Fourier Transform Infrared Spectroscopy for ProteinSurface Studies

24 Fourier Transform Infrared Spectroscopy for Protein—Surface Studies R. M . G E N D R E A U , R. I. LEININGER, S. WINTERS, and R. J. JAKOBSEN

Downloaded by CORNELL UNIV on August 9, 2016 | http://pubs.acs.org Publication Date: July 27, 1982 | doi: 10.1021/ba-1982-0199.ch024

Battelle Columbus Laboratories, Columbus, O H 43201

Fourier transform infrared spectroscopy (FTIR) was applied to the study of protein interactions with surfaces. In addition, the various spectroscopic techniques and methods used to in terpret adsorbed protein spectra are discussed, including transmission and attenuated total reflection (flowing) experi ments using both aqueous solutions of single proteins and aqueous solutions of protein mixtures. Also included are spec tral subtraction, spectral derivation, and spectral deconvolution. Use of these techniques showed that in transmission stud ies of albumin, a conformation change occurred when the protein concentration reached approximately 3% (w/v) in sa line.

Spectral

evidence also indicated the possibility

of

hydrogen-bonded polymerization of albumin as concentration increased. Studies of albumin-fibrinogen mixtures allowed to adsorb completely onto a surface illustrated that albumin ad sorbed initially, followed by fibrinogen adsorption and dis placement of albumin. The infrared band at 1400 surface-adsorbed

γ-globulins

cm

-1

of

correlated quantitatively with

the amount of protein adsorbed, as shown by radiolabeling techniques.

The biological spectroscopy program in our laboratory was designed to provide molecular level studies of blood-surface interactions primarily using Fourier transform infrared spectroscopy (FTIR). In addition, the pro gram utilizes flowing blood from a living dog equipped with a shunt to follow molecular level adsorption of blood proteins onto various surfaces in real time. To accomplish these aims, we faced two problems, stated in the follow ing list, together with the studies needed to overcome these problems. 0065-2393/82/0199-0371$07.00/0 ©1982 American Chemical Society

Cooper et al.; Biomaterials: Interfacial Phenomena and Applications Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

BIOMATERIALS: INTERFACIAL PHENOMENA AND APPLICATIONS

372

D e v i s i n g m e t h o d s for o b t a i n i n g q u a l i t y F T I R spectra o n • A q u e o u s solutions,

flowing

systems

S i n g l e m o d e l p r o t e i n s a n d mixtures of m o d e l proteins S e r u m , p l a s m a , w h o l e b l o o d (bagged) • E x v i v o studies i n v o l v i n g live animals Dogs Sheep I n t e r p r e t i n g spectra o f a d s o r b e d proteins b y • T r a n s m i s s i o n spectra Single m o d e l proteins


M i x t u r e s of m o d e l proteins • A t t e n u a t e d total r e f l e c t i o n (ATR) (flowing) S i n g l e m o d e l proteins M i x t u r e s of m o d e l proteins W e have c o m p l e t e d the first series o f shunt studies u s i n g live dogs to devise methods for o b t a i n i n g spectra. T h e e x p e r i m e n t a l details of this w o r k have b e e n d e s c r i b e d (1 ) p r e v i o u s l y a n d n e e d not be r e p o r t e d here. D e m o n strations of the q u a l i t y of the spectra o b t a i n e d o n aqueous solutions of m o d e l proteins are g i v e n i n s u c c e e d i n g sections. T h u s , this chapter emphasizes the progress made towards o v e r c o m i n g the second p r o b l e m — t h a t of l e a r n i n g to i n t e r p r e t the F T I R spectra of the adsorbed b l o o d proteins. T h i s progress i n c l u d e s i n f r a r e d t r a n s m i s s i o n studies o f single m o d e l proteins a n d mixtures, and attenuated total r e f l e c t i o n ( A T R ) studies of flowing solutions of single proteins as w e l l as o f m i x t u r e s of proteins.

