Langmuir 1987,3,433-438 the a-Ni(OH)2to 7-NiOOH transition. Other questions to be answered are the role of Co(OH12and the ingress of electrolyte cations into r-NiOOH. The EXAFS technique should yield valuable new information on these effects.
to Dr' D' Acknowledgment' We Koningsberger (Eindhoven University, The Netherlands) for providing the programs for data analysis and for giving guidance on their use. We are grateful to Dr. L. M. Moroney and C. Y. Yang (North Carolina State University)
433
for their help and discussions. This research was carried out at the National Synchrotron Light Source, Brookhaven National Laboratory, which is supported by the Department of Energy, Division of Materials Sciences and Division of Chemical Sciences. The Department af Energy, Division of Materials Sciences, provided operation funds for beam line x-11under Contract DE-1C05-80ER10742. Registry No. Ni(OH)2,12054-48-7;NiOOH, 12026-04-9;Co(OH),, 21041-93-0; NiO, 1313-99-1.
Intrinsic Fluorescence Characteristics of Apomyoglobin Adsorbed to Microparticulate Silica C. H. Lochmuller* and Steven S. Saavedra P. M. Gross Chemical Laboratory, Duke University, Durham, North Carolina 27706 Received September 3, 1986 The intrinsic fluorescence of sperm whale apomyoglobin was utilized as the probe in a study of protein adsorption, desorption, and conformational behavior on a series of microparticulate silica ,gels. The results indicate that adsorption is rapid and largely irreversible. The degree of protein unfolding that takes place upon adsorption is dependent on the pH of the contact buffer. The tryptophans in the pH 4.0 surface conformer are more exposed and interact to a greater extent with the interfacial environment than in the pH 7.5 surface conformer. Partial, reversible refolding of the protein in the sorbed state is observed with a change in the pH of the contact buffer. Introduction The interaction of proteins with solid surfaces is a subject of considerable interest in several fields, such as biomedical engineering, controlled release of pharmaceuticals, and liquid chromatographic separations. The two principle foci of research in this area have been quantification of the amount of adsorbed protein and characterization of the changes in protein conformation, if any, accompanying the sorption process. Evidence of altered protein structure in the adsorbed state has been reported by several groups. Walton and co-workers utilized circular dichroism (CD) to show that the a-helical content of albumin and fibrinogen decreases after long adsorption times on polyamino acids' and that clotting factor XI1 undergoes a major reorganization when adsorbed on quartz.2 Iwamoto et al. correlated a shift in the total internal reflectance fluorescence spectrum of fibronectin sorbed on hydrophobic silica with a change in protein c~nformation.~ Attenuated total reflectance fourier transform infrared spectroscopy has been used by Castillo et ale4and Gendreau et al.5to show that albumin conformation is altered after adsorption for long periods to soft contact lens material and germanium crystals, respectively. A series of studies by Karger, Benedek, and coworkersH have reported on the properties of proteins eluted from (1) Soderquiet, M. E.; Walton, A. G. J. Colloid Interface Sci. 1980, 75, 386-397. (2) McMillin, C. R.; Walton, A. G. J. Colloid Interface Sci. 1974,48, 345-349. (3) Iwamoto, K.; Winterton, L. C.; Stoker, R. S.; Van Wagenen, R. A.; Andrade, J. D.; Mosher, D. F. J.Colloid Interface Sci. 1986,106,454-464. (4) Castillo, E. J.; Koenig J. L.; Anderson, J. M.; Lo, J. Biomaterials 1984.5. ~ ,. -,319-325. -.- - ~ - ~ ( 5 ) Gendreau, R. M.; Leininger, R. I.; Winters, S.; Jakobsen, R. J. Adu. Chem. Ser. 1982, No. 199, 371-394.
(6) Cohen, S. A.; Benedek, K. P.; Dong, S.; Tapuhi, Y.; Karger, B. L. Anal. Chem. 1984,56, 217-221. (7) Benedek, K.; Dong, S.; Karger, B. L. J. Chromatogr. 1984, 317, 227-243.
(8)Lu, X. M.; Benedek, K.; Karger, B. L. J. Chromatogr. 1986,359, 19-29.
