Adsorption of ovalbumin on calcium hydroxyapatite crystals

Colloids oncl Surfaces, 18 (1986) l-8 Elsevier Science Publishers B.V.. Amsterdam

ADSORPTION CRYSTALS

OF OVALBUMIN

M.R. CHRISTOFFERSEN’

- Printed

in The Netherlands

ON CALCIUM

, J. CHRISTOFFERSEN’

HYDROXYAPATITE

, P. IBSEN2

and H. IPSEN3

*Medicinsk-Kemisk Institut, Panum Institute, University of Copenhagen, Blegdamsvej DK-2200 Copenhagen N (Denmark) 2The Department of Pathology, Frederiksberg Hospital, DK-2000 Copenhagen (Denmark) ‘The Protein Laboratory, University of Copenhagen, Sigurdsgade 34, DK-2200 Copenhagen (Denmark) (Received

10 May 1985; accepted

in final form

11 October

3,

1985)

ABSTRACT The adsorption of ovalbumin (OVA) onto calcium hydroxyapatite (HAP) crystals at concentrations up to 1.5 g 1.’ at 25°C and pH = 7 can be described by a simple Langmuir isotherm with an adsorption constant K = 10.5 + 1.5 1 g-l and a binding capacity of 1.53 f 0.02 mg me’. The binding capacity of OVA is similar to that found previously for bovine serum albumin (BSA), but the adsorption constant of OVA is approximately 30 times smaller than that of BSA. The dissolution rate of HAP is found to be correspondingly less inhibited by OVA than by BSA. Despite the inhibition of the dissolution rate of HAP caused by adsorbed proteins, HAP may be a potential adjuvant/depot substance for allergen extract vaccines, the normally used depot substance, AI(O being less biologically compatible and less soluble.

INTRODUCTION

Calcium hydroxyapatite (HAP), Ca10(P04)6(OH)2, plays an important role both as a model compound for bone formation/resorption [l, 21 and as a chromatographic medium in the purification of biological molecules [3]. Mineralization of bones presumably occurs in a collagen matrix, and proteins adsorbed onto the surface of the bone mineral may interfere with the bone turnover. A number of molecules of biological interest bind to the surface of HAP crystals [4-121 and inhibit the rate of growth [7-91 and dissolution [ 11, 121 of HAP. Further, inorganic compounds such as A1(OH)3 are currently used as an adjuvant/depot substance for allergen extract vaccines, and since HAP is presumably a more biologically compatible substance, it may be possible to use it instead of Al(OH),, provided the binding capacity for complex mixtures of proteins is sufficiently high. The aims of this study were to investigate the adsorption of ovalbumin (OVA) onto the surface of HAP crystals and the effect this adsorption has on the dissolution of HAP.

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o 1986 Elsevier

Science

Publishers

B.V.

2 THEORY

The adsorption of small ions or molecules onto HAP crystals can often be described by a Langmuir adsorption isotherm [5, 11, 121, Eqn. (1):

’

K=

(1 - x)C where K is the adsorption constant; x is the fraction of the adsorption sites occupied by the adsorbate; and C is the concentration of the adsorbate in the solution. For larger molecules, such as proteins, one cannot to define the adsorption sites as single entities, because the probability for an adsorbate molecule to react with a single free site in the crystal surface depends on the fraction of neighbouring sites being vacant or occupied by adsorbate molecules. In this case the adsorption may still be described by a Langmuir isotherm [4, 6, 91, as expressed by Eqn. (2): B

K=

Rnax

BI4mx

X

- B)C = (1 - B/B nl,x)C=(l-3c)C

where B is the mass of protein adsorbed per mass of adsorbent; Bmax is the maximum value of B, i.e. the binding capacity; C is the concentration of protein in the solution; and x is the ratio BIBmax. In the present case, B is the mass of OVA adsorbed per mass unit of HAP. Equation 2 can be rearranged to give Eqn. 3 :

c

-_=-+_ B B,,

c

1 KB,,,

(3)

