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Archs oral Bid. Vol. 37, No. 7, pp. 549-557, 1992 Printed in Great Britain. All rights reserved

copyright 0 1992 Pqamon

ADSORPTION FROM SALIVARY FRACTIONS AT SOLID/LIQUID AND AIR/LIQUID INTERFACES El.VASSILAKOS,~J.

RUNDEGRRN,’T. ARNEBRANT*~~ and P.-O. GLANTZ'

‘Department of Prosthetic Dentistry, Dental School, Carl Gustafs viig 34, S 214 21, Malmo, 2Biosurface AB, Box 839, S-201 80, Mahnii and ‘Department of Food Technology, University of Lund, Box 124, S 221 00, Lund, Sweden (Received 30 August 1991; accepted 25 February 1992) Summary-Ellipsometry and the drop-volume technique were used to study the interfacial behaviour of fractions obtained from unstimulated whole saliva. Fractionation was by gel filtration on a Superdex 200 Hiload column equilibrated with 10 mM potassium phosphate buffer, pH 6.8, containing 0.15 M NaCl. The fractions were reconstituted to have the same absorbance at 215 nm (estimated molecular-weight range, F,>76046OK, F2 20%39K, F3 144SK, F, 4.5-2.5K, Fs 1.5-0.85K, F6 0.857205-39 14-4.5 4.5-2.5 1.5-0.85 0.85~GO.5

*The values shown for saliva are those calculated for the unfractionated saliva diluted to 49%. The fractions are estimated to give final A,,, values corresponding to 49% saliva (1.55).

The solution was agitated by a magnetic stirrer operating at the bottom of the cell at a constant rate of 325 rev/mm. The temperature was kept constant at 37°C by circulating thermostated water within the holder of the cell. Each experiment was done twice. RESULTS

Chromatograms at Azso and A,,, from the saliva fractionation are shown in Fig. 1. The A,,, absorbance showed that fraction F2 contained two peaks and that F4 appeared as a shoulder. The F, fraction, which appeared as a small shoulder peak at A 2w, absorbed strongly at A,,, . Preparatory data and estimated molecular-weight ranges for the fractions are shown in Table 1. Before lyophilization of the F6 fraction an unsuccessful attempt was made to concentrate this fraction on an YCOS (Amicon) membrane with a cut-off of 500 mol. wt. The hydrophobic/hydrophilic ratio was highest for fraction 3, with a value of 3.21 (Table 2), followed by fraction 2 and 4 at 1.57 and 1.22, respectively. Fractions 1 and 6 had the lowest hydrophobic/ hydrophilic ratios, both 0.62.

The amino

acid composition

of the fractions

is

shown in Table 3. The largest amounts of proline were detected in F3 (25.6/1OOmol) followed by F, (23.1/100 mol) and F, (22.1/100 mol). Fraction 1 was rich in serine (12.4/1OOmol) and threonine (11.7/100 mol). Fractions 5 and 6 were rich in histidine with values 7.8 and 8.3/1OOmol, respectively.

Table 2. Results from the hydrophobic interaction chromatography of the salivary fractions Sample

4 F2 F3

F, FS F6

ws

Ratio 0.62 1.57 3.21 1.22 0.94 0.62 3.36

Numbers represent the ratio of hydrophobic/hydrophilic area in the obtained chromatogram. WS, whole saliva.

552

N.VA.WLAKOS

et al.

Table 3. Amino acid composition of salivary fractions and whole saliva (WS) expressed in mol/lOOmol Ala Arg Asx CYS Glx Gly His Ile Leo LYS Met Phe Pro Ser Thr TYr Val

