Biophysical Journal Volume 68 May 1995 2091-2097

Absorption Spectra Indicate Conformational Alteration of MyoglobinAdsorbed on Polydimethylsiloxane

Aron B. Anderson* and Channing R. RobertsonDepartment of Chemical Engineering, Stanford University, Stanford, California 94305 USA

ABSTRACT To assess the effects of adsorption on protein structure, ultraviolet optical absorption spectra of myoglobin (Mb)bound to polydimethylsiloxane (PDMS) were measured. A flow cell, which enabled adsorption under controlled hydrodynamicconditions, was used in conjunction with a conventional spectrophotometer to obtain the spectra. Adsorption to PDMS reducedsignificantly the absorbance in the Soret region of the Mb spectrum, whereas the spectrum in the region near 280 nm wasessentially unaffected. This result showed that disruption of the native structure of Mb occurs following interaction with PDMS.Furthermore, the change in the absorption spectrum may indicate loss of heme from the heme pocket of the adsorbed protein.Mb structure was altered from its solution configuration within fifteen min of contact with the surface. Exchange of adsorbedMb with Mb in solution had little or no effect on the absorption spectrum of the surface-confined protein, indicating that exchangeoccurs only between conformationally altered species or between native species.

INTRODUCTION

Protein adsorption

Protein adsorption at the solid-liquid interface plays an im-portant role in many applications (Cooper and Peppas, 1982;Andrade, 1985; Horbett and Brash, 1987). One key elementto understanding the physical and chemical properties of ad-sorbed proteins is elucidation of the effects of adsorption ontheir three-dimensional structure. Many previous studies ofadsorbed protein conformation have involved proteins (forexample, fibrinogen, fibronectin, and serum albumin) whosestructures are incompletely characterized in solution, makinginterpretation of adsorbed protein structure difficult. Further-more, the limitations of current structure-sensitive tech-niques have prevented precise examination of adsorbed pro-tein conformational issues. Consequently, the relationshipbetween structure and function of adsorbed proteins remainsincompletely understood.To address issues of adsorbed protein structure, we have

chosen to study, as a model system, adsorption of spermwhale myoglobin (Mb) onto polydimethylsiloxane (PDMS).In three previous studies of this system (Darst et al., 1986;Darst et al., 1988; Anderson, 1991) results were obtained thatcould be interpreted as evidence for conformational changesof the protein after adsorption. In the first study, nonlineari-ties of the time-dependent fluorescence of fluorescein-labeled Mb and increasing tenacity of the PDMS-bound Mbwith adsorption time were postulated to involve structuralchanges of the adsorbed protein. In the second study, the

Receivedfor publication 15 November 1993 and infinalform 21 February1995.Address reprint requests to Dr. Channing R. Robertson, Department ofChemical Engineering, Stanford University, Stanford, CA 94305. Tel.: 415-723-4986; Fax: 415-725-7294; E-mail: chanbo@rio.stanford.edu.Dr. Anderson's present address: BSI Corporation, 9924 W. 74th Street, EdenPrairie, MN 55344.X) 1995 by the Biophysical Society0006-3495/95/05/2091/07 $2.00

binding of monoclonal antibodies to PDMS-bound Mb wasinhibited compared to binding to Mb in solution. It was notpossible, however, to distinguish conformational changesfrom non-random orientation of the adsorbed Mb. In the thirdstudy, the fluorescence of a Mb analog containing a chlo-rophyll derivative instead of heme was reduced after ad-sorption of the protein to PDMS. One interpretation of thesefluorescence results was that the conformation of the proteinwas altered following adsorption to PDMS such that thequantum yield of the chlorophyll was decreased. Because ofthe complexity of interpreting antibody binding and fluo-rescence results in terms of adsorbed protein conformation,it was necessary. to examine this system with other tech-niques to obtain more definitive information concerning thestructure of PDMS-bound Mb.