A plot of C/B versus C should thus, for this type of adsorption, give a straight line with slope l/B,, and C/B-axis intercept l/(B,,K). Adsorption isotherms can be complicated both by interactions between adsorbed molecules on the crystal surface, and by complex formation between the adsorbate and ions in the solution. For adsoprtion of OVA onto HAP crystals, we may expect negatively charged groups in the protein to associate with calcium ions in the HAP surface or in the solution. Further, the protein may release hydrogen ions as adsorption or complex formation in solution takes place. We have discussed [ll] a number of cases of Langmuir-like adsorption isotherms for which the kinetic effects of inhibitors can be described by an equation of the type J,jJL

= 1 + KkinC

(4)

where JL and Jo are the crystal growth or dissolution rates determined with and without the inhibitor present, keeping all other parameters constant as far as possible; C is the concentration of inhibitor in the solution; and

3

Kkh is a constant, which can be related to the equilibrium adsorption constant. Generally, Kkh will increase as equilibrium, with respect to growth or dissolution process, is approached. EXPERIMENTAL

The HAP crystals were prepared as described previously [ 131. Chicken egg albumin (OVA) and bovine serum albumin (BSA) were obtained from Sigma Chemical Company, U.S.A. (Nos. A5503 and A4378, respectively). All other chemicals used were of analytical grade. The concentration of OVA solutions was determined by light absorption measurements at 280 nm using a Carl Zeiss PMQII spectrophotometer equipped with a BM 7820 digital processor unit detection system. The absorption coefficient both of filtered and unfiltered OVA solutions at 280 nm was determined to be 0.61 f 0.01 1 g-l cm-’ at pH 6-7 in the presence of calcium and phosphate up to 0.5 mM. Calcium concentrations were determined by atomic absorption spectrometry using a Perkin-Elmer 4000 instrument, and the specific surface area of the HAP crystals, 49 m2 g-’ , was determined by the BET gas adsorption technique using a Quantasorb instrument. All adsorption experiments were carried out at 24 * 1°C. Ovalbumin was dissolved carefully in deionized water, filtered through a Millipore GS 0.22~pm filter and mixed with the HAP crystal suspension at pH 6.5 or 7.0. No important change in pH was observed when the crystals and the protein were mixed. After 1-3 days, with occasional careful mixing, the suspension was filtered (Millipore GS 0.22~pm filter) and the concentration of the protein in the filtrate was determined. The mass of the HAP crystals was determined from the measured calcium content of the acidified suspension. In these experiments with 5-10 ml crystal/protein mixtures, containing approximately 10 mg HAP per ml (surface area 0.49 m2 ml-‘), equilibrium was reached after 1 day. The effect of OVA on the dissolution rate of HAP crystals was studied at pH 7.00 * 0.01, 25.0 + O.l”C, using the pH-stat technique described previously [ 131. The HAP crystals (- 10 mg) were added to a solution (- 0.9 1) of OVA and the pH was kept constant by addition of 2 mM nitric acid (Radiometer pH-stat equipment); a slow stirring rate could be used without affecting the normal dissolution rate of the HAP crystals. During and after experiments, samples of the reaction mixture were filtered and the calcium concentration of the filtrate was determined. For the experiments reported here, with a maximum protein concentration of 30 pmol 1-l and maximum calcium concentration of 50 pm01 l-l, the calcium concentration of the filtered solution agreed with the calcium concentration calculated from the acid consumption to within 5%, and the latter concentrations are reported here. The remaining reaction mixture was acidified to dissolve the crystals completely and measurements of the calcium content of this solu-

4

tion enabled the initial mass of crystals to be determined. Similar kinetic measurements were carried out with BSA, the adsorption of which onto the HAP has been recently reported [ 41. RESULTS

Adsorption The results obtained for the adsorption of OVA on HAP are shown in Fig. 1, in which C/B, the concentration of OVA in solution divided by the mass of OVA adsorbed per mass of HAP crystals, is plotted against C, see Eqn. (3). From the gradient we obtain B,,, = 0.075 f 0.001 g(OVA) per g(HAP), equivalent to 1.53 mg(OVA) rne2. From the gradient and the inter-