Experimental All spectra were run on a Digilab FTS-10 FTIR system equipped with fast-scan capabilities, a Hycomp 32 data array processor, and a nitrogen-cooled mercury-cadmium-telluride detector. Transmission spectra were obtained using CaF windows with a 6-μπι spacer. For each transmission spectrum, 500 scans were co-added at 4-cm resolution. Smoothing was not needed on these spectra. All the spectra in this chapter (both transmission and ATR) are subtracted spectra, that is, they are the resultef subtracting a saline (H 0) spectrum from the spectra of the aqueous protein solutions. ATR techniques were utilized to follow the adsorption of proteins onto various surfaces using 60° germanium crystals mounted in a liquid cell specially designed for flowing liquids by Harrick Scientific Co. The infrared depth of penetration using such crystals is approximately 2000 A at 1600 c m . The scanning sequence during such a flowing protein solution run (flow rate of 15 mL/min) was: 2

-1

2

- 1

1. The 25-scan, 8-cm~ resolution spectra were collected every 5 s for the first 110 s of flow. 2. The 50-scan, 8-cnrT resolution spectra were collected every 11 s for the next 8 min. 1

1

3. The 200-scan, 400-scan, and then 1000-scan, 4-cm resolution spec tra were collected during the remaining 3 h of the adsorption experi ment. -1


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373

All proteins were used as received from Sigma Chemical (albumin, 97% pure; fibrinogen, 95% clottable; and 7-globulin, 99% pure). All solutions were made in 0.152V NaCl, and in experiments where the pH was regulated, HC1 and NaOH were used to hold the pH at 7.4. For experiments where the pH was allowed to vary, it ranged from 6.3 to 7.4. For the tagging experiments, proteins were labeled with I by the lac toperoxidase procedure using the Radio-iodination System Kit produced by New England Nuclear. After termination of the reaction, the sample was chromatographed on a 0.7 x 29-cm column of G-10 Sephadex and eluted with 0.152V NaCl. Fractions of the protein peak were dialyzed overnight against 0.152V NaCl before use. Protein concentration and specific activity were determined from aliquots of the chroma tographed, dialyzed protein solution. The labeled protein solution was flowed through the ATR cell with protein adsorption (onto the ATR crystal) monitored by infrared. After a protein film had built up on the ATR crystal, 0.152V saline was substituted for the protein solution, and desorption commenced. Desorption con tinued until infrared indicated that no further desorption was occurring. The liquid ATR cell was then drained of saline, and the ATR crystal was removed. This crystal was soaked overnight in a 2% Countoff solution (New England Nuclear) to remove the protein. The solution was then counted using a Searle Series 1185 7-counter adjusted to the blank to determine the number of counts per minute. The amount of protein adsorbed ^g/cm ) was then calculated from the value of counts per minute, the concentration of the solution, and the area of the ATR crystal.


125

2

Transmission Studies—Single Proteins To i n t e r p r e t the i n f r a r e d spectra of proteins, transmission studies of aqueous solutions of single p r o t e i n s w e r e u s e d , because p h y s i c a l parameters such as c o n c e n t r a t i o n , p H , heat, etc. can be v a r i e d easily, i n d u c i n g structural changes that are r e f l e c t e d i n the i n f r a r e d spectra. T h u s , spectral changes can be related to p r o t e i n s t r u c t u r a l changes

i n d u c e d b y p h y s i c a l parameter

changes. A n e x a m p l e is g i v e n i n F i g u r e 1 w h i c h shows transmission spectra of v a r y i n g concentrations of a l b u m i n i n saline solution. O f special significance are the changes w i t h c o n c e n t r a t i o n o c c u r r i n g i n the A m i d e III spectral r e g i o n (1200-1350 c m ) . C h a n g e s (with concentration) occur i n the n u m b e r , shape, - 1

and f r e q u e n c y of the bands near 1300 c m , f o l l o w i n g a definite pattern w i t h - 1

concentration as i l l u s t r a t e d i n Table I. T h e s e changes i n v o l v e o n l y those bands near 1300 c m

