aqueous solution interface

297 - 306 B.V., Amsterdam

Colloids arrd Surfaces. 9 (1984)

Elsevier Science

Publishers

297 -

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in The Netherlands

IN SITU ADSORPI’ION OF BOVINE SUBMAXILLARY MICA/AQUEOUS SOLUTION INTERFACE E. PEREZ,

J-E. PROUST,

A. BASZKlN

and MM.

MUCIN AT TI1E

BOlSSONNADE

Loboratoirc de PhysicoX%inlic des Surfaces et dcs blctnbrancr. Equipe de Hechrrehe du CNRS mm&e ci Wnit~crriti Park V. UKR Riomhdicate, 45 rue dcs Saints-P&es. 75270 Paris cfidcx 06 (France) (Received

11 June

1983;

accepted

in final form 1 November

1983)

ABSTRMX An ha situ adsorption measurement method at the mica/protein solution interface is described. An apparatus erpccially constructed tar this purpose permits direct and continuous measuronrcnt of total adsorption (reversible and irreversible) of “C-labelled proleh~. Bobinc ruhtnaxillary muctn (BSM), extracted from salivary glands, was acetylatcd with (CH, “CD), 0. The rrsults show thr increase of BShl adsorbed on mica surfaces with its concentration in solution and adsorption time. Pseudo plateaux are obtained for all concentrations studied, indicating the formatlon of thick layers. The loosely tround fractlon of adsorbed mudn is proportional to the hulk concentration in solution. The amounl or BSM adsorbed increases in lhc neighbouthood of the isoelectric point of X3M (pi1 3).

During recent years various ceramics have been applied as medical and dental implant materials [I $21 t The common feature of all these bioceramics is that SiOz forms their primary network. Adding AlaO, or P,O, to a glass composition can result in formation of alumina-silicate or calcium phosphate layers on top of a silica-rich substance. These protective layers arc supposed to increase the durability of hioglassos In alkali and acid solutions 131. When such ceramics are used as biomaterials they interface with the tissue so that the interactions with different physiological constituents such as collagen, mucopolysaccharides and mineml salts take place. All these interactions determine whether an implanted material may be considered as biocompatible or not in a given physiological environment. The concept of this work is to study adsorption at the mica-bovine submaxillary mucin solution interface, to get some insight into the behaviour of certain bioglasses in contact with mucosal surfaces. Muscovite mica is a layer structured aluminosilicate and its basic chemical formula is KA12(AISiSO~a)(OH)2 [S] . Mucins, gtycoprotc!ins in the mucous secretions of submaxillary glands, have been shown to consist of a polypeptidc chain with many relatively small 0166.6622/84/$03.00

6 1984

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oligosaccharide side chains linked to the peptide through glycosidic bonds i5]. Their function is to lubricate epithelial cells in the mouth. respiratory, gastro-intesttnal and reproductive tracts and to protect them from intimate contact with the external environment [Sl . Bovine submaxiltary mucin (IBM) is a large, highly asymmetric, rod-like molecule, with a molecular weight of 4 X IO6 [S] . In this !.aper, which is an extension of our earlier investigat,ion of protein adsorption on polymer surfaces [7,8), we describe a new technique of in situ adsorptionjdesorption measurements of ‘?XabeH& proteins on mica surfaces. Direct relationship of these experiments with the measurements of forces between two mica surfaces separated by mucin aqueous soluMon, which are now being carried out in our laboratory using lsraelachvili technique 191, can be ant,icipated. EXPERIMENTAL

BSM isolatiotr and mdiulabellillg BSM was isokted from fresh salivary glands obtained at a slaughter house by a procedure dcscribti by Tettamanti and Pigman [IO]. The lyophtlized mucin was analysed to determine the sialtc acid content which specifically clraracterizcs glycoprotcins, Using the periodate-resorcinol method [ 111, the mucln was found to contain about 30% sialic acid, a value in good agreement with thut reporled in Ref. tl0], 12ndiolabclling of t31c lyophilized mucin by acotylation with [l-“‘C J acelic anhydride is described elsewhere (121. The amount of NH3 groups acctylated during this reaction and determined by a colorimettric ninhydrin reaction nccording to Yemm and Cocking (131 was found to be 40%. The total protein concentration after dialysis of acetyIat& mucin found by t-he Lowry method [ 141 was 0.55 mglml. Finally its specific activity, estimated by comparison with 1“Cl hcxadccyltrhnet-hyl ammontum bromide solution of known spccific activity, was about 4.4 pCi/mg of dry protein. The surface tension measurements of acetylated and nonacctylated mucin show that acetylation did not change the surface activity of BSM 1121.