In the present study, an optical property peculiar to heme-proteins was exploited to investigate the structure of Mbbound to PDMS. The absorbance of Mb in the ultraviolet andvisible wavelengths depends directly on the physical envi-ronment of the heme and, consequently, the structure of theheme pocket. For this reason, the absorption spectrum ofPDMS-bound Mb can serve as a useful indicator of confor-mation and is more directly interpreted in terms of proteinstructure than the antibody binding (Darst et al., 1988) andfluorescence (Darst et al., 1986; Anderson, 1991) results re-ported previously. Experiments designed to measure the ab-sorbance of PDMS-bound Mb described here were per-formed to challenge these previous results and to providemore evidence concerning the time-dependence of alterationof adsorbed Mb structure.

Absorbance of Mb in solution

Absorption spectra of Mb in solution have been extensivelycharacterized (Antonini and Brunori, 1971; Hanania et al.,1966; Eaton and Hochstrasser, 1968) and are known to berich in information concerning the physical state of the pro-

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tein. Mb absorption bands in the ultraviolet and visible wave-lengths can be used to reveal physical properties such as theoxidation state of the heme iron (Antonini and Brunori,1971), the identities of ligands bound to the heme (Eaton andHochstrasser, 1968), and the structural integrity of the pro-tein (Schechter and Epstein, 1968). This final property iscritical for the present study, but the other features of theabsorption spectra would be useful for examining the func-tion of Mb in the adsorbed state.The iron atom contained within the heme can exist in either

an oxidized (ferric) or reduced (ferrous) form. In all experi-ments discussed in this work, the heme iron is in the ferricstate. This form of Mb, often referred to as metMb, can bindvarious ligands from solution, including cyanide, azide, fluo-ride, ammonia, and formate (Hanania et al., 1966; Eaton andHochstrasser, 1968). In the absence of specific ligands,metMb contains a water molecule or a hydroxide ion boundto the heme, depending on the pH of the solution. For allstudies described herein, solutions are maintained at pH 7.4and the metaquo-Mb (i.e., water-liganded) form dominates.For convenience, and because heme iron is in the ferric form,pH is kept constant at 7.4, and no specific ligands are in-volved in these experiments, the term Mb will be usedthroughout to refer to metaquo-Mb.The absorption spectrum of Mb in phosphate-buffered sa-

line (PBS) at pH 7.4 contains two strong bands in the ul-traviolet region and several weaker bands in the visible re-gion (Antonini and Brunori, 1971; Gouterman, 1978). Theextinction coefficients of the visible bands are too low toallow their measurement for adsorbed Mb with the experi-mental system used here. For this reason, discussion is lim-ited to the two ultraviolet bands.The peak centered at 280 nm in the Mb absorbance spec-

trum is due to aromatic amino acids (primarily tryptophanand tyrosine) in the polypeptide and to the heme. Its locationin the Mb spectrum is characteristic of most proteins. Theother absorption peak centered at approximately 410 nm isdue entirely to the heme. This narrow, intense band is char-acteristic of all heme proteins and is commonly referred toas the Soret band. The position, intensity, and shape of thispeak depend strongly on the oxidation state of the heme ironand the identity of any ligand bound to the heme.The Mb absorption spectrum also is sensitive to the struc-

ture of the protein. Shown in Fig. 1 is a comparison of thespectra of Mb in PBS and Mb in PBS and 8 M urea (proteinconcentration is identical for the two spectra). This concen-tration of urea disrupts significantly the secondary and ter-tiary structure of Mb (Schechter and Epstein, 1968; Hers-kovits et al., 1970b). The loss of native protein structurecauses a profound decrease in the extinction coefficient anda blue shift of the absorption maximum of the Soret band,whereas the absorbance of the 280 nm band is relativelyunaffected.The spectral shift shown in Fig. 1 is specific to urea de-

naturation; it is recognized, however, that the spectrum un-dergoes qualitatively similar changes when Mb encountersother denaturing conditions, including high temperature

1.0 -.

0.8 -

a)c 0.6-

m0(0. 0.4-

0.2

25;0 300 350 400 450 500 550

Wavelength (nm)

FIGURE 1 Absorption spectra for Mb in PBS and in PBS containing 8M urea. The protein concentration in both solutions was 85 ug/ml (4.8 ,uM).

