Proteins at Interfaces II - American Chemical Society

Chapter 26

Adsorption of Human Low-Density Lipoprotein onto a Silica—Octadecyldimethylsilyl (C ) Gradient Surface Downloaded via TUFTS UNIV on July 11, 2018 at 12:44:38 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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Chih-Hu Ho and Vladimir Hlady Department of Bioengineering, Center for Biopolymers at Interfaces, University of Utah, Salt Lake City, UT 84112

Adsorption kinetics of human low density lipoprotein (LDL) onto a silica-octadecyldimethylsilyl (C18) gradient surface was studied using Total Internal Reflection Fluorescence (TIRF) and autoradiography. The fluorescein-labeled LDL adsorption rate onto the negatively charged silica surface was transport-limited. On the hydrophobicC18silica end of the gradient surface the adsorption rate was slower than the transport-limited rate. A simple adsorption model was used to determine the adsorption and desorption rate constants. The LDL adsorption was equal to a sum of adsorption processes on available hydrophilic and hydrophobic adsorption sites on either end of theC18gradient region. The middle part of the C18 gradient displayed a retardation of the adsorption rates. The lipid -labeled LDL adsorption experiments resulted in a fluorescence adsorption pattern that resembled the protein-labeled LDL adsorption, indicating that initially both protein and lipid components of LDL remain adsorbed on the surface.

Human lipoproteins are lipid-protein complexes responsible for the transport of water insoluble lipids in the circulation. The presently accepted structure of lipoproteins depicts them as an apolar core surrounded by polar and amphiphilic components (1,2). Interest in lipoproteins arises primarily from their association with coronary artery disease. The popular interpretation of low density lipoprotein (LDL) as "bad" and high density lipoprotein (HDL) as "good" lipoprotein is based on their use as risk markers for atherogenesis and coronary artery disease. A n interrelation between an atherosclerotic plaque formation and arterial thrombosis has been shown to be due to the action of lipoprotein (a), Lp(a), a variant of L D L which in its structure contains an additional protein, apolipoprotein(a), homologous to plasminogen (3,4,5). It has been shown recently that adsorbed L D L or Lp(a) may promote a procoagulant state (6).

0097-6156/95/0602-0371$12.00/0 © 1995 American Chemical Society Horbett and Brash; Proteins at Interfaces II ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