0743-7463/87/2403-0433$01.50/0
reversed-phase liquid chromatography (RPLC) columns. Multiple and/or distorted peaks were seen to arise from injection of a single protein. Analysis of the peaks by measurements of UV absorbance and enzymatic activity showed that partial denaturation, which was attributed to adsorption on the silica packing material, had occurred. Interpretation of the results is complicated, however, due to the low pH and presence of organic modifier in the mobile phase. In contrast, results from these same studies and others could be interpreted as evidence that adsorbed proteins can retain native secondary tertiary, and quaternary structure. Walton and Maenpag found that the fluorescence spectrum of albumin adsorbed to copolypeptide substrates did not differ from the solution spectrum. McMillin and Walton2 reported that the CD spectrum of fibrinogen on quartz is unchanged from that in solution. Native proteinslOJ1 and a synthetic, dimeric peptidelo have been recovered from hydrophobic interaction chromatography and active papain6 has been purified by RPLC. However, these sometimes conflicting results are not surprising when one considers variations in (a) the lability of the proteins investigated; (b) the physical and chemical properties of the sorbent; (c) the composition, ionic strength, and pH of the contact solvent; and (d) the experimental technique employed. In addition, the degree of reorganization in the sorbed structure may range from minor6 to substantial,2 may be a relatively slow process,l and may be reversible.8J2 The latter phenomenon particularly complicates the characterization of proteins eluted from chromatographic columns due to the possibility that a fast, reversible change in conformation may occur after (9) Walton, A. G.; Maenpa, F. C. J. Colloid Interface Sei. 1979, 72, 265-278. (10) Ingraham, R. H.; Lau, S. Y. M.; Taneja, A. K.; Hodges, R. S. J. Chromatogr. 1985,327, 77-92. (11) Wu, S.; Benedek, K.; Karger, B. L. J. Chromatogr. 1986, 359, 3-17. (12) Hearn, M. T. W.; Hodder, A. N.; Aguilar, M. I. J. Chromatogr. 1985,327,47-66.
0 1987 American Chemical Society
434 Langmuir, Vol. 3, No. 3, 1987
Lochmiiller and Saauedra
Table I . Characteristics oP Microparticulate Silica Gels" particle size, surface area, pore diam, % silica Cm t m2/g A typeb carbon encapped A 10 323 93 0.9 B 10 60 300 monomeric butyl 2.4 Yes C 5-6 350 70-80 monomeric octadecyl 14.6 Yes D 10 323 93 polymeric octadecyl 17.4 no E 10 323 93 polymeric octadecyl 18.5 Yes " Carbon analyses performed by M-H-W Laboratories, Phoenix, AZ; remainder of the information provided by the suppliers. * Indicates functionality (mono or trichloro) and chain length of silane used to chemically modify the silica surface. desorption but prior to detection. The paucity of data describing the structure of adsorbed proteins, particularly on the microparticulate silica supports used in RPLC, has led us to apply techniques previously developed in this lab~ratory'~ to study the intrinsic fluorescence behavior of apomyoglobin sorbed to these materials. This report is a continuation of a preliminary paper14and reinforces the conclusions drawn there. The results show that apomyoglobin undergoes a relatively fast conformational change upon adsorption on both hydrophilic and hydrophobic silicas and that the extent of structural reorganization is dependent on the pH of the contact buffer. The existence of at least two surface conformers, which differ in their degree of interaction with the sorbent, has been established. Partial interconversion between them can be induced by a change in the pH of the contact buffer.