- 1

a n d not those near 1250 c m " . T h u s , they are not l i k e l y 1

to be c o n f o r m a t i o n a l changes of the type i n v o l v e d i n changing f r o m (for instance) an α-helix to a β-pleated sheet or a r a n d o m c o i l conformation. C o n f o r m a t i o n changes such as these w o u l d be i n d i c a t e d b y a change i n the ratio of the 1 3 0 0 - c m " the 1 2 5 0 - c m "

1

1

and 1250-cm"

1

complexes, a n d f r e q u e n c y shifts i n

r e g i o n . Rather, these changes appear to be t y p i c a l of h y

d r o g e n - b o n d d i l u t i o n effects, a n d thus appear to i n v o l v e changes i n the h y d r o g e n b o n d structure of the p r o t e i n s . M o s t l i k e l y , these changes involve the formation of h y d r o g e n - b o n d e d d i m e r s or p o l y m e r s as the concentration increases. F o r the solutions s h o w n i n F i g u r e 1 a n d Table I, the concentration was v a r i e d , b u t no a t t e m p t was m a d e to h o l d the p H constant. F o r these solutions the p H v a r i e d f r o m about 6.3 to 7.4. A n o t h e r series of transmission runs w e r e



374

BIOMATERIALS: INTERFACIAL P H E N O M E N A A N D

1

500

1400

1

1

1300 1200 WAVENUMBERS

I

1100

APPLICATIONS

i-

1000

Figure 1. Transmission spectra of albumin in isotonic saline. Concentrations are 0.5, 0.625, 1.25 and 5% (w/v)from bottom to top. Spectra are water-subtracted and plotted at different scale expansions to illustrate differences. made w h e r e the c o n c e n t r a t i o n was v a r i e d (as before), but the p H was titrated to 7.4 (buffers w e r e a v o i d e d to p r e v e n t any potential interference w i t h the p r o t e i n spectra). F o r b o t h series o f solutions, t h e intensities o f various i n frared bands w e r e m e a s u r e d . T h e intensities o f various pairs o f adsorption bands w e r e ratioed, a n d this ratio was p l o t t e d against c o n c e n t r a t i o n as shown i n F i g u r e 2. I n b o t h the


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375


constant a n d variable p H solutions, a n d for b o t h the 1300-cm" /1250-cm" 1

1

ratio a n d the 1400-cm ~ 7 1 3 0 0 - c r r T ratio, major changes i n slope o c c u r r e d i n 1

the i n t e r v a l b e t w e e n 1 a n d 3 % (w/v) concentration. Because infrared bands of the A m i d e III r e g i o n are i n v o l v e d i n the ratios (just given), a n d because these A m i d e III bands have b e e n shown to be sensitive to conformational chang;es, the change i n slope n o t e d indicates that a conformational change occurs w i t h c o n c e n t r a t i o n changes i n the a l b u m i n solution. T h e effects of ρ H are i n d i c a t e d i n F i g u r e 3 w h i c h shows spectra obtained for solutions of a l b u m i n (5% w/v) i n w h i c h the p H v a r i e d f r o m 1.35 to 10.5. In this figure, the ratio o f the 1 4 5 0 - c m " i n f r a r e d b a n d to the 1 4 0 0 - c m " b a n d 1

1

changed as the p H was l o w e r e d . A t a p H o f 1.35, the 1 4 0 0 - c m " b a n d almost Downloaded by CORNELL UNIV on August 9, 2016 | http://pubs.acs.org Publication Date: July 27, 1982 | doi: 10.1021/ba-1982-0199.ch024

1

disappeared. K o e n i g (2) assigned a R a m a n b a n d at this frequency to the s y m m e t r i c C O O " s t r e t c h i n g v i b r a t i o n of the carboxylate groups of amino acid side chains. T h i s assignment is c o n f i r m e d b y the infrared data shown i n F i g u r e 3. F o r basic solutions (high p H ) the carboxylate frequency (1400 c m ) - 1

is strong, b u t as the p H is l o w e r e d the carboxylate b a n d disappears because the protonated f o r m is generated. A t l o w p H distinct changes occur i n the A m i d e III spectral r e g i o n as c o m p a r e d to the spectra at h i g h e r p H values. These changes ( F i g u r e 3, p H 1.35) involve a m a r k e d intensity increase i n the 1250-cm

-1

b a n d as c o m p a r e d to the 1 3 0 0 - c m

b a n d , i n d i c a t i n g an a l b u m i n

-1

conformational change at this l o w p H .