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The aim of this work was to investigate the adsorption behavior of L D L in relation to the hydrophobicity of the adsorbing surface. Rather then using a series of partially hydrophobic surfaces we have utilized hydrophobicity gradient surface, originally described by Elwing (7). This linear gradient surface prepared on flat silica can be used in combination with the Total Internal Fluorescence Reflection (TIRF) technique as a convenient tool for characterizing protein surface adsorption (8). Here, we have used a surface density gradient of octadecyldimethylsilyl groups (silica-C18 gradient surface) which had a several millimeters long gradient region of increasing surface density of octadecyldimethyl-silyl groups between a clean silica end and a selfassembled CI8 monolayer end. The advantage of a silica-C18 gradient surface is that one can evaluate protein adsorption and desorption rates from a flowing solution as a function of an average surface density of silica bound-C18 chains under otherwise identical experimental conditions. Our results showed that the apparent affinity of L D L decreases with increasing surface hydrophobicity. By performing two identical TIRF adsorption experiments, one with protein-labeled L D L , the other with lipidlabeled L D L , we confirmed that both protein and lipid components of L D L became adsorbed on the silica-C18 gradient surface. Experimental Isolation of Lipoprotein. L D L was isolated by the method of ultra-centrifugation (9). Blood was drawn from ten healthy human donors in 5 ml Vacutainer™ evacuated blood collector tubes which contained 0.05 ml of 15% (75 mg) E D T A solution (Becton Dickinson) and centrifuged at 3,000 rpm and 4°C for 30 minutes. Plasma from ten different donors was pooled. In the first step, V L D L was separated from plasma. The density of the remaining plasma was adjusted to 1.063 g/ml by adding 4.778 M NaBr solution (d = 1.3199 g/ml at 20°C). Three ml of this solution was loaded into a centrifuge tube and 2 ml of 0.844 M NaBr solution (d = 1.063 g/ml at 20°C) was floated on the top. Eighteen tubes were put in an ultracentrifuge rotor (50.3Ti, Beckman) and spun at 40,000 rpm, 20°C and vacuum for 20 hours. After 20 hours, L D L floated to the top of the tube was removed by the tube slice method and pooled. The purity of L D L was checked by one dimensional polyacrylamide gel electrophoresis (Phast, Pharmacia). Half milliliter aliquots of L D L stock solution were placed in 0.65 ml vials and frozen at -20°C. For each adsorption experiment, a desired amount of L D L was thawed. A modified Lowry's method (10,11) was used to determine the concentration of the L D L apoprotein. The L D L concentration was estimated by assuming 25% of protein and 75% of lipid in each L D L particle (72). Preparation of C18 Gradient Surfaces. The silica plates (2.54 x 7.62 x 0.1 cm, ESCO Products, Inc.) and all glassware were cleaned in a hot chromic acid at 80°C for 30 minutes, thoroughly rinsed in purified deionized water and dried in an oven at 120°C for more than 2 hours. The silica contact angle was measured by the Wilhelmy plate technique to check the cleanliness of the surface (13). The silica-C18 gradient surface was prepared by a two phases diffusion method in which a high density solvent with a silane reagent is layered below a low density solvent in a container with clean silica plates. A solution mixture of 125 ml dichloromethane (DCM, density, d = 1.325 g/cm , E M Science), 1 ml pyridine (J.T. Baker) and 2 ml 3

Horbett and Brash; Proteins at Interfaces II ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

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Adsorption of Human Low-Density Lipoprotein

octadecyldimethyl-chlorosilane (ODS, Aldrich) was layered below 150 ml /^-xylene (d = 0.86 g/cm , Fluka). During the surface modification process, the silane diffuses into the low density solvent forming a concentration gradient between the two phases. The silanization reaction proceeded for 4 hours. The silanized silica plates were rinsed by D C M , ethanol and deionized water. The contact angles of silica-C18 gradient surface were measured by the Wilhelmy plate technique. 3

Labeling of Lipoprotein. Fluorescein isothiocyanate (FITC, Aldrich) was covalently bound to the L D L particle following the method of Coons et al. (14). Three mg of apolipoprotein (12 mg of LDL) were thawed and diluted in 3 ml of Dulbecco phosphate buffer (DPBS, 0.05 M phosphate, 0.145 M NaCl, 0.90 m M CaCfe, 0.88 m M MgCl2, 2.7 m M KC1, p H 7.4) (15). 0.6 ml of 1 mg/ml FITC in carbonate bicarbonate buffer (CBB, pH 9.2) was added to 3 ml of the L D L solution (1 mg apolipoprotein/ml) and incubated at room temperature for 3 hours. Separation of FITC-labeled L D L (FITC-LDL) from free FITC was performed on a PD-10 column (Pharmacia). 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO, Molecular Probes Inc.) was used to label the L D L lipids following the procedure described by Stephan and Yurachek (16). 1.5 ml of DiO solution (3 mg/ml DiO in dimethylsulfoxide) was added into 2 ml L D L solution (1 mg apolipoprotein/ml) and incubated at 37°C for 15 hours. The DiO labeled L D L (DiO-LDL) was then separated from the free DiO by filtration (0.8 Jim filter, Millipore) (16). The technique of I-iodination of L D L was a modified IC1 iodination protocol (17) performed at pH 10 to minimize the iodination of lipids. The free I was removed from I-labeled L D L ( I - L D L ) by ion exchange chromatography on the QAE-Sephadex A-25 column (Pharmacia). The I L D L was stored in the refrigerator at 4°C and used within a week. 125