Experimental Section Materials. All materials were reagent or spectrophotometric grade and, except myoglobin, were used without further purification. Sperm whale myoglobin was purchased from Sigma, St. Louis, MO. PRODAN (6-propionyl-2-(dimethylamino)naphthalene) was purchased from Molecular Probes, Junction City, OR. Buffers were prepared with 20 mM NaH2P04. Solvents and buffers were passed through a 0.45-pm filter before use. The microparticulate silica substrates were underivatized Whatman Partisil 10 (A), Hypersil Butyl WP-300 (B), Du Pont Zorbax ODS (C), and Whatman Partiail10 ODs-2 0); a fifth silica (E) was prepared by endcapping D with a 5 M excess of trimethy1~hlorosilane.l~ Silica A was drawn from the Duke Standard Substrate Collection while the others were obtained commercially. Apomyoglobin Preparation. Heme extraction was performed by using the methyl ethyl ketone method.15 Stock solutions were prepared by reconstituting the protein in buffer and adjusting the pH to 4.0, followed by filtration through a 0.45-pm filter. The absolute concentration of apomyoglobin stock solutions and the efficiency of heme removal were assessed spectrophotometrically by using published molar absorptivities.ls Sample solutions were prepared by dilution with buffer or mixed solvent to a final protein concentration of 2-4.8 pM. Apparatus. Technical fluorescence data were acquired with a Perkin-Elmer Model MPF-66 spectrofluorimeter. Excitation and emission monochromator slit widths were set at 3 nm. Apomyoglobin samples were excited at 295 nm, PRODAN samples at 363 nm. Except where noted, the excitation shutter was closed between measurements of adsorbed protein fluorescence to prevent excessive photodegradation. Background subtraction was necessary in all cases except PRODAN solution spectra. Signal to noise was enhanced by signal-averaging multiple emission scans and application of a smoothing function to the resulting spectrum. Samples were thermostated at 22 O C . A syringe pressurized with a mechanical drive was used to pump buffers and protein solutions through the quartz column flow cell.I3 (13)Lochmiiller, C.H., Colborn, A. S.; Hunnicutt, M. L.; Harris, J. M. Anal. Chem. 1983,55, 1344-1348. (14)Lochmtiller, C.H.; Saavedra, S. S. J. Am. Chem. Soc. 1987,109, 1244-1245. (15)Antonini, E.; Rossi-Bernardi,L.; Chiancone, E. Methods in Enzymology: Hemoglobins; Academic: New York, 1982;Vol. 76,pp 74-75. (16)Harrison, S.C.;Blout, E. R. J . Biol. Chem. 1965,240, 299-303.
I
4%
440
4Y)
YD
470
ml
J
490
WAVELENGTH (nm)
Figure 1. Fluorescence spectra of PRODAN adsorbed to silicas A-E from saturated pH 4.0 buffer. The heights do not reflect relative intensity. The cell was dry packed under vacuum applied to the outlet. The packing was equilibrated by pumping 5 mL (57 empty column volumes) of buffer through the cell. After each measurement, the cell was emptied and repacked with fresh silica. Apomyoglobin Adsorption and Desorption. Adsorption from pH 4.0 buffer was followed by continuously monitoring the change in 340-nm emission intensity as a 4.7 pM solution was delivered to the flow cell at 0.1 mL/min. The cell was packed to a height equal to the top of the excitation light path. Desorption was measured as follows. After the background intensity was recorded, the excitation shutter was closed and a solution of 4.5 pM apomyoglobin in pH 4.0 buffer was pumped through the cell at 0.1 mL/min until the sorptive capacity of the silica was attained. Flow was resumed from a syringe filled with 5 mL of fresh buffer while the intensity at 340 nm was measured periodically. Spectra of Adsorbed Molecules. Apomyoglobin was adsorbed by pumping a solution ranging in concentration from 2.6 to 4.7 pM through the flow cell at rates ranging from 0.1 to 0.4 mL/min. The low concentrations were necessary to minimize the contributionto fluorescence emission from the protein in the void volume. The volume paased through the cell was varied depending on the sorptive capacity of the particular silica. When adsorption had reached 70-100% of capacity, flow was stopped and the fluorescence spectrum acquired. Additional spectra were acquired periodically for up to 18 h. In other experiments, the dynamic behavior of the protein in the sorbed state was examined. The procedure employed in these instances has been described previ~usly.'~ Fluorescence spectra of adsorbed PRODAN were also acquired. A saturated solution buffered at pH 4.0 was pumped through the cell at a rate ranging from 0.1 to 0.4 mL/min while emission intensity, indicating probe sorption, was monitored. When sufficient PRODAN was adsorbed to enable measurement, flow was stopped and the spectrum recorded.