(Acampora and Hermans, 1967), low pH (Acampora andHermans, 1967; Breslow and Gurd, 1962), guanidine-HCl(Schechter and Epstein, 1968), and alcohols (Herskovits etal., 1970a). The Soret peak alteration results from changesto the physical environment of the heme after unfolding ofMb. Transfer from a hydrophobic to an aqueous environ-ment, scission of the iron-nitrogen bond, interaction withdenaturant (if present), and contact with other heme mol-ecules can alter heme absorbance. The Mb absorption spec-trum, therefore, is a direct indicator of heme environmentand, consequently, integrity of the protein's structure.

Specific structural changes that occur to Mb after dena-turation have not been characterized completely at the atomiclevel. It is known, however, that: a-helical structures are

disrupted, based on circular dichroism studies (Schechter andEpstein, 1968); buried histidine residues become exposed,based on acid titration measurements (Breslow and Gurd,1962); fluorescence of tryptophan residues is altered(Schechter and Epstein, 1968; Hochstrasser and Negus,1984); and intrinsic physical properties, including heat ca-

pacity (Privalov et al., 1989) and intrinsic viscosity (Privalovet al., 1989) are modified. Such results indicate that dena-turation ofMb in solution encompasses significant alterationof its three-dimensional structure. Although the Mb absorp-tion spectrum alone does not contain this level of detail, itis possible nevertheless to determine whether alteration ofheme environment occurs. Indeed, the similarity betweenspectra of isolated heme in solution (Gouterman, 1978) anddenatured Mb suggests that the heme may be lost entirelyfrom the polypeptide during denaturation. Additional evi-dence exists to support this hypothesis (Schechter and Ep-stein, 1968).The goal of the work described here is to measure the

absorption spectrum ofMb bound to PDMS. Mb absorbancecan be interpreted directly in terms of the environment of theheme and, therefore, the integrity of the heme pocket. For this

0 Mb1&c 0 Mb/8M urea00

0 0

0

0 0

0o 000

00

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Absorption Spectra of Adsorbed Myoglobin

reason, absorbance measurements are considered to be amore straightforward indicator of Mb structure than previousfluorescence or antibody binding measurements.

MATERIALS AND METHODS

The flow cellSeveral specific criteria governed design of the flow cell. First, it was desiredto conduct adsorption experiments under laminar flow conditions similar tothose used during previous total internal reflection fluorescence (TIRF)studies of adsorbed Mb. Second, it was necessary that the flow cell fit withinthe optical train of a conventional ultraviolet/visible spectrophotometer.Third, it was essential that the spectrophotometer beam sample enoughadsorbed protein to allow sufficient signal to be obtained. Finally, it wasnecessary that the adsorbing surfaces be optically transparent to wavelengthsat least as low as 260 nm to allow adequate measurement of the 280-nmband.

These specifications were used to design and construct a cell consistingof a stack of seven PDMS-coated quartz slides alternating with six Teflonspacers, each of which was machined to form a chamber when bounded bya slide on each side. The cell is shown schematically in Fig. 2. Not shownin this figure (for purposes of clarity) are the end caps which couple theentrance and exit ports at the ends of the Teflon chambers to a flow systemand the hardware used to compress the stack of slides and spacers. Whenassembled, the flow cell contains six parallel thin slits through which proteinsolution can flow and twelve surfaces onto which protein can adsorb. This

OptcalBeam

Assembled Stack

FIGURE 2 Schematic diagram of the flow cell used for the absorptionspectra measurements. (Top) Exploded view of the spacer/slide stack. (Bot-tom) Assembled stack. The stack was compressed with end plates (notshown) aligned with bolts threaded through the holes in the spacers. Capswere added to the top and bottom of the stack to couple the ends of the Teflonspacers to the flow system. The flow cell was placed in the beam of thespectrophotometer.

cell is similar in design to a multiple-surface cell used previously (McMillinand Walton, 1974) to measure circular dichroism spectra of adsorbedproteins.