1 2 5

125

125

1 2 5

Protein T I R F Adsorption Experiments. A l l details of the custom-built TIRF apparatus and the flow cell are described elsewhere (8). TIRF protein adsorption experiments were performed in a flow cell containing two identical rectangular flow channels so that two experiments could be performed on a same silica-C18 gradient surface. The concentration of L D L were equal to 1/100 of its respective concentrations in normal plasma: apolipoprotein concentration was 0.01 mg/ml, the total concentration of L D L was 0.04 mg/ml. In the adsorption segment of the TIRF experiment, L D L solution flowed for 11 minutes through the channel with the rate of 0.84 ml/min initially displacing the DPBS buffer. In the 11 minutes desorption segment the flow was by switched back to the buffer solution. The fluorescence from the adsorbed L D L (FITC-LDL or DiO-LDL) along the silica-C18 gradient surface was excited by a spatially-filtered and expanded A r M o n laser beam (10 mW, @ 488 nm). The emitted fluorescence was passed through a monochromator (1681C, Spex Inc.) and recorded along the silica-C18 gradient surface every second by a charged couple device camera (Photometries Inc.). A l l TIRF experiments were performed at room temperature. 1 2 5

Protein Autoradiography Adsorption Experiments. In this experiment, IL D L was measured instead of FITC-LDL (or DiO-LDL). I - L D L was adsorbed onto the silica-C18 gradient surface from DPBS buffer. The concentration of protein solution, the flow rate and the adsorption-desorption cycle were the same as in the 1 2 5



374

1 2 5

TIRF experiments. The L D L solution contained a 1 : 4 ratio of I - L D L and unlabeled L D L . After the adsorption-desorption cycle, the adsorbed L D L on the silicaC18 gradient surface was fixed with 3 ml of 0.6% glutaraldehyde solution in DPBS buffer. A calibration plate with a set of known amounts of I - L D L was prepared separately as an autoradiography standard for the quantification of adsorbed protein. The silica-C18 gradient silica plate with the adsorbed and fixed I - L D L and the calibration plate were placed in a polyethylene bag and brought in contact with an autoradiography film ( X - O M A T A R , Kodak) in a light tight cassette. The film was exposed at low temperature (-70°C) for 21 days. The exposed film was processed in an automated developer system and its optical density was recorded by a custom-built densitometer (18). The adsorbed amount of L D L was calculated as a function of C-18 gradient position (19). 1 2 5

1 2 5

Results Characterization of Silica-C18 Gradient Surface. The advancing, 0 d , and receding, 0 , water contact angles of the silica-C18 gradient surface are shown in Figure l a as a function of the gradient surface position. The maximum angles, 0 dv = 104°, 0 = 85° were found at positions greater than 4.6 cm indicating the hydrophobic C18-silica surface is at the end of the gradient region. The contact angle hysteresis, i.e. the difference between 0 dv and 0 c, AO = 19°, indicated that the hydrophobic CI8-silica was not a defect free CI8 monolayer. The contact angles decreased smoothly towards the silica end of the gradient. At the positions smaller that 3.45 cm from the silica end the angles remained unchanged at 0 dv = 10°, O ec = 0°, indicating that the surface is a hydrophilic silica. The fractional surface coverage of the CI8 chains along the gradient, 0/0max(Cl8)> was calculated from the advancing contact angles using the Cassie equation (20). a

v

r e c

a

r e c

a

re

a

#

COsOadv = (©/©max(C18)>cos0ci8 + (1 - 0/@max(C18)) COsOsiiica

r

(1)

where the contact angle of a fully packed monolayer of CI8 chains, 0c18 = 112° (21) and clean silica 0 iiica = 0°. Figure lb shows the CI8 fractional surface coverage as a function of the gradient surface position. The arrows indicate the four positions along the gradient region where the fractional surface coverage was 0.12, 0.22, 0.33 and 0.72 respectively. s