Results and Discussion Properties of Silica Substrates. Characteristics of the silica gels are presented in Table I. On the basis of carbon content, they range from hydrophilic (A) to very hydrophobic (E). However, we felt a better measure of relative surface polarity would be provided by examination of the fluorescence characteristics of an adsorbed probe
Langmuir, Vol. 3, No. 3, 1987 435
Fluorescence of Adsorbed Apomyoglobin Table 11. Fluorescence Emission Maxima of Molecules in Solution and Adsorbed to Silicaa apomyoglobin emission PRODAN max, nmg emission max, mediumb nm' pH 4.0 pH 7.5 water 522.2 f 0.d 335.4 f 0.2 330.4 f 0.4 50% methanol 509.5 339.7 50% acetonitrile 501.5 339.5 50% propanol 501.2 339.3 silica A 480.0 f 4.5 337.8 f 0.8 335.6 f 0.3 silica AC 340.0 f 0.8 338.1 f 0.5 silica B 476.6 f 7.3 337.1 f 0.5 336.9 f 0.8 silica C 459.0 f 3.5d 330.4 f 0.4 334.0 f 0.5 silica D 481.5 2.8 331.3 f 0.3 332.3 f 0.9 silica E 439.3 f 1.7 327.8 f 0.8 330.7 f 0.8
*
'Error limits denote 95% confidence level of the mean of at leaat three measurements. Where no limits are listed, only a single measurement waa made. Mixed solvents prepared according to mass by buffer dilution, reducing the overall NaH2P04concentration. Contact solvent for surface measurements is pH 4.0 or 7.5 buffer. e2-3 h after adsorption. dEmission max in contact with pH 7.5 buffer is 462.7 nm. 'Solutions and contact solvents buffered at pH 4.0. Concentration is 2.2 pM in mixed solvents; water solutions are saturated. 'Emission max in pH 7.5 buffer is 522.3 nm. Maxima were unaffected by variations in solution concentration, flow rate, and amount of adsorbed protein (relative to sorptive capacity of silica). f
molecule exhibiting high sensitivity to its environment. The fluorophore PRODAN is such a mo1ecule;l' it displays a very large shift in wavelength of maximum emission from cyclohexane to water solutions and does not possess a permanent charge. Fluorescence spectra of PRODAN adsorbed to the five substrates from pH 4.0 buffer are displayed in Figure 1. The emission maxima are listed in Table I1 along with values for the probe dissolved in various solvents. All of the silica surfaces are less polar than solutions of 50% methanol, propanol, or acetonitrile, which is not surprising except in the case of underivatized A. Lochmuller and co-worker~13J8 have established that the silanol moieties on the surface of this material are clustered into patches of high density, leaving the remaining areas relatively "bare". The large blue shift in the PRODAN emission maximum relative to water is presumably due to sorption on the areas largely devoid of, in addition to areas rich in, surface silanols. Another anomaly is the emission maximum on D, which is approximately equal to that on A. Although D is 17.4% carbon, it is prepared by reaction with a trichlorosilane and is not endcapped. The resulting high concentration of unreacted silanols, both on the surface and in the polymeric bonded phase, may impart a more polar character than the carbon analysis would otherwise indicate. We believe this is the case because the emission maximum is blue shifted more than 40 nm when D is endcapped with trimethylchlorosilane (silica E). A single measurement of the emission maximum on C in contact with pH 7.5 buffer yielded a value of 462.7 nm. This demonstrates that the polarity of the surface is unaffected by pH over the range of interest. Kinetics of Apomyoglobin Adsorption and Desorption. In Figure 2 are shown rate curves for apomyoglobin adsorption from pH 4.0 buffer to silicas A, B, and D. Rapid adsorption takes place on each although the initial rate is lower on A than on the hydrophobic B and D. Similar behavior has been reported for f i b r ~ n e c t i n . ~ (17)Weber, G.;Farris, F. J. Biochemistry 1979,18, 3075-3078. (18)Lochfiller, C. H.;Colbom, A. S.;Hunnicutt, M. L.; Harris, J. M. J. Am. Chem. SOC.1984,4077-4082.