8 9 2.4 6.9 2.7 10.6 10.4 1.9 2.4 4.9 2.7 0.3 4.2 10.6 12.4 11.7 1.4 4.5

F2

F,

F,

Fs

F6

WS

2.6 3.6 8 0.6 22.4 17.1 1.7 1.6 3.3 3.5 0.2 2 22.4 5 1.4 0.6 3

2.8 4

1.5 5.4 49 0:6 20.4 14.7 2.5 1.2 3.4 3.8 ND 3.5 23.1 5 1.1 6.8 1.5

3.4 6.7 59 1:2 16.7 13.1 7.8 1.5 3.3 5.6 ND 4.2 14.3 5.2 1.2 7.3 1.7

7.4 3.3

3.2 5.3 78 0’6 17:1 13.7 2.6 2.5 4.7 4.5 0.4 3 19.7 5.2 2.2 3.7 3.1

8’: 18:7 16.5 1.3 1.9 2.9 3.9 0.2 2.4 25.6 5.6 1.5 2.2 2.5

I% 21.6 18.3 8.3 1.2 3.4 3.3 ND 4.3 7.6 6 1.1 6 3.2

ND, Non-detected. The interfacial tension decay for the different fractions is shown in Figs 2(a) and (b). After 30 min F2 and F3 had decreased the interfacial tension more than 20% whole saliva. Fz quickly reached a plateau value at 52 mN/m. The surface tension of 20% saliva after 30min was about 54mN/m. F, slowly lowered the interfacial tension and it reached 59mN/m at 30min. The corresponding value for F4 was about 55 mN/m. Fractions 5 and 6 only caused small decreases in the surface tension. The adsorption kinetics for the different salivary fractions on the hydrophilic and hydrophobic surfaces are depicted in Figs 3(a) and(b), respectively; the curves are representative ones out of two replicate exper-

iments. Table 4 summarizes values obtained at different stages of the adsorption for all of the fractions. On hydrophilic surfaces F, gave the largest adsorbed amounts compared to the other fractions [Fig. 3(a)]. After rinsing, a small part of the film desorbed. Fractions 2 and 3 reached lower adsorbed amounts and small parts of the formed films desorbed upon rinsing. The adsorption curves revealed that F2 very quickly reached a plateau value and no further adsorption took place. F3 rapidly formed a fdm of 0.02 pg/cm* and then a small increase in the adsorbed amount slowly took place. Fraction 5 adsorbed in larger amounts than F2 and 4. From the adsorption curve, however, it is obvious that the kinetics of this

65

0

500

1000

1500

2000

Time (s) Fig. 2. Surface-tension reduction measurements for the unfractionated saliva and fractions 1,2. A, Whole saliva; 0, fraction 1; n , fraction 2.

Adsorption from salivary fractions

553

0 0

0

0

0

OQI

n n

m

50 0

500

2000

1500

1000 Time (s)

Fig. 3. Surface-tension reduction measurements for the unfractionated saliva and fractions 3,4,5 and 6. A, Whole saliva; A, fraction 3; n , fraction 4; 0, fraction 5; 0, fraction 6.

process were slow and that there was tendency for further adsorption. Upon rinsing no desorption took

DISCUSSION

place. Fractions 4 and 6 did not adsorb at all on the hydrophilic surfaces. During 30 min of adsorption the greatest amounts adsorbed on hydrophobic surfaces were recorded for fraction 3 [Fig. 3(b)]. Upon rinsing, however, a large part of the film desorbed. Fl adsorbed with similar kinetics as on the hydrophilic surfaces. A small part of the film desorbed after rinsing and the remaining amounts were the greatest recorded on hydrophobic surfaces. F2rapidly reached a plateau value and no further adsorption took place. Upon rinsing a small part of this film was found to desorb. F, adsorbed slowly in increasing amounts and no desorption took place after rinsing. Fraction 5 adsorbed with slow kinetics during 30 min and also exhibited a tendency for further adsorption. Fedid not adsorb at all on the hydrophobic surfaces. In general terms comparatively larger amounts of the fractions were adsorbed on the hydrophobic surfaces than on the hydrophilic ones [Figs 3(a) and (b) and Table 21.