To alleviate problems with leaking, a thin layer of vacuum grease wasused to couple each slide to the Teflon spacers and to couple the end capsto each end of the slide/spacer stack. Under these conditions, leaking couldbe avoided, but only at low peristaltic pump flow rates. This limited therange of shear rates that could be examined with this cell.

Flow system and spectrophotometer

The flow system used for these experiments was similar to that used forprevious TIRF experiments (Lok et al., 1983). The system consists of tworeservoirs containing protein solution and buffer solution, respectively, heldin a constant temperature bath. Before addition of Mb to the protein res-ervoir, buffer solutions were sparged with helium to reduce oxygen contentand to inhibit bubble nucleation in the flow system. After 30 min of heliumflow, the spargers were lifted above the fluid surface and the gas flow wasswitched to nitrogen. This protocol provided a gaseous blanket over theprotein and buffer solutions, reducing contact with ambient air. Fluid waspumped by a peristaltic roller pump (Rainin, Middleton, WI) and directedto the flow cell or a waste receptacle by a four-way valve near the entranceto the flow cell.

The flow system and cell were coupled with a HP8452A diode-array,UV/Vis spectrophotometer (Hewlett Packard, Palo Alto, CA) in conjunctionwith a Vectra personal computer (Hewlett Packard). The flow cell wasoriented vertically in the spectrophotometer with the entrance port at thebottom. Data were collected and stored digitally on the Vectra using thespectrophotometer controller software. The HP8452A measures a completespectrum (from 190 to 820 nm) approximately every 0.1 s. For the experi-ments conducted here, a spectral averaging time of 25 s was used. Thus, eachresulting spectrum was the average of 250 consecutive measurements takenover a 25-s interval. The signal-to-noise ratio in the spectra was observedto decrease with decreasing averaging times.

Myoglobin

Sperm whale Mb was obtained from Sigma Chemical Co. (St. Louis, MO)and used as received. Stock solutions of Mb were prepared by dissolvingthe protein in water purified with a standard MilliQ system (Millipore,Bedford, MA). Mb solutions were centrifuged to remove insolubles andstored at 4°C. Protein concentration was determined spectrophotometrically.A few crystals of KCN were added to a diluted aliquot and the absorbanceat 540 nm was measured. An extinction coefficient of 10.4 mM-1 cm-'(Hanania et al., 1966) and a Mb molecular weight of 17,800 were used forall concentration calculations. Protein solutions used in the adsorption ex-periments were prepared by diluting an aliquot of stock solution into PBSsparged previously with helium (as described above).

PDMS-coated quartz slides

CO-quartz slides (1 inch X 3 inches X 1 mm) were obtained from ESCOProducts (Oak Ridge, NJ). Slides were cleaned using procedures identicalto those described previously (Lok et al., 1983; Cheng et al., 1987). Becauseit was necessary to coat the slides on two sides with PDMS, spin-coatingprotocols were modified as follows. PDMS was deposited on the first sideof the slide as described previously (Cheng et al., 1987). Immediately afterthe 1-min spin on the photoresist spinner, the coated slide was inverted,placed again on the spinner, and coated with PDMS on the opposite side.After PDMS had been applied to both sides, the slide was then placed undera heat lamp for curing. This procedure was found to yield slides of superioroptical quality compared to slides coated on one side and cured and sub-sequently coated and cured on the second side. PDMS-treated slides wereused within 2 weeks. Fresh slides were used for each experiment.

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Volume 68 May 1995

SolutionsProtein solutions were prepared in PBS having the same composition de-scribed previously (Darst et al., 1986). Buffer and protein solutions weretreated as described above before adsorption experiments.

Experimental conditions

All adsorption experiments were conducted under the following conditions:temperature, 25°C; wall shear rate, 10 s-1; protein concentration, 8.5 mg/ml(480 nM). It was not possible to examine adsorption at shear rates higherthan 10 s-' because increased flow rates through the cell resulted in leaking.

