content at 100 and 0% RH was obtained by subtracting the spectrum of the random coil (denatured BSA), shown in Figure 6a, from the difference spectra in Figure 5 (Figure 6, b and c) and integrating the intensity between 1320 and 1260 cm-I (linear base line between these points). We find the dry film contains 34% helix and 66% random coil. As the water content within the film increases, the amide I11 integrated intensities increased, indicating an increase in helical conformation. At 100% R H (Figure 6c), the intensity ratio corresponds to 45% helix and 55% disordered structures. Using the same procedure, our estimate of the two conformations in solution (4 wt 7%)was 54% helix and 46% disordered. These later values agree with those obtained from optical rotation dispersion measurements of BSA solution, i.e., 55% helical and 45% random coi1.4 I (40) Savitsky, A.; Golay, M. J. E. Anal. Chem. 1964, 36, 1627. (41) Shechter, E.; Blout, E. R. Proc. Natl. Acad. Sci. USA 1964, 51,695.
The Journal of Physical Chemistry, Vol. 93, No. 22, 1989 7639
Spectra of Bovine Serum Albumin
I
-
6 . .
61-OX 31
*'ii
RH
- o x nn
17-OX
I((
1-OXIH I
1500
1400
368
.1
I
Figure 7. Raman spectra of the hydrated carboxylate ion at different % RH (a) and difference spectra (spectrum at % RH - spectrum at 0% RH) (b).
The intensity of the skeletal C-C stretcing mode near 940 cm-' has been correlated with cy-helical content.'* We observe that the intensity of this band increases by 43% from 0% R H to 100% R H which also supports the conclusions drawn from our analysis of the amide I and amide I11 regions. When the film was dehydrated (0% RH) we found (from amide 111 intensities) a higher amount (=40%) of cy-helix than what was present initially. After one hydration/dehydration cycle the helical content was 6% more than the initial film which is in agreement with the lower refractive index and greater film thickness obtained from our waveguide measurement. Sites of Hydration. Raman difference spectra can be used to determine small differences in protein conformation as well as the location of sites of water/protein interaction with increasing water content. Figure 7a shows spectra of BSA films in the 1400-cm-' spectral region from 0% to 64% relative humidity. Again, the 1004-cm-' band was used as an internal intensity standard. We note that the intensity of the doublet at 1450 cm-l increases, and a new band forms at 1406 cm-I, with increasing hydration. The 1450-cm-I doublet is attributed to CH2 scissoring and CH3 bending modes from the side chains of aliphatic groups such as alanine, valine, or leucine.42 We have no explanation for the increase in band intensity with increasing hydration, observed as low as 2% RH, but it may reflect the increasing hydrophilic environment of these side chains. The difference spectra (wet BSA film - dry BSA film) at several humidities is shown in Figure 7b. The band at 1406 cm-' has been assigned to the symmetric stretching mode of the ionized side chain carboxyl (COO-) of glutamic and aspartic acids.I8 This band is seen distinctly at 2% R H and the intensity increases with water content. It is believed that one of the first events of protein hydration involves ionization of acid side chains and that these residues are saturated by water at about 0.30 g of H 2 0 / g of p r ~ t e i n . ~To ?~~ follow the hydration of the carboxylate group we obtained the difference spectra of several BSA films at increasing humidity. First, we determined the integrated intensity of the carboxylate group (1380-1435 cm-I) in a 4 wt % solution. This solution spectrum was scaled and a dry BSA film spectrum substracted from it. The integrated intensity of the 1406-cm-' (1380-1430 cm-I) band was taken to represent maximum carboxylate hydration. Next, the integrated intensities (1406 cm-l) of several BSA difference spectra (wet film - dry film) at varying water content were determined. The ratio of these integrated intensities to the intensity of the 4% solution band reflects the fraction of hydrated sites. The % of hydrated carboxylate residues versus water concentration for several BSA films are shown in Figure 8. We note that 50% of the COO- groups (50 sites) are hydrated at 0.07 g of H 2 0 / g of BSA (approximately 20% RH, 258 H,O/BSA) which, if all waters are associated only with these groups, implies the association of about 5 waters per site. This (42) Thomas, G . T., Jr.; Prescott, B.; Day, L.A. J. Mol. Biol. 1983,165, 321. (43) Bull, H. B.; Bresse, K. Arch. Eiochem. Eiophys. 1968, 128, 488.
B mol S
736
1104
A
1472
I
l!!LA
200
.2
.3
.4
.5
g H20/ g BSA
1300
"1
H ~ ~O ~
Figure 8. % of COO- sites which are hydrated as a function of the water concentration in the film.
2800
2400
2000
cm"
Figure 9. Raman difference spectra of D20(% RH spectrum - 0% RH spectrum) in BSA films at different humidity. The upper spectrum is
that of liquid D20at ambient temperature.
level of site hydration is constant down to 3% RH. This result is in accord with previous determinations of 4-8 waters for charged group sites.' At 70% R H (0.17 g of H 2 0 / g of BSA), the ratio of water to COO- sites is about 7:l and at 90% R H (0.3 g of H*O/g of BSA) it is about 11:1. If the maximum site hydration involves 4-8 waters/site, other sites must be involved in hydration above 70% RH. While there are other classes of hydrophilic groups available for potential water interaction (i.e., aliphatic or aromatic hydroxyls and amino groups), we could not extract direct evidence of such interaction from our experimental data. We note no apparent change in intensity or frequency of the N-H stretching (amide A) at low hydration levels (