Cl

9/

j: O0

I

0.5

1

C

1.0

1 .5

9/L Pig. 1. The concentration, C, of OVA in the aqueous phase divided by the mass of OVA adsorbed per mass of HAP crystals, plotted against C. The points can be approximated by a straight line which shows that the adsorption of OVA onto HAP, at least phenomenologically, can be described by a simple Langmuir adsorption isotherm in this concen(mg ml-‘): tration range. (A, x, @) pH = 6.5; (0, +) pH = 7.0. HAP mass concentrations (A) 9.8; (x) 10.2; (~3) 9.0; (0) 10.0; (+) 9.8.iO mg HAP has surface area 0.49 m*.

5 0.075 n-@Y

n

Q

II-@-

/;y a’+ /x ‘@

0.050-

l

/ B

4 9

0.025

I 01 0

05

c

1.0

15

g/L Fig. 2. The mass of OVA adsorbed per mass of HAP crystals plotted against the concentration of OVA in the aqueous phase. The curve drawn through the experimental points is calculated from the values of the adsorption constant and the binding capacity determined from the Langmuir isotherm in Fig. 1. 20

Jo/J,

BSA

/

10

OVA

Fig. 3. The ratios J,/JL and JL/J, plotted against the concentration of protein in solution: (A) BSA; (0) OVA. Jo and JL are the rates of dissolution per unit mass HAP in the absence and presence of protein, and all other parameters affecting the rate being constant. The rates are measured at pH 7 and 25% saturation.

6

cept of the line with the C/B-axis we obtain K = 10.5 + 1.5 1 g-‘, and, with the molecular mass of OVA (43 kD), we have K = (4.5 f 0.6) X lo5 1 mol-’ . These values of B,,, and K were used to calculate the curve in Fig. 2, in which the experimentally determined values of B are plotted against C.

Kinetics The results of the kinetic experiments of the effects of BSA and OVA on the dissolution rate of HAP are given in Fig. 3, in which J,,/JL and JL/Jo are plotted against C, the concentration of protein in solution, at pH 7.0 and 25% saturation. It can be seen from this plot that the retarding effect of BSA is much greater than that of OVA, and that Eqn. (4) can be used to describe the inhibition of the dissolution rate. From the slopes of the lines in the plot we obtain Kkh = 1170 1 g-* for BSA and Kkir, = 36 1 g-’ for OVA. DISCUSSION

AND CONCLUSION

The adsorption of proteins onto HAP crystals is often not reversible [lo], corresponding to a very slow rate of desorption. The adsorption of proteins can, however, often, at least phenomenologically, be described by a Langmuir adsorption isotherm. No adequate theory for protein adsorption onto mineral surfaces has yet been developed. That this adsorption process can be described by a Langmuir isotherm may partly be explained by the proteins existing in different states when adsorbed on a surface [14, 151. At very low concentration, the protein molecules may spread over the adsorbent surface (state 1) and, at higher concentration, the protein molecules may be in a condensed form on the adsorbent surface (state 2). Roughly, such a description is equivalent to an elipsoidal protein molecule being able to adsorb side-on or end-on, states 1 and 2, respectively. With low protein concentration on the adsorbent surface, protein molecules may spontaneously change from state 1 to state 2 (side-on to end-on adsorption), with the production of free adsorption sites on the surface. Even in the case of the whole adsorbent surface being covered by molecules adsorbed in state 1, due to the possible change-over from state 1 to state 2, and the subsequent production of vacant adsorption sites, adsorption may increase, if the protein concentration is increased in the aqueous phase. The amount of surface sites or fraction of the surface which is available for adsorption of molecules in state 2, in this case, is proportional to the fraction of the surface covered by molecules in state 1. Such a model will, for low protein concentration, behave quite similarly to a Langmuir adsorption model. The binding capacities of BSA [4] and OVA (present work) onto HAP are 1.9 mg mm2 (0.028 pmol mw2) and 1.5 mg mm2 (0.035 pmol mv2), respectively. Assuming the protein molecules cover the entire HAP surface at the plateau adsorption value (binding capacity), we calculate from the specific surface area of the crystals that one BSA molecule covers 60 nm2