Experiments were conducted initially as follows. After degassing ofPBSsolutions and addition of Mb to the protein reservoir, PBS was passedthrough the flow cell. After several minutes of flow, a blank spectrum wasrecorded. This spectrum served as the baseline for all subsequent spectra.Shortly thereafter, the PBS solution was replaced with a Mb solution (t =0). Because the protein solution and the adsorbing surfaces were both in thepath of the spectrophotometer beam, it was not possible to measure spectraof adsorbed Mb continuously as a function of time. Therefore, it was nec-

essary to replace the Mb solution with PBS again to record the spectrum ofadsorbed Mb alone. The time at which the Mb solution was replaced withPBS is considered to be the adsorption time. After -10 min of PBS flow,the adsorbed protein spectrum was recorded.

During experiments using this protocol, it was determined that spectro-photometer lamp intensity decreased slowly with time. Because absorbanceof adsorbed Mb was sufficiently low, the drift in lamp intensity influencedthe resulting spectra. To account for intensity drift, the baseline spectrumwas measured at 5- or 10-min intervals for 1 h before starting an adsorptionexperiment. The rate of drift in baseline absorbance was steady over severalhours but varied from day to day. Therefore, it was necessary to measure

the absorbance drift before each experiment and subtract the time-dependentbaseline from the adsorbed protein spectra (extrapolating drift for experi-ments longer than 1 h). Because the magnitude of absorbance driftwas substantial and the drift rate was not constant over tens of hours,long adsorption experiments were more susceptible to error and, therefore,were discontinued. It was determined, however, that absorption spectra ofPDMS-bound Mb changed little after about 15 min of adsorption (see below)and, thus, experiments lasting more than several hours were not essential tothis study.

RESULTS AND DISCUSSION

The spectrum of Mb is affected significantlyby adsorption

Adsorption to PDMS was determined to alter the absorptionspectrum of Mb. Shown in Fig. 3 is a comparison of twospectra, of Mb adsorbed to PDMS and of Mb in solution.Each spectrum shown is for an equivalent number of Mbmolecules in the path of the spectrophotometric beam. Theadsorbed Mb spectrum shown was recorded after adsorptionof Mb to PDMS for 2 h. The optical density of the Mb so-

lution spectrum was adjusted to represent 12 surfaces cov-

ered with native, intact Mb with a surface concentration,determined previously (Darst et al., 1986), of 94 ng/cm2.If no change to the optical properties of Mb occurred afteradsorption, then the two spectra in Fig. 3 would beidentical. It is clear, however, that the spectra are signifi-cantly different.Two qualitative features are apparent in Fig. 3. First, the

optical density and shape of the Soret peak are altered afteradsorption. The shape of this portion of the spectrum is com-

0.008 I

0.006 -

a)

0

.0(Aon0.004 -

0.002

0.000 -25 300 350 400 450 500 550

Wavelength (nm)

FIGURE 3 Comparison of the absorption spectra of Mb in solution andMb adsorbed to PDMS. The solution spectrum was recorded in PBS, pH 7.4at the protein concentration given in Fig. 1. The adsorbed Mb spectrum wasmeasured after 2 h of adsorption to PDMS under conditions described in thetext.

parable to the shape of the spectrum of Mb exposed to urea,although the reduction in intensity is more substantial. Sec-ond, the 280-nm peaks, in contrast to the Soret peaks, appearsimilar in terms of location, shape, and optical density. Thisobservation is also consistent with the behavior of the280-nm peak during denaturation of Mb in solution (seeFig. 1).

It is difficult, however, to extrapolate a change of hemeenvironment to predict precise conformational changes ex-

perienced by the polypeptide portion of Mb following ad-sorption. Indeed, one could postulate various mechanisms toaccount for altered heme environment, ranging from a minorchange involving relaxation of the folded structure of theheme pocket which allows exposure of heme to either theaqueous or PDMS phase to a drastic change involving lossof heme from the polypeptide and complete disruption of alltertiary and secondary structure. Previous study of the Mb/PDMS system (Darst et al., 1988) suggests that alteration ofMb structure is not as extreme as implied by the latter mecha-nism, but does not relieve entirely the ambiguity surroundingadsorbed Mb structure.