It is well established that mass accumulation at solids in contact with biological fluids is preceded by the spontaneous formation of a proteinaceous biofllm (Baier, 1970). Such films determine the outcome of interfacial events taking place, for example, during the implantation of artifical devices in tissues, marine fouling, the formation of dental plaque and the contamination of contact lenses (Baier et al., 1984; Meyer ef al., 1988; Glantz, 1980). Biological flhns in the mouth, commonly called acquired pellicles (Meckel, 1965), form very quickly (Baier and Glantx, 1978). When the kinetics of salivary pellicle formation were studied by X-ray photoelectron spectroscopy (Kuboki, Teraoka and Okada, 1987), the main body’ of the pellicle was formed in 30 min and that the adsorption process levelled off in 2 h. Siinju and Rolla (1973) earlier reported similar findings. Therefore, 30min were considered as sufficient to monitor salivary adsorption and the reduction in surface tension. Ellipsometry, being a non-destructive technique, has been used frequently in investigations of thin-film

Table 4. Adsorbed amounts on hydrophilic and hydrophobic surfaces recorded by elhpsometry before and 30 min after end of rinsing Adsorbed amount fig/cm2 After rinsing

Before rinsing Surfaces

F,

FZ

F3

F,

HPHILIC HPHOBIC (0.05)

Fs 0.11 (0.14) 0.17 (0.18)

Records of replicate experiments are shown in parentheses.

F, 0 (0) 0 (0)

FI 0.18 (0.17) 0.24 (0.25)

F2 0.04 (0.04) 0.11 (0.09)

4 0.015 (0.010) 0.17 (0.15)

F4 0.05 (0.05)

g.3

F,

0.11 (;;;)

-

(0:17)

-

554

N.~ASSILAKOS et al.

0.3 R

Time (s)

Fig. 4. Recordings from the adsorption of the fractions on hydrophilic surfaces. R indicates rinsing with PBS. formation in a variety of scientific fields (Azzam and Bashara, 1977; Baier and Glantz, 1978; Ivarsson and Lundstrom, 1986). The instrument we used allows for in situ measurements with fast resolution. Therefore, the method was considered very suitable for

investigation of fast adsorption processes, such as salivary film formation. Vassilakos, Amebrant and Glantz (1992), using the same instrument, found that the coefficient of variation of ellipsometric measurements was 10.5%

A Fl l

F2

w F3 A F4

0 F5 o F6

0 0

500

1000

1500

2000

2500

3000

3500

4000

Time (s)

Fig. 5. Recordings from the absorption of the fractions on hydrophobic surfaces. R indicates rinsing with PBS.

Adsorption from salivary fractions

when fresh whole saliva was used as the adsorbate. Fractionated salivary samples should be more homogeneous than fresh whole saliva donated individually before each experiment. Therefore, even though we did not study the ellipsometric accuracy and precision because of the limited amounts of the saliva available, it is likely that our coefficients of variation will have been at least at the same level as those observed for fresh whole saliva. Thus we considered the precision of the determinations to be satisfactory. By not allowing any muscle activity during the collection of saliva, we sought to minimize contamination in order to reduce selective adsorption of proteins on to dislodged cells. There was’s correspondence between the Azsa and the A,,, gel-filtration chromatograms for whole saliva and thus the A,,, reconstitution of the salivary fractions selected from the A,,, chromatogram was considerd justified. The A,,, evaluation for reconstitution waS chosen to increase the sensitivity for conjugated proteins in the fractions. The retention time of the salivary fractions on the Phenyl-Superose column was similar, and they generally eluted at around SO-70% of the gradient. To obtain a comparison of the relative hydrophobicity of the fractions the ratio between hydrophobic peak areas and the area of the hydrophilic peak was used to express the relative hydrophobicity of the fractions. The drop-volume method, used to study the surface-tension decay, has been satisfactorily used for investigations of the interfacial behaviour of a large number of proteins (Tomberg, 1978; Tomberg, 1987). We used double-distilled water throughout in order to check the accuracy of the instrument. Thus, it was unlikely that the surface-tension measurements were influenced by contamination from impurities. Our results indicate that there are distinct differences in the interfacial behaviour of the various salivary fractions. A common observation, however, for all of the fractions was that great amounts were adsorbed on hydrophobic than on hydrophilic surfaces. These observations are in agreement with those from studies of adsorption from whole saliva (Vassilakos et al., 1992) and from parotid saliva (Simonsson, Amebrant and Petersson, 1991). A major part of pellicle material is net negatively charged (Eggen and Rolla, 1982), and under our experimental conditions the silica surfaces were alSo negatively charged [isoelectric point 2 (Parks, 1965)]. As a consequence, electrostatic repulsion between the substrate and the salivary molecules or between the already adsorbed molecules and those that approach the interface might thus have limited a’dsorption of larger amounts on the hydrophilic surfaces. Adsorption on the hydrophobic substrates might, on the other hand, have involved interactions between the surfaces and hydrophobic patches located in the exterior of the salivary molecules. Such interactions might also have taken place with hydrophobic domains originally located in the interior but exposed after the conformational changes of the molecules that are known to occur upon adsorption (Norde, 1986). The high molecula.r-weight fraction F, adsorbed in large amounts on both types of surfaces. The presence of charged glycoproteins in this fraction is