Transmission Studies—Mixtures of Model Proteins O u r previous transmission studies of aqueous solutions o f single proteins demonstrated that spectral changes can be related to structural and/or con formational variations i n single p r o t e i n systems. N o w w e must show that this can be a c c o m p l i s h e d for mixtures of proteins a n d most i m p o r t a n t l y , that

Table I. Transmission Spectra of Aqueous Solutions of Albumin Concentration 0.5 0.625 1.0 1.25 2.5 3.0 5.0 10.0 15.0

(%)

Frequency 1311 1312 1313 1314 - 1 3 1 5 W , Sh 1317 S h 1312 S h 1317 S h possible 1312-16 S h

(cm' ) 1

0

1287 1290 1295 Sh 1286 S h

b

b

1300 1301 1306 1302 1305 1305

"Computer-generated frequencies. Resolution was 4 cm \ with data points collected every 1 cm" . Sh = Shoulder, W = Weak. 1

h


Cooper et al.; Biomaterials: Interfacial Phenomena and Applications Advances in Chemistry; American Chemical Society: Washington, DC, 1982. J

Figure 2. Plots of band intensity ratios for albumin in isotonic saline ^water-subtracted transmission spectra). Key; 1300/1250: •, no pH control; 0, pH 7.4; 1400/1300 cm ; ·, no pH control; O , pH 7.4.


C/5

δ ζ

r η

α >

m Ζ Ο M Ζ > > Ζ

κ

•β

η > r

?

Ζ H M 50

CD

r

2 >

PI

δ

2

-a σ>


24.

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J

1500

— 4

1400


1

1300

1

1200

ι

ItOO

377

L

O00

WAVENUMBERS

Figure 3. Water-subtracted transmission spectra of albumin in isotonic saline with varying pH. All solutions were 5% (w/v). The pH was adjusted with HCl and NaOH to (from bottom to top) pH i . 3 5 , 3.25, 4.65, and 10.5. i n d i v i d u a l proteins can b e i d e n t i f i e d i n a m i x t u r e . W e approached this i d e n t i fication p r o b l e m i n t h r e e ways. W e w o r k e d c o n s i d e r a b l y o n the first approach (spectral subtraction), and are just b e g i n n i n g w o r k on the second a n d t h i r d approaches (deconvolution o f spectra and second derivative spectra), but all three techniques show great p r o m i s e for certain applications.


378


T h e first approach is illustrated i n F i g u r e s 4, 5, and 6. F i g u r e 4 shows spectra of 7-globulin (top), a l b u m i n (bottom), and a 1:1 m i x t u r e of a l b u m i n and 7-globulin (middle). T h e presence of both components c o u l d possibly b e d e d u c e d f r o m the spectra

o f the m i x t u r e b y b o t h the bands

i n the

1 4 0 0 - 1 4 5 0 - c n T region and b y the bands i n the A m i d e III region. T h e ratio


1

1500

1200

900

WAVENUMBERS

Figure 4. Water-subtracted transmission spectra of 2% (w/v) solutions of albumin (bottom), y-globulins (top), and a 1:1 mixture of albumin and y-globulins (middle).


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FTIR Spectroscopy for Frotein-Surface Studies

of the 1 4 0 0 - c m / 1 4 5 0 - c m _1

_1

379

bands i n the s p e c t r u m of the m i x t u r e is inter-

m e d i a t e l y b e t w e e n that o f p u r e a l b u m i n and that o f p u r e 7-globulins. A l s o , the b a n d near 1320 c m

- 1

i n a l b u m i n is i n the s p e c t r u m of the m i x t u r e as is

the 1 2 5 0 - c m " b a n d o f 7-globulins. H o w e v e r , i f this w e r e an u n k n o w n m i x 1


ture, the only real p r o o f of the i d e n t i t y o f the components o f the m i x t u r e

1400

1200

1000

WAVENUMBERS

Figure 5. Result of subtracting albumin from albumin-globulins mixture shown in Figure 4 (bottom) ana reference spectrum of 7-globulins (top).