A new apparatus and technique have been used to measure the adsorption of mdiolahellcd proteins. The principle of t-he apparatus is illustrated in Fig. 1. A specially constructed circular glass container with fixing screws enables one to form a cell with a mica window at the bottom. The mica windows are freshly cleaved under a laminar flow cabi::& and their thickness is ca. 10 pm. Molten paraffin is spread onto the flat ground part of the glass cell and the glass container is tightly sealed by means of Viton “0” rings. The cell is filied with “C-labelled protein (ca. 3 ml) and placed in a special support above the

299

Pif 1. In situ adsorption measuring dcvicc. (I), (2). (8) ~upport5ensuring rcproducibilitg of gcomctrical conditions; (3) mica window; (4) cover; (5) glaxs cell; (6) “0” ring; (7) ceil as5cmbling screws.

gas flow counter corder

which measurc~ the radioactivity

13s a function

and displays

it on a re-

of time.

Thus, the in situ aclsorl,tion/dcsorption of proteins on solid surfaces can he measured. mntmry to other commonly used techniques to measure prctcin adsorption on solid surfaces, our method enables one to distitqpish ixttvecn the reversibly and irreversibly adsorbed fractions of the protein. Two calibrations are required to know the quantity of adsorbed protein mole-

cules. Because the mica windows varied in thickness it was necessary, bcforc each measurement, to determine how much radiation they absorb. This was

done using the solid polymer source, in our case [“Cl poly(methy1 methacrylate). The absorption coefficient for mucin windows varied within 30--50% range of total radioactivity originating from the source. Allowance has also to be made for the radioactivity originating from the hulk protein molecules, nonadsorbed at the mica/solution interface but con-

tributing to the total measured radioactivity. This value is proportional to the BSM concentration and may be obtained when instead of [ “C) BSM a nonadsorbing substance containing the same : adioactive element 14C is used. Aqueous solutions of potassium thiocyanate (K’*CNS) which were isotopb tally diluted to give the same specific activity as that of [**Cl CSIU were used for this purpose. At low BSM concentrations < f X 10” mg/ml the bulk radioactivity contribution to the total measured radioactivity is negUgible and for adsorption at the highest bulk concent.ration studied (2 X 10-l mgl ml) this contribution represented about 2G% of t.he total adsorption value. The total amount of BSM adsorbed on the mida sample was calculated by means of a calibration graph, previously determined by depositiug and drying on mica surfaces known am0unt.s of labelled protein which, when counted, yielded a factor per unit amount of BSM. Knowing the conversion factor and the area of the mica window, the amount of BSM adsorbed (H/cm’) was obtained. All experiments were carried out in triplicate with each sample beitlg counted three times; an overall mean value was then taken. The amount of mucin irreversibly adsorbed on the mica surfaces was abtnined by replacing the BSM solution with water or with a buffer sole JOII in the measuring cell. It has been recently shown [ 151 that thr?reductive methylation of chicken eggwhite lysozymc with ‘ECHO has significant effects on its adsorption bchaviour on hydrophilic and hydrophobic surfaces - for example, on both surfaces the labclled mzrtcrial adsorbs preferentially to the unlabelled. The feasibdity af using ‘*‘I- and P9mTc-lnbeIld albumin for adsorption studies on different. surfaces has also been questioned [ lG] .To test if such artefacts

Fig. 2. Test 4wwing the equivalence of adsorption of labellcd and unlabclled BSII. Tatal BSM concenkration of the labclled~unlabelled mixlurc in solution 0.1 mg/ml; [NaCl J = lO“.‘If;

adsorplion

time 5 IL

30s

involved in the adsorption measurements which we have performed on mica, the percentage of labelled protein was varied from 20 to 100% with constant bulk concent.ration of 0.1 mg/ml and 0.16 ASNaCI. The results shown in Fig. 2 indicate that neither the labclled nor the unlabelled mucin molecules are preferentially adsorbed.

are

RKSULTS

AND DISCUSSION

Rates of adsorption have been determined for various mucin concentrations in bulk solution at constant ionic strength {NaCI] = lo-‘M. The resu1t.s are represented in Fig. 3. The first meaningful p0int.s of adsorption kinetics registered with out experimental technique are those after about 16 min from the beginning of adsorption. The curves in Fig. 3 show also that initial rates of adsorpt.ion, for ah concentrations st.udied, are high and then a more gradual increase of adsorption takes place. In all cases the adsorption process did not attain the steady-state values and the pseudo plateau was observed on all curves. If the surface concentrations of these adsorption experiment-s were plotted versus square root of adsorptIon time, they would yield the curves shown in Fig. 4. The dashed portions of Fig. 4 are the ext.rapolation of the cu~yes to the zero adsorption time. Assuming the dtffusion-controlled process, the diffu-

IO”mq/ml

ZQ.4 0 .C E;: “, 0.2.