Adsorption of L D L onto the Silica-C18 Gradient Surface. The L D L TIRF adsorption-desorption patterns are shown in Figure 2. The two markers indicate the CI8 gradient region. The flow of the protein solution was from the hydrophilic end towards the hydrophobic end of the gradient. The FITC-LDL adsorption results are shown in Fig 2a. The initial fluorescence increase was linear in the region of the hydrophilic silica surface. After this initial increase the fluorescence intensity reached a steady-state level. A very small decrease of fluorescence intensity in the desorption segment of the experiment indicated a slow desorption. The initial fluorescence in the C18-gradient region and at the CI8 end of the gradient increased slowly with time. The D i O - L D L adsorption results are shown in Fig 2b. Although the fluorescence intensity of the adsorbed D i O - L D L and the signal-to-noise ratio was lower than in the


HO & HLADY


3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 distance from hydrophilic end, (cm) Figure 1. (a) Water contact angles of silica-C18 gradient surface and (b) fractional C18 surface coverage, ®/®max(C18> shown as a function of gradient position.


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Figure 2. Spatially-resolved TIRF adsorption fluorescence pattern showing the fluorescence intensity vs. gradient position vs. time, (a) FITC-LDL, (b) DiOLDL.


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case of the FITC-LDL, the overall fluorescence pattern resembled the results of the FITC-LDL adsorption experiment. Quantification of Lipoprotein Adsorption. A quantitative analysis of the L D L adsorption kinetics required that the amount of adsorbed L D L is known as a function of time. Two independent quantification schemes were performed and the results compared. In first, the initial FITC-LDL adsorption (Fig 2a) on the hydrophilic silica was assumed to be limited by transport, i.e. by the availability of protein molecules at the adsorbing surface and the flux of protein molecules to the surface, dA/dt, was compared with the initial increase of adsorbed protein fluorescence, dF/dt (22,23). As described previously (8,20), when the desorption rate is very small or zero, this comparison can be used to calculate the adsorbed amount from the initial linear fluorescence increase since the flux of protein molecules to the surface, dA/dt, in rectangular flow channel can be computed from the Leveque equation: dA/dt = (r(4/3))-^9-l/3.(6q/b2 w 1 D L D L ^ - D L D L - C L D L

(2)

where T is the gamma function, q is the experimental volumetric flow rate (0.84 ml/min), b is the thickness of the TIRF flow cell (0.05 cm), w is the width of the TIRF flow cell (0.5 cm), 1 is the distance from the entrance of the flow chamber (2.8 7

2

1

cm), D L D L is the diffusion coefficient of L D L (1.8xl0' c m s" ) (75), and C L D L is the

bulk concentration of L D L . In the L D L adsorption experiment (Fig 2a) dA/dt amounted 1.84 x 10" pg cm" s" and the factor which relates the protein flux and the fluorescence increase, Z = (dA/dt)/(dF/dt) iii a (M£ c m count ) was 1.01 x 10" pg c n r count . A l l experimental FITC-LDL fluorescence results were converted to the surface density of LDL, TLDL> using the same conversion factor Z . In the second quantification scheme, autoradiography was used to measure the adsorbed amount of I - L D L along the silica-C18 gradient surface. Since in the autoradiography experiment the signal from the surface adsorbed I - L D L can not be recorded independently from the solution I - L D L , only the final adsorbed amount of I - L D L was measured after the unbound protein was washed out of the flow cell. The comparison between the two L D L adsorption quantification schemes is shown in Figure 3. Given the fact that the protein label was different, the agreement between the two quantification schemes was remarkable, especially at the both ends of the CI8 gradient, but worsened in the middle of the gradient. Accordingly, the analysis of L D L adsorption kinetics was limited to several positions close to the ends of the silica-C18 gradient surface (indicated with arrows in Fig lb). Figure 4a shows the respective experimental adsorption-desorption kinetics. On the hydrophilic silica surface the L D L adsorption reached a maximum of T L D L = 0.40 pg cm" after four minutes. The initial rate of L D L adsorption decreased and the linearity of the initial slope disappeared as the hydrophobicity of the surface increased. 2