It is also not clear from this result whether all heme re-

mains at the interface with the Mb polypeptide or whethersome of it is lost to solution. It was shown previously (Darstet al., 1986) that the surface concentration of the polypeptideportion ofMb adsorbed on PDMS is constant between 30 minand 10 h of adsorption. If the heme were lost from the surfaceand carried away by the flowing solution, reduction of theabsorbance of the Soret band, as well as the 280 nm bandwould be expected. Because absorbance of the latter is notdecreased significantly by adsorption, it is likely that reducedabsorbance in the Soret band is due primarily to alteration ofthe environment of the heme.

* 2 hours adsorbedo Mb in solution

0000° o0

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o

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5 0

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Absorption Spectra of Adsorbed Myoglobin

Mb structure is altered before 15 min

The absorption spectrum of Mb was monitored initially fortimes ranging from 2 to 8 h to ascertain whether changesoccurred during the same time period as the fluorescence ofFITC-Mb (Darst et al., 1986) and chlorophyll-substituted Mb(Anderson, 1991). Because of the problems associated withlamp intensity drift, it was difficult to measure spectra ac-curately at these longer times. The spectra (not shown) didnot appear to change significantly, however, over this timeperiod. This result highlighted the need for examination ofMb absorbance at shorter surface-contact times.Shown in Fig. 4 are absorption spectra of Mb adsorbed to

PDMS for times ranging from 15 min to 2 h. These resultsindicate that alteration of heme environment and Mb struc-ture, discussed in the previous section, occurs before 15 minof adsorption to PDMS. Also, based on the similarity be-tween the 15 min and 2 h spectra, it is concluded that nosignificant changes in heme environment occur betweenthese two times. These findings are in contrast with fluo-rescence results (Darst et al., 1986; Anderson, 1991), whichindicated that structural changes to PDMS-bound Mb mayhave occurred after 2 h of adsorption, although the fluores-cence data measured in these studies were not unambigu-ously influenced by conformational changes alone.

It would have been revealing to examine the spectrum ofMb adsorbed for times shorter than 15 min to determine moreprecisely when heme pocket structure is first affected, butexperimental protocols and the nature of PDMS-bound Mbimposed this time as a lower limit. This time was necessaryto allow complete replacement of PBS with Mb solution inthe flow cell and to ensure that Mb was bound with sufficienttenacity to the surface so that no significant desorption oc-curred during the PBS flush of the flow cell after adsorption.Mb has been shown to unfold in solution in times rangingfrom milliseconds to minutes (Shen and Hermans, 1972). It

0.005 -

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coL.0

0.003 -

0.002 -

0.001

15min* 1 hour° 2 hours

0.000 .-,I I250 300 350 400 450 500 550

Wavelength (nm)

FIGURE 4 The absorption spectra of Mb adsorbed to PDMS for timesranging from 15 min to 2 h. The time shown in the legend corresponds tothe start of the PBS flush of the flow cell.

is conceivable, therefore, that unfolding of surface-boundMb could occur on a similar time scale, although confine-ment to the surface may inhibit molecular motions within theprotein. To examine adsorbed Mb conformation before 15min, it would be necessary to devise a method or apparatuswhich allows spectra to be measured during adsorption. Aninternal reflection, UV/visible spectrophotometer would beuseful for such measurements.

Exchange has no effect on adsorbedMb absorbance

Adsorption of a protein to a surface is a dynamic process. Forexample, although the surface concentration of Mb reachesa maximum early in the adsorption process (after a few min-utes for the conditions used in these experiments), it is stillpossible for Mb in solution to exchange with surface-boundMb. Indeed, adsorbed Mb does exchange with Mb in solution(Darst, 1987); the rate of exchange, however, decreases as afunction of time, eventually approaching 0 after severalhours of adsorption. Therefore, during the range of timesover which Mb absorbance was measured in this study, it islikely that Mb in solution was exchanging with adsorbed Mband, thus, the time that an individual protein was actually incontact with the surface varied among the adsorbed proteinpopulation.To examine whether exchange had an effect on absor-

bance, Mb was adsorbed to PDMS from solution for 15 minand then PBS was re-introduced into the flow cell and theabsorption spectrum of surface-confined protein was moni-tored as a function of time. With this protocol, the absorptionspectrum at each time was due only to protein that was ad-sorbed at 15 min, because no exchange could occur afterreplacement of the Mb solution with PBS.The results of this experiment are shown in Fig. 5. There