7

and one OVA molecule 48 nm’. If OVA and BSA molecules have similar shapes (not necessarily spheres) and densities, the area covered by one molecule should be proportional to the molar mass to the power 2/3. With an error of less than lo%, this is found to be the case here. The Stokes radius for serum albumin is 3.6 nm and for OVA 2.8 nm [16, 171. The radii of circles with areas 60 nm’ (BSA) and 48 nm* (OVA) are 4.4 nm and 3.9 nm, respectively. This indicates that some structural rearrangement could take place during adsorption, and gives no indication of multilayer adsorption. Moreno et al. [4] found the value 2.49 X 10’ 1 mol-’ (360 1 g-l) for the adsorption constant of BSA on HAP. This is considerably larger (34 times) than the value found in the present work for the adsorption constant of OVA on HAP. The kinetics experiments reported here confirm this large difference in adsorption behaviour of BSA and OVA on HAP. Kinetic adsorption constants, Kkh, increase as the solution composition approaches equilibrium and cannot be directly compared with equilibrium constants. This has been discussed in Ref. [12]. However, kinetic constants determined at constant solution composition give information about the relative inhibitory effect of additives. The ratio of the kinetic constants determined here for BSA and OVA at* 25% saturation is 33, in agreement with the results of the equilibrium measurements. This indicates that BSA and OVA interact differently with the HAP surface under the conditions applied. This difference can apparently only be explained as due to the difference in molecular structure of the proteins. Further investigations are needed before the groups in BSA and OVA which are responsible for this difference can be identified. ACKNOWLEDGMENTS

We thank Mrs Nobuko Christiansen, Eva Hald Hansen and for technical assistance. Grants for equipment, laboratory travel from the Danish Medical Research Council (12-3527, Carlsberg Foundation (35/IV) and NOVO’S Foundation are with thanks.

Majbritt Glar$ assistance and 12-4077), the acknowledged

REFERENCES 1 2 3 4 5 6

M.J. Glimcher, in A. Veis (Ed.), The Chemistry and Biology of Mineralized Connective Tissue, Elsevier, Amsterdam, 1981, p. 617. H. Fleich, in G.H. Nancollas (Ed.), Biological Mineralization and Demineralization, Springer-Verlag, Berlin, Heidelberg, New York, 1982, p. 233. G. Bernadi, M-G. Giro and C. Gaillard, Biochim. Biophys. Acta, 278 (1972) 409. E.C. Moreno, M. Kresak and R.T. Zahradnik, Caries Res., 11, Suppl. 1, (1977) 142. M. Kresak, E.C. Moreno, R.T. Zahradnik and D.I. Hay, J. Colloid Interface Sci., 59 (1977) 283. E.C. Moreno, M, Kresak and D.I. Hay, J. Biol. Chem., 257 (1982) 2981.

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E.C. Moreno, K. Varughese and D.I. Hay, Calc. Tissue Int., 28 (1979) 7. E.C. Moreno and K. Varughese, J. Cryst. Growth, 53 (1981) 20. T. Oaba, E.C. Moreno and D.I. Hay, Calc. Tissue Int., 36 (1984) 651. V. Hlady and H. Fiiredi-Milhofer, J. Colloid Interface Sci., 69 (1979) 460. J. Christoffersen and M.R. Christoffersen, J. Cryst. Growth, 53 (1981) 42. J. Christoffersen, M.R. Christoffersen, S.B. Christensen and G.H. Nancollas, J. Cryst. Growth, 62 (1983) 254. J. Christoffersen, M.R. Christoffersen and N. Kjaergaard, J. Cryst. Growth, 43 (1978) 501. D.K. Chattoraj and H.B. Bull, J. Am. Chem. Sot., 81 (1959) 5128. P.G. Koutsoukos, C.A. Mumme-Young, W. Norde and J. Lyklema, Colloids Surfaces, 5 (1982) 93. C. Tanford, Physical Chemistry of Macromolecules, Wiley, New York, 1963, p. 359. W.J. Moore, Physical Chemistry, Lowe and Brydone, Thetford, 1972, p. 945.