Excess adsorption of lysozyme and water at solid-liquid interfacesα

C~~~ff~~~and Surfaces 0927-7165/94/$07.00

I3 ~zoi~~er~~c~~, 2 ( 1994) 41 i-417 0 1994 - Elsevier Science B.V. All rights reserved.

Excess ~ds~~pti~n of lys~~yrn~ an interfaces”

(Received

2 April 1993: accepted

411

water at solid-liquid

22 July 1993)

Abstract Adsorption isotherms of lysozyme at solid-water interfaces have been studied as a function of protem con~nt~atlon~ iomc strength of the medium, pH and tem~~atur~ using silica, a~urn~na, carbon, chromium and Sephadex as solid surfaces. Ad~o~~tjo~ of lysozyme is affected strongly by change of pH, tem~rat~re and ionic strength. In mast cases adsorption isotherms attained a state of ad~~rpt~o~ saturation. On chromium, lysozyme is either expanded laterally or negatively adsorbed. In some cases, adsorption isotherms were S shaped, showing the existence of some kind of interactions within the adsorbed protein layer. Adsorption of lysozyme on Sephadex at pH 5.0 and 7.5 is negative due to the excess adsorption of water by this material. The standard free energies (AG” ) of positive and negative adsorption of lysozyme per square meter, sigmfymg the relative affimty of adsorption m the state of monolayer saturation, have been calculated. The magmtude of the standard free energy of transfer (AG,) of one male of protein from solution to the surface IS observed to be 40.3 kJ mol-’ and the value is ~~de~ende~t of pH_ iomc s~r~n~tb~ nature of the surface and temperature. Kep words. Adsorption;

Lysozyme;

Solid-llqmd

interface;

Water

The study of the interaction of lysozyme wit different types of model hydrophobic, hydrophilic and metallic surfaces may be of some importance in revealing the mechanism and physicochemical nature of lyso~yme-dell surface ~~t~raction~ The spread and adsorbed mono~ayers of ~ysozymes at air-liquid interfaces have been studied in detail by Yamashita and Bull [l]. Sarkar and Chattoraj [ 2] have recently studied the kinetics of adsorption of lysozyme at solid-liquid interfaces and obtained some important information about the mechanism *Corresponding author. “NCe Deepa Ganguly. “The prehminary form of this paper was presented at the 7th International Conference on Collold and Surface Science held in Compikgne, France, 7-13 July 1991. and was coordinated for publication by Professors D. X/fuller and D. Labarre.

of adsorption of this enzyme at interfaces. Recently, equilibrium adsor~tiu~ measurements of di~e~~~t proteins at diRerent solid-liquid interfaces have been carried out by various workers [3-61. In the present paper, we have presented our data on the adsorption of lysozyme on hydrophobic, hydrophilic and metallic interfaces at various values of pH, ionic strength, temperature and protein concentrations in solution. T affinities of this protein for these surfaces under different conditions have also been compared on the basis of a unified scale of free energy of adsorption.

Experimental Lysozyme from chicken weight 14 000) was obtained

egg white (molecular from Sigma Chemical

Co. Highly

pure

powdered

alumina

(chromato-

powdered graphite graphic grade), Germany), chromium powder (BDH, grade), Sephadex

powder

powder (Qualigens,

(Fluka. standard

(G- 100, Sigma) and silica

India) were used as adsorbents.

All salts used were of analytical

grade.

Distilled

water was used throughout. Alumina, silica. graphite and chromium powders were heated for 12 h at 250-3OO’C before being used

for adsorption

experiments.

dried in a vacuum acid for 3 days

desiccator

Sephadex

containing

to constant

weight.

was

sulphuric The surface

area A of each solid powder except graphite was measured by the palmitic acid adsorption from benzene

method

surface

areas

Sephadex

described of silica,

as

elsewhere alumina,

measured

by

[ 71. Average

chromium

and

method

are

this

102 + 1 m* g-l, 20.1 +_0.2 m2 g-‘. 80.6 +_ 1.5 m* g-i and 50 &- 1 m2 g 1 of solid respectively. The surface area of powdered graphite was determined by adsorption

of acetic acid from aqueous

described

elsewhere

surface

area

media

as

in detail

171. The

specific

thus

determined

was

A

50.0 F 1.0 rn’ gg ‘. Thus, the area of all types of surfaces used were calculated from monolayer adsorption of fatty acids oriented perpendicular to the surface plane. Hence values of surface areas in all cases are relatively

comparable

Using the isopiestic method measurement at water activity

to each other. of vapor pressure 0.95, the numbers

of moles of water vapor adsorbed alumina, powder

silica, Sephadex, have

been

found

charcoal

of

and chromium

to be 48.7, 33.9. 30.1,

0.221 and 0.0185 respectively. alumina porosity relative

per kilogram

This indicates

that

has the highest and chromium the lowest in terms of water holding capacity at 0.95 humidity. The powdered particles were