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0.001-

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250 300 350 400 450 500 550

Wavelength (nm)

FIGURE 5 The time-dependent absorption spectra of Mb adsorbed toPDMS for 15 min followed by PBS flow through the cell. The time shownin the legend indicates the total time of contact between the protein and thePDMS.

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Anderson and Robertson 2095

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2096 Biophysical Journal Volume 68 May 1995

is little change to the spectrum of Mb confined to PDMSbetween 15 min and 2 h. Indeed, this figure appears verysimilar to Fig. 4 except for the slight decrease in absorbancein the Soret peak between 15 min and 2 h. This decreasecould indicate either that the average environment of hemechanged (for example, more dimers formed) or that someheme was lost from the interface over this time period. Theresults of this experiment indicate that the absorbance datapresented above were not affected substantially by exchangewith the surrounding protein solution. This is not to say thatexchange did not occur; it indicates only that, if exchangeoccurred, it did not alter the absorption spectrum of the ad-sorbed proteins.

CONCLUSIONS

The primary conclusion from these results is that the nativestructure of Mb is affected by adsorption to PDMS. Theabsorbance measurements indicate that the environment ofheme in the adsorbed protein is altered, which can only occurif the structure of the protein changes to expose the heme tothe aqueous or PDMS phase or the heme leaves the pocketaltogether. This result is consistent with a recent study (Araiand Norde, 1990) which concluded, based on thermody-namic arguments, that Mb structure should be altered afteradsorption from aqueous solution to a low energy surface,like PDMS. The nature and extent of conformational changesof adsorbed Mb were not specified in that work. In addition,these results eliminate the ambiguity associated with previ-ous fluorescence studies of adsorbed Mb (Darst et al., 1986;Anderson, 1991). For example, one issue that is now clearis that the structure of Mb is altered significantly within 15min of contact with PDMS.The absorbance measurements suggest that the heme

group may be lost from Mb adsorbed to PDMS. As discussedin the Introduction, loss of heme from Mb in solution isaccompanied by significant changes to the three-dimensionalstructure of the protein. Results of a previous study (Darst etal., 1988) of PDMS-bound Mb using site-specific mono-clonal antibodies to probe the conformation of the adsorbedprotein indicated that, if structural changes to the proteinoccurred following contact with the surface, they were smalland confined to local regions. A cursory examination indi-cates these two results are contradictory.The two interpretations can be rendered consistent, how-

ever, by consideration of the following. First, it is known thatapoMb (Mb without the heme) shares considerable three-dimensional structure with native Mb. Based on circular di-chroism measurements (Schechter and Epstein, 1968), it hasbeen shown that apoMb retains -80% of the a-helical con-tent of Mb. Therefore, loss ofheme in solution does not resultin complete disruption of the native secondary structure ofthe protein. Second, confinement of Mb at the polymer sur-face may limit the extent of structural change by restrictingthe protein to a subset of the dynamic conformations nor-mally sampled in solution after removal of the heme. Third,it has been demonstrated that the affinities of the antibodies

used previously (Darst et al., 1988) to probe adsorbed Mbstructure are not substantially altered by removal of the hemefrom the protein. The affinities for apoMb of two of theantibodies used in that study are reduced by a factor of -2-3compared to their affinities toward Mb (Jay A. Berzofsky,National Cancer Institute, National Institutes of Health, per-sonal communication). It was observed that affinities forthree of the antibodies were reduced slightly toward PDMS-bound Mb, whereas the affinities for the other two were re-duced by greater than two orders of magnitude. The resultswith the first three antibodies are consistent with the inter-pretation of the present study (i.e., loss of heme from ad-sorbed Mb), whereas the results with the other two antibodiessuggest more substantial disruption of the antibody-bindingsites in adsorbed Mb or orientation of the adsorbed proteinsuch that the antibody-binding sites are inaccessible. Theseresults taken together suggest that Mb may retain a structuresimilar to apoMb on the PDMS surface.