555

indicated by the high content of serine and threonine. Adsorption on hydrophilic surfaces might take place through electrostatic interactions or entropically favourable conformational changes involving increased disorder in the protein molecules by, for example, loss of the secondary structure (Norde, 1986). Adsorption on hydrophobic surfaces is expected also to involve hydrophobic interactions between adsorbing molecules and the substrates. The results from the drop-volume experiments for the I;; suggest that after 30 min the reduction of the surface tension was rather small. A tendency for further decay was, however, observed. It has been suggested that the process of protein adsorption at the air/liquid interface is the result of a number of subprocesses (Tomberg, 1978; Tomberg, 1987; De Feijter and Benjamins, 1986). They involve diffusion of the proteins to the subinterface region, adsorption at the interface and rearrangements of the molecules within the adsorbed 6lm. Furthermore, the behaviour of proteins at the air/liquid interface has been related to their adsorption on hydrophobic substrates (Amebrant et al., 1984). The small and slow decay in surface tension by F, might, therefore, be explained in terms of the low rate of diffusion of these large molecules and a longer time-scale for rearrangements taking place at the interface compared to those for the lower molecular-weight fractions. In addition, a longer time might be necessary to optimize the adsorption at the air/liquid interface than at the solid/liquid interface, owing to different mass-transport conditions in the two systems. The ellipsometric system is vigorously stirred whereas the drop-volume system is virtually unstirred. Further, the low relative hydrophobicity of this fraction points out to low amphiphilicity of the contained molecules, which is in line with the observed small reduction in the interfacial tension. Fraction 2 quickly reached a plateau value on both types of surface showing high affinity for the interfaces. The ellipsometric observations are in agreement with the drop-volume data, where a large reduction in the surface tension that reached a steadystate value within 10 min was observed. The desorbable parts upon rinsing suggest that molecules with different binding strengths had adsorbed within the film. This may be due to multiple adsorption states of one type of molecules (Horbett and Brash, 1987) or to adsorption of different types of molecules. The presence of two peaks in this fraction supports the latter. The proline-rich fractions 2, 3 and 4 were also found to have the highest relative hydrophobicities among the fractions. Proline-rich proteins readily adsorb on to enamel and are part of the salivary pellicles (Bennick ei al., 1983). These findings, which indicate that proline-rich proteins are surface active and have high affinity for hydroxyapatite, are in agreement with ours which show that the proline-rich fractions have high amphiphilicity. Fraction 3, which was the most proline-rich fraction, showed remarkable differences in the adsorption behaviour on the different substrates. Small amounts were adsorbed on the hydrophilic surfaces, whereas on the hydrophobic very large amounts were recorded. This is in good agreement