c
i
o
m
un
bxI
Em
la0
Im)
la
IKa
Ism
TIME ( s e d
Figure 2. Apomyoglobin adsorption to silicas A, B, and D from pH 4.0 buffer. Flow rate = 0.1 mL/min; protein concentration = 4.7 p M . Curves were scaled for illustration and are presented
for qualitative comparison only. Although a strictly quantitative measure of protein adsorption to a solid surface cannot be made with the present experimental system, a relative comparison is possible among equal volumes of silica gels of equal particle size. The relative sorptive capacities of A, D, and B are 6.5:2.5:1.0. These values were calculated as where Ib is the background fluorescence intensity, I,,,is the intensity at the sorptive capacity, and G is the ratio of the intensities in the spectrum of sorbed apomyoglobin a t the emission maximum to that a t 340 nm. The assumptions made in these calculations are that the quantum yields of the silicas a t 340 nm are equal and the quantum yield of adsorbed protein is invariant with respect to sorbent. The lower capacity of B is expected due to its smaller surface area. We suspect that the capacity of D is less than that of A because the polymeric octadecyl phase excludes the protein from some fraction of the pore volume. Desorption of apomyoglobin from these three silicas occurs slowly, if a t all. The decrease in fluorescence intensity observed upon flushing the cell with 5.0 mL of pH 4.0 buffer ranged from 6% on A to 16% on D. However, these values cannot be interpreted quantitatively because the decline is due to a combination of three processes: (a) desorption; (b) on-column incubation; (c) UV photodegradation. The individual contribution of desorption to the overall decrease cannot be discerned but the measurements show that adsorption is essentially irreversible when water is used as the eluent. RPLC of myoglobin demonstrates that desorption is easily effected from similar surfaces by using an organic modifier such as propanol or acetonitrile.1°J9 The fluorescence intensity of sorbed apomyoglobin declined slowly with time on all of the substrates. An example is found in panel M of Figure 3 where a drop of 7.7% a t 340 nm was observed over a period of 13.4 h. During this time, the cell was kept in the dark a t stopped flow. The process is n o t accompanied by a change in emission maximum, which argues against but does not preclude a slow conformational change. The intensity decline is greatly accelerated when the sample is continuously irradiated with 295-nm light, as shown in panel N of Figure 3. We interpret the 18.6% decrease in 340-nm emission over 51 min as evidence of photodegradation. Photochemical decomposition of adsorbed proteins with UV light has been noted previously20 (19)Sadler, A. J.; Micanovic, R.; Katzenstein, G . E.; Lewis, R. V.; Middaugh C. R. J . Chromatogr. 1984,317,93-101.
Lochmiiller and Saavedra
436 Langmuir, Vol. 3, No. 3, 1987
334
'
, 0
WAVELENGTH ( n d
1
I
I
I
I
10 20 30 40 K Organic Modifier (wlw)
I
50
Figure 4. Emission maxima of apomyoglobin dissolved in mixed aqueous/organic solvents ( 0 ) methanol; (A)acetonitrile; (0) propanol. Protein concentration 2 pM, aqueous portion buffered at pH 4.0. Solventa were prepared by buffer dilution, reducing the overall NaH2P04concentration.
-
E
Min: 6 . 0 :
I Ya
!ob3
Iyo
mlo
Ish,
m
D
A1
A2
lsbo
TIME (seC)
Figure 3. (M) Fluorescence spectra of apomyoglobin adsorbed to silica B: (1)immediately postsorption; (2) 13.4 h postsorption. The emission maxima of 1 and 2 occur at 337.0 and 337.2 nm,
respectively. Between spectra, the cell was kept in the dark at stopped flow. (N) 340-nm emission from continuous 295-nm excitation of apomyoglobin adsorbed to silica B. Adsorption (flow) was stopped at approximately 550 s; intensity decline thereafter is due primarily to photodegradation.