It is often assumed in the literature that proteins adsorbedto hydrophobic surfaces experience conformational changes.To understand the function or to control the behavior of ad-sorbed proteins, however, it is essential that the nature andextent of conformational change be elucidated. The presentwork has revealed several conclusions concerning the natureand extent of the alteration of the three-dimensional structureof adsorbed Mb. Nevertheless, many unanswered questionsremain about this system and other protein/surface combi-nations. It is likely that the answers to these questions willbe unattainable without the advancement of current tech-niques and the development of novel methodologies for ex-amining the three-dimensional conformation of adsorbedproteins. This should be a major focus of future studies ofproteins at interfaces.

The authors thank Professor Alice Gast of the Department of ChemicalEngineering at Stanford University for providing the spectrophotometerused in this study.This work was supported in part by National Institutes of Health grantHL27187.

REFERENCESAcampora, G., and J. Hermans, Jr. 1967. Reversible denaturation of sperm

whale myoglobin. I. Dependence of temperature, pH, and composition,J. Am. Chem. Soc. 89:1543-1552.

Anderson, A. B. 1991. Examination of adsorbed myoglobin conformationusing absorbance spectroscopy and total internal reflection fluorescence.Ph.D. dissertation. Stanford University, California.

Andrade, J. D. 1985. Principles of protein adsorption. In Surface and In-terfacial Aspects of Biomedical Polymers, Vol. 2. Protein Adsorption.J. D. Andrade, editor. Plenum Press, New York. 1-80.

Antonini, E., and M. Brunori. 1971. Hemoglobin and Myoglobin in TheirReactions with Ligands. North Holland, Amsterdam. 436 pp.

Arai, T., and W. Norde. 1990. The behavior of some model proteins atsolid-liquid interfaces. 1. Adsorption from single protein solutions. Col-loids Surf 51:1-15.

Breslow, E., and F. R. N. Gurd. 1962. Reactivity of sperm whale metmyo-globin toward hydrogen ions and p-nitrophenyl acetate. J. Biol. Chem.237:371-381.

Cheng, Y., S. A. Darst, and C. R. Robertson. 1987. Bovine serum albumin

Anderson and Robertson Absorption Spectra of Adsorbed Myoglobin 2097

adsorption and desorption rates on solid surfaces with varying surfaceproperties. J. Colloid Interface Sci. 118:212-223.

Cooper, S. L., and N. A. Peppas, editors. 1982. Biomaterials: InterfacialPhenomena and Applications, ACS Advances in Chemistry Series 199.American Chemical Society, Washington, DC.

Darst, S. A. 1987. The adsorption of myoglobin and the interactions ofmonoclonal antibodies with the adsorbed antigen on a polydimethylsi-loxane surface. Ph.D. dissertation. Stanford University, California.

Darst, S. A., C. R. Robertson, and J. A. Berzofsky. 1986. Myoglobin ad-sorption onto crosslinked polydimethylsiloxane, J. Colloid Interface Sci.111:466-474.

Darst, S. A., C. R. Robertson, and J. A. Berzofsky. 1988. Adsorption of theprotein antigen myoglobin affects the binding of conformation-specificmonoclonal antibodies. Biophys. J. 53:533-539.

Eaton, W. E., and R. M. Hochstrasser. 1968. Single-crystal spectra of fer-rimyoglobin complexes in polarized light. J. Chem. Phys. 49:985-995.

Gouterman, M. 1978. Optical spectra and electronic structure of porphyrinsand related rings. In The Porphyrins, Vol. III. Physical Chemistry, PartA. D. Dolphin, editor. Academic Press, New York. 1-165.

Hanania, G. I. H., A. Yeghiayan, and B. F. Cameron. 1966. Absorptionspectra of sperm-whale ferrimyoglobin. Biochem. J. 98:189-192.

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