556

N. V~~LAKOS et al.

with the results from the hydrophobic interaction chromatography, which indicate a large hydrophobic contribution in the fraction. After rinsing a large part of the film desorbed from the hydrophobic surfaces. This finding suggests again the presence of proteins with different binding strengths within the film. The reduction of surface tension after 30min was the largest amongst all of the fractions. This observation agrees well with the fact that before rinsing this fraction gave the largest amounts adsorbed on the hydrophobic surfaces and that this fraction showed the greatest relative hydrophobicity. For fraction 4, which is also rich in proline, no adsorption took place on the hydrop~lic surfaces. It is possible that electrostatic repulsion between the substrates and the molecules of this fraction prevented adsorption. On the hydrophobic surfaces, adsorption of small, slowly increasing amounts were recorded. The molecules were obviously firmly bound to the surfaces, as no desorption took place upon rinsing. The driving force for such adsorption might be a dehydration of exposed hydrophobic parts of the molecule. Further, the kinetics of adsorption on the hydrophobic surfaces correspond with the surface tension, which decreased slowly. For fraction 5, slowly increasing amounts were adsorbed on both hydropholic and hydrophobic surfaces. Relatively large amounts were adsorbed on hydropholic surfaces compared to the other fractions. This might be attributed to the relative’high content of basic amino acids (histidine, arginine, lysine) in this fraction. No desorption took place upon rinsing. A slow decrease in the surface tension was also observed, which is in line with the low relative hydrophobicity of this fraction. The kinetics of the interfacial events suggest that either the molecules might self-associate before or upon adsorption or that they have low affinity for the interfaces. Finally, the smallest molecular species in fraction F6 did not adsorb on either of the surfaces, probably due to a low amphiphili~ity of the molecules, which is further supported by a low surface-tension reduction. The low relative hydrophobicity ratio of this fraction is an additional indication of low amphiphilicity. Our results show large differences in the biophysical properties among the various proteinaceous components of saliva. Furthermore, the adsorption ~haviour of each one of them obviously is related to the physicochemical properties of the interfaces. Therefore, it is likely that the physicochemical characteristics of a surface that comes into contact with the pool of salivary proteins will influence, to a large extent, the ‘race’ for the interface of the different proteins present. This is in agreement with tidings of differences in the amino acid composition of salivary pellicles formed on solids with different physicochemical characteristics (SSnju and Glantz, 1975). There are also differences in composition between salivary pellicles extracted from enamel and from exposed root surfaces (Ruan, Paola and Mandel, 1986). This influence of the substrate characteristics on the adsorbed proteins has also been described for the type of proteins retained on surfaces in contact with blood (vroman et al., 1971). Interactions between the different proteins in saliva are expected to alter their individual interfacial

behaviour. It has, for example, been demonstrated that the presence of other proteins influences the adsorption behaviour of proline-rich proteins (Bennick et al., 1981) and that coadsorption with phosphoproteins slows down the rate of adsorption of salivary mucins (Amerongen et al., 1990). Tabak et al. (1985) have also shown that presence of cysteine-containing salivary phosphoproteins inhibited the adsorption of salivary mucins. We show that, due to differences in their individual diffusion kinetics, the proteins arrive at the interfaces at different times and that their varying binding strengths might then lead to replacement processes. This finding is in agreement with those which indicate that certain proteins in the fresh pellicle gradually disappear in aged pellicles (Rennick et al., 1983). Embery, Heaney and Stanbury (1986) found lower molecular-weight proteins in the more tightly bound part of salivary pellicles, whereas higher molecularweight proteins were adsorbed in the more readily removable layers. These data are in line with our results from the surface-tension reductions, which show that fractions 2 and 3 form films at the air/liquid interface much faster than the large molecular-weight proteins and also that they result in much greater reductions in surface tension than F,. However, in clinical studies, the critical surface tension of ‘mature’ pellicle covered teeth was found to be similar for a large number of persons (Jendresen and Glantz, 1980). Our findings thus suggest that the adsorption behaviour of salivary proteins depends on the physicochemical properties of the interface. There are distinct differences among the different fractions. Electrostatic and hydrophobic interactions are probably important for the adsorption processes. For all salivary fractions, larger amounts were adsorbed on the hydrophobic substrates than on the hydrophilic. REFERENCES

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liquid interfaces.

Ellipsometry and the drop-volume technique were used to study the interfacial behaviour of fractions obtained from unstimulated whole saliva. Fraction...
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