2

1

- 2

s

2

-1

4

C

-1

1 2 5

1 2 5

125

1 2 5

2

Modeling kinetics of the lipoprotein adsorption. A simple protein adsorption model, to which the experimental data were fitted, comprised of two opposing processes: adsorption and desorption (24): dr(t)/dt = w a - r(t)/r )-c(0,t) - k H r ( t ) / r max

o f

m a x

)


(3a)

378


Figure 3. Comparison between the FITC-LDL adsorption quantified by using Eq 2 and the I - L D L adsorption measured by autoradiography, both shown as a function of fractional CI8 surface coverage. 1 2 5


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time, (sec)

Figure 4. (a) Experimental FITC-LDL adsorption-desorption kinetics, (b) Comparison between the experimental FITC-LDL adsorption (symbols) and the "adsorption sum" model (Eq 5, solid lines) for several positions along the silicaCIS gradient surface. Fractional C18 surface coverage, ® / © m a x ( C 1 8 > is indicated.


380


where k and k ff are the intrinsic adsorption and desorption rate constants, T is the maximum adsorption, T(t) is the adsorbed amount per unit surface at time t, (1 r ( t ) / T ) is the fraction of unoccupied adsorption sites and c(0,t) is the protein concentration right next to the adsorbing surface. A numerical computer routine was used to calculate c(0,t) from the Fick's diffusion law across the unstirred layer close to the surface and to model the adsorption using Eq 3a. The model also allowed that the intrinsic adsorption and desorption rate constants are defined as exponential functions of protein surface concentration, T: Q n

0

m a x

m a x

k k

o n

o f f

= k xp(-ar) = k exp(pD

(3b) (3c)

i e

1

where kj is the initial intrinsic adsorption rate constant, k_j is the initial intrinsic desorption rate constant and a and p are the "cooperativity" adsorption and desorption constants, respectively. Fitting of the experimental results of L D L adsorption was carried out for the hydrophilic (0 dv = 0°, ®/©max(C18) = 0) d hydrophobic CISsilica surface (0 dv = 104°, ®/©max(C18) = 0.90). k_j was found first by fitting the desorption segments (t > 11 minutes, Fig 4) to the Eq 3 using the condition: c(0,t) = 0. Tmax was assumed to be 0.4 pg c n r at the hydrophilic surface and 0.3 jag c m at the hydrophobic C18-silica surface. These two T values agreed very well with a previous study (75). The comparison between the model (solid line) and the experimental results (• symbols) is shown in Figure 5. The parameters used to achieve the fits shown in Fig 5 are listed in Table I. a n

a

a

2

- 2

MAX

Table I. The L D L adsorption parameters obtained by fitting the experimental results to the model given by E q 3

Oadv ®/®max(C18)

a P max (^gcnr ) k (cm pg" s ) r

2

3

1

_1

1

hydrophilic silica 0° 0

hydrophobic C18-silica 104o 0.9

-2 0 0.4 7.5* 102.9»10"

2.5 0 0.3 4>102>10"

4

4

1

Ki (s" ) K (M- )

6.5 *109

1

hypothetical CI8 monolaver 1210 1.0 2 0 0.26 2.2* 10-5

5

2.3* 10"

4

4.9 • l O

2.4 • l O

8

4

8

1

The apparent affinity constant, K, (in M " ) for each surface was calculated from the ratio k /k_ assuming that the M L D L = 2.6 • 10 Da. The apparent affinity of L D L for the negatively charged silica surface (K = 6.5 *10 M ) was larger by an order of magnitude compared to the hydrophobic C18-silica surface (K = 4.9 »10 M " ) . This difference was due to the intrinsic adsorption rates differences since the respective desorption rates were quite similar (Table I). 6

x

v

W

9

_ 1

8

1


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|

0

110

220

330

440

550

440

550

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time, (sec)

0

110

220

330

time, (sec) Figure 5. Comparison between the experimental FITC-LDL adsorption (•) and the model (Eq 3, solid line) for the hydrophilic silica (a) and hydrophobic CISsilica surface (b), respectively.