380

BIOMATERIALS: INTERFACIAL P H E N O M E N A A N D APPLICATIONS

w o u l d be i f the c o m p o n e n t s w e r e separated a n d the s p e c t r u m of a separated c o m p o n e n t m a t c h e d a reference s p e c t r u m o f that c o m p o n e n t . O n e m e t h o d to obtain this result is t h r o u g h spectral subtraction. B y subtracting a reference s p e c t r u m o f one c o m p o n e n t f r o m the m i x t u r e , a s p e c t r u m o f the other c o m p o n e n t is generated. T h i s other c o m p o n e n t t h e n can be i d e n t i f i e d b y c o m p a r i s o n to reference spectra. T h i s result is i l l u s t r a t e d for the 1:1 a l b u m i n - g l o b u l i n s m i x t u r e i n F i g u r e s 5 and 6. F i g u r e 5 shows the result (bottom) of subtracting a s p e c t r u m of a l b u m i n f r o m the s p e c t r u m of the m i x t u r e . T h i s


subtracted s p e c t r u m matches the reference s p e c t r u m o f g l o b u l i n s shown at

1400

1200

1000

Figure 6. Result of subtracting globulins from albumin-globulins mixture shown in Figure 4 (top) and reference spectrum of albumin (bottom).


24.

GENDREAU ET AL.


the top (differences b e l o w 1050 c m "

1

381

were spectral artifacts, and w e r e not

important i n this identification). T h e reverse subtraction is shown i n F i g u r e 6 w h i c h identifies the other c o m p o n e n t as a l b u m i n . T h e second approach to i d e n t i f y i n g proteins i n mixtures is shown i n


F i g u r e 7. A t the top o f the figure, a second derivative o f the infrared spec-

'

1 — 1800

1

1

1500

1200

1 — r — 900

WAVEN UMBERS

Figure 7. (Top) Spectrum obtained by taking second derivative of spectrum at bottom. Arrows point to second-derivative bands that fall at frequencies where albumin and y-globulins are known to have second-derivative bands. (Bottom) Albumin-globulins mixture from Figure 4.


382


t r u m of the 1:1 m i x t u r e of a l b u m i n a n d 7-globulins is c o m p a r e d to the adsorbance s p e c t r u m o f this m i x t u r e (bottom). A l t h o u g h the A m i d e I b a n d (1650 c m " ) shows a slight l o w - f r e q u e n c y a s y m m e t r y , p r o v i n g the presence 1

of two components, m u c h less i d e n t i f y i n g t h e m , w o u l d be difficult. H o w e v e r , the second-derivative s p e c t r u m o f the m i x t u r e shows four peaks (marked w i t h arrows) i n the A m i d e I r e g i o n . T h e two h i g h - f r e q u e n c y peaks corre spond to the frequencies

o f the two peaks w e observed i n the second-

derivative s p e c t r u m of p u r e a l b u m i n , w h i l e the two l o w e r - f r e q u e n c y peaks of the m i x t u r e c o r r e s p o n d to the two peaks seen i n the second-derivative s p e c t r u m of 7-globulins.

A l b u m i n tends to be a m i x t u r e of α-helix a n d


β-pleated sheet conformations (3). T h e h i g h - f r e q u e n c y A m i d e I peaks i n the second-derivative s p e c t r u m o f the m i x t u r e agree i n frequency w i t h the fre quencies assigned (4) to α-helix (1655-cm" ) and β-pleated sheet ( 1 6 8 5 - c m ) 1