and was attributed to photolysis of aromatic amino acids and buildup of the reaction products. Conformation of Apomyoglobin in Solution and Adsorbed on Silica. Fluorescence spectra were acquired for apomyoglobin dissolved in buffers and mixed aqueous/organic solvents and adsorbed to silica gels A-E. Listed in Table I1 are the wavelengths of maximum emission; with the exception of silica A, no change in these values was observed for a t least 12 h after adsorption. The emission maxima in solutions buffered a t pH 4.0 and 7.5 are 335.4 and 330.4 nm, which agree with the values reported by Kirby and Steinera21 The 5-nm red shift observed upon acidification is associated with destruction of the heme-binding pocket and loss of approximately half of the a-helical content of the protein.22~a However, the protein is far from fully denatured at pH 4.0. The position of the fluorescence maximum, the helical content, and polarized emissiona show that the tryptophan residues are still part of an organized structure. Since the PRODAN data indicated that the silica surfaces were less polar than water, we felt it instructive to examine the fluorescence behavior of apomyoglobin in mixed aqueous/organic solvents. In Figure 4 are plotted the emission maxima in solutions with the aqueous portion buffered a t pH 4.0 (at pH 7.5, the protein is insoluble in the presence of low concentrations of organic modifier). A red shift to about 340 nm is observed in the presence (20)Andrade, J. D.Surface and Interfacial Aspects of Biomedical Polymers; Plenum: New York, 1985; Vol. 2,pp 113-116. (21)Kirby, E. P.; Steiner, R. F. J. Bid. Chem. 1970,245,6300-6306. (22)Colonna, G.;Irace, G.; Parlato, G.; Aloj, S. M.; Balestrieri, C. Biochem. Biophys. Acta 1978,532,354-367. (23)Irace, G.;Balestrieri, C.; Parlato, G.; Servillo, L.; Colonna, G. Biochemistry 1981,20, 792-799.
i m
WAVELENQTH (nm)
Figure 5. Fluorescence spectra of apomyoglobin adsorbed to silicas A, D, and E from pH 4.0 buffer: (Al) immediately postsorption; (A2) 3 h postsorption The heights do not reflect relative intensity. of 50% (w/w) organic modifier. Similar results have been observed for myoglobinlg and are thought to be due to protein unfolding and extrusion of the tryptophans into the solvent. Of interest is the positive correlation of helical content with concentration of organic m ~ d i f i e r , ' ~im,~~ plying that transition to a highly ordered, extended structure takes place. Fluorescence spectra of apomyoglobin adsorbed to silica A from pH 4.0 buffer are displayed in Figure 5. The emission maximum immediately after adsorption is 337.8 nm (Table 11),a red shift of approximately 2 nm from solution. This indicates that the protein has undergone a conformational change to a structure in which the tryptophan residues me more exposed relative to the native form. During the next 2-3 h, a red shift to 340.0 nm occurs, suggesting a slower transition to an even more unfolded state. A structural change involving increased exposure also occurs upon adsorption to the hydrophobic silica B from pH 4.0 buffer, as evidenced by a similar red shift to 337.1 nm. In contrast, Figure 5 and Table I1 show that the fluorescence of apomyoglobin sorbed to silicas C-E from pH 4.0 buffer is blue shifted 4-8 nm from the solution emission. This implies that the protein is either folded into a more compact configuration upon adsorption or is unfolded to allow interaction between the exposed fluorophores and the octadecyl chains chemically bound to these substrates. We think the latter possibility is the (24)Herskovita, T.T.;Gadegbeku, B.; Jaillet, H. J. Biol. Chem. 1970, 245, 2588-2598.
Langmuir, Vol. 3, No. 3, 1987 437
Fluorescence of Adsorbed Apomyoglobin
435
445 455 465 475 PRODAN Emlsslon Max (nm)
405
Figure 7. Emission maxima of apomyoglobin vs. emission maxima of PRODAN on silicas A, B, C, and E. and A denote protein adsorption at pH 4.0 and 7.5. Apomyoglobin maxima on A are 2-3 h postsorption.