The L D L adsorption kinetics indicated that the adsorption onto the hydrophilic silica surface takes place via a mechanism that is different from the mechanism of adsorption onto the hydrophobic CI8 silica. To answer the question whether the L D L adsorption on a mixed hydrophobic-hydrophilic surface behaves as a simple addition of two independent adsorption processes or the hydrophobic-hydrophilic neighboring sites affect each other, the experimental L D L adsorption to the silica-C18 gradient region was fitted to an "adsorption sum" model. The model adds the adsorption on the hydrophobic (subscript (C18)) and hydrophilic (subscript ( u)) surface sites, respectively, and weights each adsorption process according to the probability of finding a given adsorption site: S

dT(t)/dt = (1 - 0/0max(C18)) {k n(sil) (l - r(t)/T x(sil)) c(0,t) - k ff(sil) (r(t)/r x(sil))} + (®/®max(C18)Wk n(C18) (l - r(t)/T ( i8)) c(0,t) - k ff(C18) (r(t)/T x(C18))} (5) #

#

O

#

ma

0

#

0

#

#

max

C

ma

#

0

ma

In order to be able to use the "adsorption sum" model (Eq 5), it was necessary to know the adsorption parameters for L D L adsorption onto a fully covered CI8 surface. Since the L D L adsorption kinetics at ®/®max(C18) = 0 and ®/®max(C18) = 0.90 were



382

experimentally determined, the hypothetical adsorption of L D L to a fully covered CI 8 surface, where 0/0 ax(C18) = 1.0, was estimated from the two experimental kinetics: m

r(t)(0/emax(C18)=l.O) = nt)(0/@max(C18)=O.9O) - OA* r(t)(@/0 (C18)=O) max

(6)

and the resulting adsorption vs. time data set was subsequently fitted to the Eq 3 to obtain the hypothetical k i , k_i and T (data shown in Table I). Eq 5 was used next to fit the adsorption kinetics measured in the C18 gradient region. The comparison between the experimental adsorption kinetics and the fits to the "adsorption sum" model is shown in Figure 4b. Included in the same figure is the hypothetical L D L adsorption fit for a fully covered CI 8 surface. The inspection of Fig 4b shows that the "adsorption sum" model (Eq 5) fits very well the experimental L D L adsorption for the CI8 surface coverage close to 1 or to 0. However, the fit worsens as one moves towards the middle of the gradient where ®/®max(C18) —> 0.5, especially in the initial adsorption stage. Notice that the adsorbed amount of L D L calculated from the fluorescence (Fig 3) showed an unexplained minimum at approximately half CI8 surface coverage. m a x