_ a .l:i-* rnq/pl 5.111”rncy .,I! 2.19”mq/ml lO”mg/m\ I

Fig. 3. Kinetics 10-111.

10 hours of BSM adsorption

20 on mica for VI rious much concentrations [ NaCl J =

502

-%erc

c is the protcin

arid I the ndsorptian

conccntralion

t inw.

in solution,

D lhc diffusion

cocfficicM

For n given protein conconlralion, the initial stope of the curve A vs. f”* is us4 to dctcrmine the diffusion coefficient I). As illustrated in Fig. 5 thz obtain4 unluc~ for D change with the BSM st+tt,ion conccntrat ion, Sucn concentration dapondenco of D may be related tcb the highly pronounced tendency totvards aggrq&on of mucin molecules in solution. Previous particle size ncasurPmtnts performed with a Coulter Nanu-Sizr?r instrument i 12 3 show that the mean radius of SSM macromolecules at- pll 7.1 and 0.154V NaCt increases from 130 nm for 10” mg/ml to 1000 nm for 4 X lomg/ml., Also, other researchers (17,181 found t.haC the size and molecular weights of mucins may be considerably different, depending on their origin, concentrstion, tonic strength and p&I. Prom surface tension measurements of BSM saline aqueous solutions, tio11y [19) reprtWI a value of 2.3 x 10 ‘)*m* 1s for the apparent diffusion cocfficicnt~ and described this value as a realistic one for macromolecules of

303

w 8 C 0 .I

m

Fig. 5. Conccntratfon dependence af ditCusbn co,offklcntof ESShf in 1Om3 M NaCI.

this size, Shogren et al. [ 171, using a light scatteringtechnique, measured the diffusion coefficient of porcine submaxillary mucin in solWon and obtainod vaiues ranging from 0.4-2.6 X 10mB2 mz/sdepending on the protein concentration In solution. If the Initial slope of BSM adsorption vs. tllz were calculated with the ap-

parent diffusion coefficient value given by Holly, then the straight hnc shown Tn Fig, 4 would be obtained. On the mica surfaces the adsorption process is much slower, According to Van Dulm anrl Norde [ 20). who studied the adsorption of albumin on different solid surfaces, the probable reason that the adsorption process does not follow the diffusion-controlied mechanism is due to the exIst.enceof a bartIer (electrostatic, orientational or steric) against deposition on the already arrived molecules. They observed such slowing down of adsorption for the system where both the adsorbing sutfttce and the protein molecules were negatively char&. This is also the case of our mica-mucin system. Furthermore, the first force/distancemeasurements between two mica surfaces covered with adsorbed BSM layers have shown that repulsion occurs in the initial stage of bringing together two mica surfaces. Such repulsion may be explained by electrostatic and steric hlndrancc 121]. Moreover, when comparing our results for BSM diffusion coefficients on mica surfaces with the value reported by Holly ( 191 for the BSM apparent diffusion coefficient, it should be borne in mind that the mucin used by Holly was Sigma commercial grade and as such should be different from our highly purified BSM. In Fig. 6 the total amounts of adsorbed mucin are plotted against the solution concentration. These results demonstrate that thick mucin layers are

built up on mica surfaces with increasing protein conccntrnt-ion in solution. Also the loosely bound fractkm of the adsorbed mucin (the amounts curtcsponding Co the difference bet.wccn two curves in Fig. 6) increaseswilh the protein conccnt,ration in the solution. Similar results were found by us for mucin adsorption on surface oxidized polyethylene films 1121 and by Wat.kins and Robertson 1221 for r-globulin adsorption on silicon rubber using an internal rcfIection fiuorescencc technique. The presence of loosely bound BSM on mica surfaces is also confirmti by force measurcrncnk between two adsorbed mucin monolayers on mica p~formcd in our laboratory by means of Isracirachvili’s technique [9] : On pressing t ht? t.wo solid surfaaces together, R part of the adsorbed mucin dcsorbs. and a part of it remains on the mica surface. The influcnco of pH has h-*en studied for the adsorption on BSM at cons stant ionic strength (10-3M). The results in Fig. 7 show that Ihe amount adsorbed depends on pI1. It can by seen that BShl adsorption rises in the neighbourhood of the isoeIectric point of BShl (pH ca. 3) [ 231. Such increase of adsorption in thr? neighbonrhood of the isoelectric point of proteins has already been reported by numerous authors studying protein adsor:.-tion on polymer films or polymer latices [24--271. It may be attributed to the fact that more BSM molecules can adsorb on a given surface due to their rearrangement to form more complex structures. The rearrangement