Stock buffer solutions of particular pH and ionic strength were prepared. Bicarbonate, phosphate and acetate buffers were used to maintain pH values

of 11.0. 7.5 and

protein

solution

definite

amount

was

5.0 respectively. prepared

of protein

A stock

by dissolving

a

in a fixed volume

of

buffer solution at a given pH and ionic strength. By mixing the above two stock solutions in appropriate proportions, K ml of protein solutions of varying molar concentrations (C’i) were prepared in stoppered bottles. To each bottle, Wg (equal to 1 g in the present case) of solid powder was added. The bottles were gently shaken for 16 h at constant temperature using an Emenvee shaker. The frothing during this mild shaking was not significant. At the end of this experiment. bottles were allowed to stand for 4 h whereby solid particles sedimented completely. The supernatant solutions were found, by spectrophotometric examination [S]. to be completely free from solid particles. The clear solution was then analysed for its molar protein concentration c, using the Folin reagent by the Lowry method [9]. The standard error for measurement of CP did not exceed 3-4s so that the standard error for rk is close to 668% only. From measured values of cb - c,, V,, W and A, the number of moles rb of protein adsorbed per unit area of the surface can be calculated

in the usual

manner. If the extent of adsorption r; is expressed in milligrams of lysozyme adsorbed per square meter of the surface, then it is equal to lOOOr~M,, where M, is the molecular weight of lysozyme. Also Ci and C, are respectively equal to lOC~/M, and 10C’,/MP where Cb and C, are percent concentrations of lysozyme before and after adsorption.

suspended in water and the size of one hundred particles examined microscopically. The average

Results and discussion

particle

At constant pH. ionic strength and temperature. ri may be given by the equation [4,10]

sizes of alumina.

silica, charcoal

and chro-

mium are 125_+8 pm. 29_+ 5 pm, 7f2 nm 9 + 2 urn respectively. In the case of alumina silica

a dye (disulphine

particles

opaque

blue) was used

for size measurement.

and and

to make

r;=-- W

1000

(111;- nip )

D Sarkar

where

und D.K. ChattorajlCol1oid.s

molalities

of the bulk

Surfaces

B. Blointerfacrs

protein

and mp before and after adsorption

solution

J 411-417

,7 ( 1994

2.0

rnb

may be taken

413

r

. . ..

as Ci and C’p respectively for dilute solutions and the total weight of the solvent in the system may be put equal to v so that ri can be calculated using experimental data as stated before. Putting WI equal to ~1:M,, n$ equal mp equal

to lOOOn,/M,n, one can obtain [4,10]

to lOOOn~/M,n:

respectively

and

in Eq. (l),

I

where n: and r$, are respectively the moles of water and protein components before adsorption (per unit surface area) and n1 and n,, are the moles of these components after adsorption (all expressed per unit surface area of the solid). M, is the molecular weight of water. If An, and An, are respectively moles of water and protein bound per square meter of the surface, then 11: and II; will be equal to n, + An, and np + An, respectively.

Fig. 1. Plot of r;r vs. pHllO,~=O.l. A. pH 5.0, p = 0.1. 0. 0, pH 5.0, p = 0.01. 28’

I

c _ I- __ 0,

I

0 16

\

I

I

0.24

C, of lysozyme m presence of dica; 28’C; A. pH7.5,~=0.1, 28~C; X’C; 0, pH 5 0, p = 0.1. 37-c; C.

08

rb = An, - An, zp “1

(3)

-An,-An,& if the solutions

008

(4) are dilute. When An, np/nl is negligi-

-

06

(r .J

04

?