Discussion We have shown previously that silica-C18 gradient surfaces can be used to study how protein adsorption depends on surface hydrophobicity (20). The objective of this study was to determine how the surface density of CI8 chains affects the L D L adsorption. The adsorption of L D L was monitored using three different L D L labels: FITC-LDL, D i O - L D L and I - L D L . The TIRF measurements (Fig 2) suggested that FITC-LDL and D i O - L D L followed similar adsorption kinetics laws. Since these two labels tag different parts of L D L particle, one can tentatively conclude that both protein and lipid components of L D L particle adsorb together to the surface. It remains to be determined whether the structural integrity of adsorbed L D L particle is affected by a longer residence time at a given surface or not. The comparison between the two quantification schemes indicated that the L D L adsorption on the hydrophilic silica is indeed transport-limited. The adsorption rates, k i , (Table 1) were calculated using a simple adsorption model (Eq 3). In order to obtain the best fit between the model and the experiment, a small positive cooperativity (a = -2) had to be assumed for hydrophilic silica and a negative cooperativity (a = 2.5) for C18 silica, respectively. The physical meaning of the adsorption cooperativity also remains to be determined. The adsorbed amount of L D L decreased with increasing coverage of CI8 chains (Figs 2 and 3). It is known that L D L binds strongly to negatively charged adsorbents; some of the commercially available LDL-apheresis devices use negatively charged dextran sulfate adsorbents (25, 26). The electrostatic interactions can explain the order-of-magnitude larger affinity of L D L for a negatively charged silica surface (K = 6.5 • l O M" ) than for a hydrophobic C18-silica surface (K = 4.9 »10 M" ). The L D L adsorption took place from a buffer containing C a ions which might act as bridges between local negative charges of L D L and negatively charged silanol groups on the silica surface. The mechanism of L D L adsorption onto the hydrophobic C18-surface is less obvious. The adsorption kinetics suggests an energy barrier to adsorption, probably 1 2 5

9

8

1

1


+ +

26.

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due to the orientation of L D L particle during the collision with the surface. The energy barrier could also have an electrostatic origin. When a charged particle arrives into the close proximity of a low dielectric surface, electrostatic image forces are created opposing further approach and contact of the particle with the surface (27). The "adsorption sum" model fitted the experimental results very well at either end of the C18-gradient region. One can expect this since a smaller fraction of given surface sites can not dramatically influence the adsorption process occurring at other surface sites present in excess. In the middle part of the C18-gradient region the "adsorption sum" model failed to fit the experimental fluorescence results. Local retardation of the fluorescence-derived adsorption rates at approximately half CI8 surface coverage has been found in a number of repeated TIRF FITC-LDL adsorption experiments. The origin of this local fluorescence decrease is not known and has not been further investigated. Summary Adsorption kinetics of human low density lipoprotein ( L D L ) onto a silicaoctadecyldimethylsilyl (CI8) gradient surface was studied using the Total Internal Reflection Fluorescence (TIRF) and autoradiography techniques. The silica-C18 gradient surface was prepared by a two phases silanization reaction. The advancing water contact angles were used to calculate the fractional surface coverage of CI8 chains, which increased from zero (clean silica) to 0.9 on the hydrophobic end of the gradient surface. The F I T C - L D L adsorption rate was transport-limited on the negatively charged silica but significantly slower on the hydrophobic CI8 silica. The lipid labeled-LDL adsorption experiments resulted in a fluorescence adsorption pattern that resembled the protein-labeled L D L adsorption. A simple adsorption model was used to calculate the adsorption and desorption rate constants. The L D L adsorption was equal to a sum of adsorption processes on available hydrophilic and hydrophobic adsorption sites at either end of the CI8 gradient region. The middle part of the CI8 gradient displayed an unexpected retardation of the L D L adsorption rates. Acknowledgments The authors are indebted to Dr. Lilly Wu (University of Utah) for teaching them how to isolate human lipoproteins. We thank all blood donors for providing us with their low density lipoproteins and J. D . Andrade and H . P. Jennissen for helpful discussions. This work was financially supported by the Whitaker Foundation, NIH grant (R01 NIH-44538) and the Center for Biopolymers at Interfaces, University of Utah.

References 1. 2. 3.

Smelie, R. M. S., Ed.; Plasma Lipoproteins; Biochemical Society Symposium No 33; Academic Press: New York, NY, 1971. Gotto, A. M. Jr., Ed.; Plasma Lipoproteins; Elsevier: Amsterdam, 1987. Utermann, G. Science 1989, 246, 904-910.


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4.