-1

conformations o f a l b u m i n . I n a d d i t i o n , the intensities of the 1685- a n d 1 6 5 5 - c m " second-derivative peaks r o u g h l y c o r r e s p o n d to the r e p o r t e d (3) 1

d i s t r i b u t i o n of α-helix c o n f o r m e r (55%) a n d β-pleated sheet

conformer

(15%). F i g u r e 8 illustrates the t h i r d approach to identify proteins i n mixtures. This figure shows the A m i d e I a n d A m i d e II regions for an absorbance s p e c t r u m o f the 1:1 a l b u m i n - g l o b u l i n s m i x t u r e (top), and the d e c o n v o l u t i o n of this s p e c t r u m (bottom). (The F O R T R A N d e c o n v o l u t i o n p r o g r a m used was generously s u p p l i e d b y D a v i d C a m e r o n o f the N a t i o n a l Research C o u n c i l o f C a n a d a . ) T h e p r o g r a m assumes a b a n d shape a n d narrows the b a n d w i d t h to resolve i n t r i n s i c a l l y o v e r l a p p e d bands. T h e authors of the p r o g r a m have p u b l i s h e d the theory of this approach recently (5). T h e l o w - f r e q u e n c y a s y m m e t r y o f the A m i d e I b a n d (1650 c m " ) is even m o r e apparent i n this ex p a n d e d s p e c t r u m , b u t still w o u l d not allow identification of the c o m p o n e n t s . H o w e v e r , i n the d e c o n v o l u t e d s p e c t r u m (bottom), two peaks are clearly discernable at 1656 c m " a n d 1643 c m " , frequencies that c o r r e s p o n d to the A m i d e I frequencies o f p u r e a l b u m i n a n d 7-globulin, respectively. T h i s frequency d i s t i n c t i o n alone w o u l d h e l p i n i d e n t i f y i n g the i n d i v i d u a l c o m ponents o f the m i x t u r e s . T h u s , spectral subtraction, d e c o n v o l u t i o n , a n d second-derivative spectra all p r o v i d e i n f o r m a t i o n h e l p f u l i n i d e n t i f y i n g p r o teins i n m i x t u r e s . H o w e v e r , because i n f r a r e d second-derivative spectra and d e c o n v o l u t i o n are r e l a t i v e l y n e w a n d u n p r o v e n techniques, we are still eval uating t h e i r usefulness. 1

1

1

ATR Studies (Flowing Systems) T h e transmission F T I R studies o f aqueous p r o t e i n solutions indicate how structural a n d c o n f o r m a t i o n a l differences i n a p r o t e i n can b e related to spectral changes, a n d that spectral features can b e used to identify proteins in mixtures. H o w e v e r , these studies i n v o l v e static systems, and our goal was to study f l o w i n g systems a n d the adsorption of proteins onto various surfaces.


24.

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383

To attain this goal, w e u s e d A T R techniques w h i c h have b e e n d e s c r i b e d p r e v i o u s l y (6). A l i q u i d A T R c e l l can be used to circulate p r o t e i n solutions (or blood) t h r o u g h the c e l l w h i l e spectrally m o n i t o r i n g the adsorption of proteins onto the surface of the A T R crystal. I n a d d i t i o n , the A T R crystal can be coated


w i t h a t h i n layer of p o l y m e r , p e r m i t t i n g us to follow the adsorption of p r o -

1750

1700

1650

Î60Ô

\55Ô

1500

Î450

WAVENUMBERS

Figure 8. Top: The 1:1 mixture of albumin-y-globulins shown in Figure 4. Bottom: Result of Fourier transform self-deconvolution. This program operates on the spectral interferogram to cause narrowing of overlapped bands so that they may be resolved. The program provides information about exact peak frequencies while trading off information about exact band shape.


384


teins onto t h e p o l y m e r surface. A l l o f the w o r k d e s c r i b e d i n the f o l l o w i n g section is c o n c e r n e d w i t h p r o t e i n a d s o r p t i o n onto the surface of a g e r m a n i u m A T R crystal. A s i n d i c a t e d earlier, w e i n i t i a l l y s t u d i e d the adsorption of proteins f r o m flowing aqueous solutions o f single proteins, w h i c h a l l o w e d us to establish a baseline for t h e t i m e - a d s o r p t i o n b e h a v i o r o f each o f the proteins. T h e p r o tein b u i l d u p was m o n i t o r e d as t h e a d s o r b e d layer increases i n thickness. A n example o f this is s h o w n i n t h e spectra o f F i g u r e 9 w h i c h show h o w the p r o t e i n layer a d s o r b e d f r o m an a l b u m i n solution changes w i t h t i m e . I n addition to an increase i n the a m o u n t o f adsorbed p r o t e i n , changes w i t h t i m e - 1