/ p8
/ m
332
334
33
ne
340
WAVELENGTH (nm)
Figure 6. Fluorescence spectra of apomyoglobin adsorbed to silicas A-E from pH 7.5 buffer: (Al) immediately postsorption; (M)3 h postsorption. The heights do not reflect relative intensity. correct interpretation because (a) a tighter configuration is rather improbable considering that most proteins unfold in hydrophobic media and (b) it is highly unlikely that unfolding would take place on silicas A and B and not take place on C-E. Note that endcapping D blue shifts the fluorescence more than 3 nm. This demonstrates that the position of the emission maximum of adsorbed apomyoglobin is a function of the polarity of the interface as well as the degree of residual structure surrounding the tryptophans. Fluorescence spectra of apomyoglobin adsorbed to the silica substrates from pH 7.5 buffer are displayed in Figure 6. Table I1 shows that, with the exception of silica E,the emission maxima are red shifted to varying degrees from the solution value. The extent of tryptophan exposure in the surface conformer(s) is therefore greater than that in the native protein. Following the reasoning stated above, we think it doubtful that a conformational change does not occur upon adsorption to silica E. It is possible that the polarities of the interfacial environment and the interior of the native protein are approximately equal; in this case, no shift would be observed. Analogous to the behavior observed a t pH 4.0, the emission maximum on silica A a t pH 7.5 is red shifted about 2 nm from the initial measurement during the 2-3 h following adsorption. However, both the initial and final maxima are blue shifted 2 nm from the corresponding measurements a t pH 4.0. This suggests the presence of nonequivalent conformations in the sorbed state at pH 4.0 and 7.5. The degree of unfolding of the protein upon adsorption appears to be mediated by the pH of the contact buffer. The tryptophans in the pH 7.5 surface conformer are less exposed to the interfacial environment than a t acidic pH.
In contrast to the behavior on A, the emission maxima for apomyoglobin sorbed on silicas C-E at pH 7.5 are red shifted from the measurements a t pH 4.0. This is the expected result, however, if the protein is less unfolded in the sorbed state a t neutral pH. A more compact conformation would allow less interaction between the tryptophans and the hydrophobic surface, yielding a fluorescence red shifted from that observed a t pH 4.0. An apparent anomaly in this interpretation is that the emission maxima of apomyoglobin adsorbed on silica B a t pH 4.0 and 7.5 are equal within experimental error. This can be explained by referring to Figure 7 in which the emission maxima of sorbed apomyoglobin are plotted against the corresponding PRODAN maxima. The polarity of the silica A surface is greater than the average polarity of the tryptophan environments in the pH 7.5 surface conformer because increased unfolding (at pH 4.0) is accompanied by a red shift. The relative polarities are reversed on silicas C-E,so a blue shift is observed upon increased unfolding. However, the greater exposure of the tryptophans in the pH 4.0 surface conformer may not be manifested as a net change in the polarity of their environments if it is balanced by increased interaction with a moderately hydrophobic surface. The pH invariance of the emission maxima on silica B may represent such a case. Dynamic Behavior of Adsorbed Apomyoglobin. In an earlier report,14it was shown that a partially reversible change in the conformation of apomyoglobin adsorbed to silica C could be induced merely by changing the pH of the contact buffer. Flushing the flow cell with 2.0 mL of pH 7.5 buffer after sorption to silica C at pH 4.0 red shifts the emission maximum 2.2 nm (mean of three determinations). Complete refolding of the pH 4.0 surface conformer to the pH 7.5 conformer apparently does not take place because the emission maximum of apomyoglobin adsorbed from pH 7.5 buffer is 334.0 nm. The red shift is coupled with an increase in fluorescence intensity of 32% a t the maximum which shows that the quantum yield of the pH 4.0 surface conformer is less than that of the refolded conformer. The correlated fluctuation of emission intensity with the pH of the flush demonstrates that repeated interconversion between conformations is p0ssib1e.l~ Similar behavior was observed on silica E as illustrated in Figure 8. Reversible refolding of the apomyoglobin heme pocket in solution, which takes place when the pH is raised from 4.0 to 5.6, involves a 50% decline in fluorescence quantum yield.21v23It is therefore unlikely that the difference between the pH 4.0 surface conformer and the partially refolded conformer (or the pH 7.5 surface conformer) is the structural integrity of the heme pocket.