Miles, L. A.; Fless, G. M.; Levin, E. G.; Scanu, A. M.; Plow, E. F. Nature 1989, 39, 301-303. 5. Hajjar, K. A.; Gavish, D.; Breslow, J. L.; Nachman, R. L. Nature 1989, 339, 303-305. 6. Simon, D. I.; Fless, G. M.; Scanu, A. M.; Loscalzo J. Biochemistry 1991, 30, 6671-6677. 7. Elwing, H.; Nilsson, B.; Svensson, K.-E.; Askendahl, A.; Nilsson, U. R.; Lundström, I. J. Colloid Interface Sci. 1988, 125, 139-145. 8. Hlady, V. Appl. Spectroscopy 1991, 45, 246-252. 9. Mills, G. L.; Cane, P. A.; Weech, P. K. in A guide book to lipoprotein technique, Laboratory techniques in biochemistry and molecular biology, Vol. 14.; Burdon, R.H.; van Knippenberg, P. H., Eds.; Elsevier: Amsterdam, 1976; pp 18-78. 10. Lowry, O. H.; Rosebrough, N. J.; Farr A. L.; Randall, R. J. J. Biol. Chem. 1951, 193, 265-275. 11. Peterson, G. L. Anal. Biochem. 1977, 83, 346-356. 12. Soutar, A. K.; Myant,N. B. In International Review of Biochemistry, Chemistry of Macromolecules IIB, Vol. 25; Offed, R. E., Ed.; University Park Press: Baltimore, MD, 1987, pp 55-119. 13. Andrade, J. D.; Smith, L. M.; Gregonis, D. E. In Surface and Interfacial Aspects of Biomedical Polymers, Vol. 1; Andrade, J. D., Ed.; Plenum: New York, NY, 1982, pp 262-290. 14. Coons, A. M.; Crech, H. J.; Jones R. N., Berliner, E. J. J. Immunology 1942, 45, 159-170. 15. Hlady, V.; Rickel J.; Andrade, J. D. Colloids and Surfaces, 1988, 34, 171-183. 16. Stephan, Z. F.; Yurachek, E. C. J. Lipid Res. 1993, 34, 325-330. 17. Shepherd, J.; Bedford, D. K.; Morgan, H. G. Clin. Chem. Acta, 1976, 66, 97109. 18. Ho, C.-H.; Hlady, V.; Nyquist, G.; Andrade, J. D.; Caldwell, K. D.J.Biomed. Mater. Res. 1991, 25, 423-441. 19. Lin, Y. S.; Hlady V.; Janatova, J. Biomaterials 1992, 13, 497-504. 20. Lin, Y. S.; Hlady V.; Gölander, C.-G. Colloids and Surfaces, 1994, in press. 21. Ulman, A.J.Mat. Ed. 1989, 11, 205-280. 22. Lok, B. K.; Cheng, Y.-L.; Robertson, C. R.J.Colloid Interface Sci. 1983, 9, 87-103. 23. Lok, B. K.; Cheng, Y.-L.; Robertson, C. R. J. Colloid Interface Sci. 1983, 9, 104-116. 24. Corsel, J. W.; Willems, G. M.; Kop, J. M. M.; Cuypers, P. A.; Hermens, W. T. J. Colloid Interface Sci. 1986, 111, 544-554. 25. Yokoyama, S.; Hayashi, R.; Kikkawa, T.; Tani, N.; Takada, S., Hatanaka, K.; Yamamoto, A. Arteriosclerosis 1984, 4, 276-282. 26. Ikonomov, V.; Samtleben, W.; Schmidt, B.; Blumenstein, M.; Gurland, H. J. Int. J. Artif. Organs 1992, 15, 312-319. 27. Israelachvili, J. Intermolecular and Surface Forces, 2nd ed., Academic Press, London, 1922. RECEIVED December 22, 1994


Proteins at Interfaces II - American Chemical Society

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