305

Fig. 7. pH dependence of BShl adsorption [NaCl] = 10”df; adsorption time 6 h.

on mlra. BShf concentration

0.1 mglml;

of protein molecules near to the isoelectric point. should be favourcd by weakening of intramolecular and lateral interactions between adsorbing molecules. The same conclusions concerning the influence of pH on adsorption of BSM molecules were reached by Evans and Blank [ZS] in their study of mucin adsorption at t.hc polarhxwl mercury/water interface using ac polarography.

REFERENCES J.F. Hulbert, J.J. Klawitter and L. Browman, Mater. Res. Bull., 7 (1972) 1233. B.A. Blenke, H. Bromer and E. Well. J. Riomed. Mater. Reh., 12 (1978) 307. L.L. Hench, in D.F. Willtams (Kd.), Fundrmcntat Aspects of Bincompatibility, CRS Press. 1981. K. Muller and C.C, Chang, Surf. Sci., 14 (1969) 39. A. Oottschafk and A.S. Bhargava in A. Oottschalk(Ed.), CZlycoproteins: Their Comporitiun, Structure and Function, Elsevier, New York, 1972. F.A. Bettclheim, Y. Hashimoto and W. Pigman, Biochim. Biophys. Acts, 63 (1962) 23s. A, Reszkin and MM. Roissonnade, C.R. Aced. Sci. Ser. C, 291 (1980) 153. J.E. Proust, A. Baszkin and M.M. Boissonnade, C.R. Acad. Sci. Ser. C, 234 (1982) 1326. J.N. Israelachvili and O.E. Adams, J. Chcm. Sot. Faraday Trans. 1,74 (1978) 976. 0, Tettamanti and $5’.Pigman, Arch. Biochem. Biophys., 124 (1968) 976.

306 11 C.W. Jourdian, L. Dean and S. Roseman, J. Biol. Chem., 246 (1971) 430. 12 J-E. Proud, A. BaszItin and M.M. Bofsso nnade. d. ColtoM Interface Sci.. 94 (1983) 421. 13 B.W. Yemm and EC. Cocking, Analyst, 80 (196S) 209. 14 0-H. Lowry, NJ. Rosebraugh, A.L. Farr and R.J. Randall. J. Biol. Chem., 193 (19Sl) 265. 15 J. Chen, D.B. Doug and J.D. Andrado, J. Coltold Interface Scl., 89 (1982) 677. 16 A. van dcr Cheer, J. Feijen, J.K. Ethorst, P.O.L,C. Krugers-Dagncaux and GA. Smolders, J. Coltoid Interface Scl., 66 (1978) 136. 17 R. Shogren, A.M. Jamieson and J. Blackwell, Polym. Prepr., 23 (1982) 33. 16 N. Payta, S. Rid and W. Pigman, Arch. Biochem. Blophys., 129 ( 1969) 68. 19 F.J. Holly, J. Colloid Interface Sci., 106 (1974) 226. 20 P. van Dutm and W. Norde, J_ Cottoid Intcrfacc Sci., 91 (1983) 248. 21 E. Perez and J.B. Proust, to bc published. 22 R.W. Watkins and Ch.R. Robertson, J. Btomed. Mater. Res., 11 (1977) 918. 23 F.A. Bettelhelm, Ann. N.Y. Acad. ScI., 106(1963) 247. 24 A. Bszkin and MM. Botssonnade In E:CMellint and P. Oiusti (Eds.), Polymers in Medicine: Biomedical and Pharmacological Applications. Plenum, London, 1983. 26 T. Surawa, II. Shlrehama and ‘I’, Fujimoto, J. -Raid Interface Sri., 86 (1982) 144. 26 W. Norde and J. Lyklcma, J. Colloid Interface Sci., 66 (1976) 267. 27 P. Bagchi and SM. Birnbaum, d. Colloid Interface tici., 83 (1981) 460. 28 E. Evans and M, Blank. J+ Colloid Interface Scl,. 86 (1982) 99,