Langmuir 1987,3,438-443
438
--
I
00
1.0
2.0
3.0
4 0
Flow Volume (mL)
Figure 8. Fluorescence intensity at the emission maximum for apomyoglobin adsorbed to silicas C and E from pH 4.0 buffer: (m) &r flushing the flow cell with 0.5 mL pH 4.0 buffer; (+) aftar flushing with 0.5 mL of pH 7.5buffer. The intensity difference between the curves is not to be interpreted. Conclusions The results of this study show that apomyoglobin is adsorbed rapidly to hydrophilic and hydrophobic silica gels. Desorption occurs slowly if at all with a water eluent. Examination of the position of the fluorescence emission maxima indicates that upon adsorption from both pH 4.0 and 7.5 buffer, the protein undergoes a conformational change involving increased exposure of the two tryptophan residues relative to the solution conformation. In contrast to other reports,'~~r~~ the structural change takes place
relatively quickly; except on the hydrophilic silica A, it is complete within 20 min (the maximum time required to reach sorptive capacity). An additional, slower transition to an even more unfolded state takes place on A during the 2-3 h following adsorption. With one exception, the emission maxima of apomyoglobin adsorbed a t pH 7.5 and 4 . 0 are not equal, which c o n f i i the existence of at least two nonequivalent surface conformers. Careful interpretation of the data indicates that the tryptophans in the pH 4.0 surface conformer are more exposed and interact to a greater extent with the interfacial environment than in the pH 7.5 surface conformer. Partial, reversible refolding of the pH 4.0 surface conformer can be induced by raising the pH of the contact buffer. Finally, we note that the interaction of a protein with a heterogeneous surface13J8such as that of silicas A-E likely yields a heterogeneous distribution of surface conformers. Consequently, the characteristics of fluorescence emission reported herein, and the conclusions inferred from these data, represent the average behavior of the protein in the interfacial environment.
Acknowledgment. This work was supported in part by the National Science Foundation under Grant CHE850658. (25)Reference 20,pp 60-63.
Association Properties of Pyrene, 1-Pyrenesulfonate,and Iodide with Ti02Particles Edward Blatt,* D. Neil Furlong, Albert W. H. Mau, Darrell Wells, and Wolfgang H. F. Sasse Division of Applied Organic Chemistry, CSIRO, Melbourne, Victoria 3001, Australia Received December 17, 1986 The adsorption of pyrene, 1-pyrenesulfonate (SPS),and I- from an aqueous solution at pH 3 onto Ti02 particles is examined by fluorescence probe, centrifugation, and electrophoresis techniques. At low probe concentrations,neither pyrene nor SPS molecules adsorb onto Ti02particles of 90-Adiameter. Adsorption of pyrene was observed when larger TiOz particles were used, and for SPS it occurs onto the 90-A-diameter particles by increasing the initial concentration of probe. Electrophoretic mobility measurements of TiOz M I- show that specific adsorption of I- onto positive TiOz surfaces does not dispersed in aqueous occur. The implication of the latter result on previously reported photochemical experiments is discussed.
Introduction Fluorescence probe techniques have found wide application in studies of the association behavior of molecules and ions with micelles and membranes,' polymers? DNA? polyelectrolytes,4 and protein^.^ Association of luminescent aromatic molecules is often inferred from changes in emission and/or absorption characteristics of the mole(1) Blatt, E.; Sawyer, W. H. Biochim. Biophys. Acta 1985, 822, 43. (2) Sumi, K.;Furue, M.; No&, S. Photochem. Photobiol. 198442, 485. (3)Atherton, S.J.; Beaumont, P. C. J. Phys. Chem. 1986,90, 2252. (4) Sassoon, R. E. Chem. Phys. Lett. 1986, 125, 74. (5) Blatt, E.; Husain, A.; Sawyer, W. H. Bioehim. Biophys. Acta 1986, 871, 6.
culeG8 as well as from lifetime and quenching data.'P8 Although in favorable circumstances standard equilibrium dialysis methods have been used s u c ~ e s s f u l l y ,most ~~~ studies do not include verification of association by other experimental techniques, The association of luminescent aromatic molecules and ions onto colloidal TiOz is an active area of current re(6)Jones, G., 11.; Jackson, W. R.; Kanoktanaporn; s.; Bergmark, W. R. Photochem. Photobiol. 1985,42,477. (7) Kalyanasundaram,K.; Thomas,J. K. J . Am. Chem. SOC.1977,99, 2039.
(8)Blatt, E.,Sawyer,W. H.; Ghiggino, K. P. J.Phys. Chem. 1984,88, 3918. (9)Sikaris,K.A.;Thulborn,K. R.; Sawyer,W. H. Chem. Phys. Lipids 1981, 29, 23.
Q743-7463/87/24Q3-0438$01.50/00 1987 American Chemical Society