r e g i o n o f t h e A m i d e III c o m p l e x . T h e adsorbed


occur i n t h e 1 3 0 0 - c m

4, 1500

1 1400

i 1 1300 1200 WAVENUMBERS

1 1100

i-tH 1000

Figure 9. Flowing ATR spectrum of 2% (w/v) albumin, adsorbing onto germanium ATR crystal (flow rate: 3 mL/min). Spectra are taken at (from bottom to top) 2, 3, 11, 28, and 119 min of flow (water-subtracted).


24.

GENDREAU ET AL.

385


p r o t e i n layer changes w i t h t i m e o f flow, a n d this behavior can b e established for each p r o t e i n . T h e s e changes are discussed i n m o r e detail i n the section on A T R studies o f p r o t e i n m i x t u r e s . M o r e i m p o r t a n t is a d e s c r i p t i o n o f the type o f i n f o r m a t i o n that can b e obtained f r o m t h e spectra o f m o r e c o m p l e x , flowing mixtures, w h i c h can b e illustrated b y c o n s i d e r a t i o n o f a 1:1 a l b u m i n - f i b r i n o g e n m i x t u r e i n saline. Representative spectra o f the p r o t e i n layer o b t a i n e d b y flowing this m i x t u r e past the A T R crystal are s h o w n i n F i g u r e 10. I n the A m i d e I I I (12001 3 5 0 - c m ) spectral r e g i o n , t h r e e types o f spectral changes occur w i t h t i m e -1

of flow. L o o k i n g o n l y at t h e series o f i n f r a r e d bands a r o u n d 1300 c m

- 1

, we


find that t h e n u m b e r o f bands, t h e frequencies o f the bands, a n d the shape

1500

1400

1300

1200

WAVE NUMBERS

1100

KXX)

Figure 10. Flowing ATR spectrum of 2% (w/v) 1:1 albumin-fibrinogen adsorbing onto a germanium ATR crystal (flow rate: 15 mL/min). Spectra are taken at (from bottom to top) 2, 5, 10, and 180 min of flow (water-subtracted). Cooper et al.; Biomaterials: Interfacial Phenomena and Applications Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

386


of the bands a l l change w i t h t i m e . T h e v e r y same type of behavior is o b s e r v e d for the series o f i n f r a r e d bands near 1250 c m . T h e t h i r d spectral change is - 1

that the ratio o f the intensities of the 1 3 0 0 - c m / 1 2 5 0 - c m _1

-1

bands also

changes w i t h t i m e o f flow. T h e significance o f these changes is discussed i n later sections after o t h e r e x p e r i m e n t a l observations are discussed. If the intensities o f various i n f r a r e d bands are m e a s u r e d a n d p l o t t e d against t i m e o f flow, the kinetics o r rate o f adsorption can be d e t e r m i n e d . T h i s is o n l y true i f i n f r a r e d bands that are not sensitive to c o n f o r m a t i o n or structural changes are u s e d ; the intensity w i l l then be related d i r e c t l y to the total amount o f adsorbed m a t e r i a l . T h e two bands that w e r e c o m m o n to all Downloaded by CORNELL UNIV on August 9, 2016 | http://pubs.acs.org Publication Date: July 27, 1982 | doi: 10.1021/ba-1982-0199.ch024

proteins s t u d i e d , a n d that w e r e i n d e p e n d e n t of conformational changes at constant p H are the bands at 1550 c m "

( A m i d e II) a n d 1400 c m " . T h e s e

1

1

bands are shown for the f i b r i n o g e n - a l b u m i n m i x t u r e i n F i g u r e 11, w h e r e 32.0-1

28.0 H

24.0·

P20.0 H

Fourier Transform Infrared Spectroscopy for ProteinSurface Studies

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