%a L

4

02

ble with respect to An,, rk is positive and equal to An,. But if the latter is greater than the former, ri becomes negative. rk stands for the Gibbs surface

excess

of protein

component

when

the

water component 1 becomes zero by suitable placement of the Gibbs dividing plane [lo] and this is indicated by the superscript in ri. In Figs. l-5, the isotherms for adsorption of lysozyme from aqueous solution to the various solid surfaces are compared. At the isoelectric pH (11.0) of lysozyme. let us compare these isotherms for the five different surfaces used, at ionic strength 0.10 and at 28’C. In all five cases, one finds that positive values of rr increase with increase of C, due to gradual replacement of surface-bound water by lysozyme. At higher values of C,, the surfacebound water is completely replaced by hydrated

Fig. 3. Plot of r; vs. C, of lysozyme in presence of alumina; pHll.O.~=O.l. 28’C: 0. pH5.0.~=0.01. 28’C: A. ,!J, pH 7.5,/1=O.l, 28 c; 10, pH 5.0, p = 0.1, 37’C; 0, pH 50,~=01, 28-C.

protein thus forming a saturated monolayer. In this situation, the value of r; approaches its maximum value rFtrn). These values obtained from the isotherms are included in Table 1. Considering the size of the ellipsoidal lysozyme molecule to be 45 x 30 x 30 A and assuming a layer of hydration of 5 A around the biopolymer

D. Sarkar undD.K.ChattorajiCollolds

0.08

Swfaces

B. Bmntrrfacrs

? ( 1994 ) 41 l-41 7

016

CP (gm%)

Fig. 3. Plot of r; vs. C, of lysozyme m presence pHllO.~~=O.l, WC: A. pH75./~=01, A. pH 5 0. /I = 0.1, 37.‘C: 0. pH50,~=01, 17. 0, pH 5.0, /I = 0.01, 18 C.

of carbon. 18’C: 28’C:

-03L Fig. 5 Plot of r; vs. C, of lysozyme m presence of Sephadex pHll.O,~=O.l, 28C, 0, pH5.0./c=O.l, WC: A. 8:‘. pH 5.0, p = 0 1, 37 ‘C; Ai. pH 7.5. I’ = 0.1. ‘8 C

06r

that

lysozyme

in

the

adsorbed

monolayer

undergoes lateral expansion by dimensional change to cover up the surface. Although both silica and alumina are hydrophilic, the observed difference in the state of this dimensional change in the two cases seems to be interesting. Even on the highly hydrophobic surface of carbon. cCrn’ is observed to be 1.0 mg m-‘, thus signifying lateral expansion of protein. It has been shown previously [ 1 l] that for completely denatured protein. only 0.4 mg of the biopolymer will be needed to cover up 1 mZ of the surface so as to form a saturated -03L Fig. 3. Plot of r; vs. C, of lysozyme in presence of chrommm; A, pH7.5,~=0.1, 28’C; 0, pH5.0.~=01. 28’C; pHll.O.k~=O.l, 28’C; ‘3, pH50,/~=01, 37’C; A. 0, pH 5.0, p = 0.01, 28 ‘C.

molecule, a complete monolayer of lysozyme with vertical or horizontal orientations corresponds theoretically to 1.86 mg and 1.35 mg respectively of the protein packed per square meter of surface area. For the silica surface, the observed G(“‘) is 1.85 mg mP2 in agreement with a vertical orientation of lysozyme in the protein monolayer. rTcrn’ for alumina is, however, 1.17 mg rn-’ which means

monolayer.

The value

of Gem’

for the metallic chromium surface is 0.51 mg rne2, indicating extensive dimensional change of protein by denaturation at the interface. For highly hydrophilic Sephadex gel. smal pores present may allow fatty acid to accumulate (during surface area measurement) but large protein molecules are unable to enter the pores. In this situation, T(m) becomes as low as 0.2 mg mm2 even though protein is not undergoing dimensional change at the interface. This low value is due to the large contribution of the second term on the right-hand side of Eq. (3) to r;. We also note from Figs. l-5, that the adsorption isotherms in all cases are found to be considerably

D. Sarkar and D.K. ChattorajiColloids Surfaces B: Blointerfaces 2 ( 1994 ) 411-417

415

Table 1 Adsorption and free energies of transfer of lysozyme at different types of soled-liquid interfaces Surface

PH

Ionic strength

f’ C)

Temp.

r; X 108 (mol mm’)

AG; (kJ mol-‘)

AG-’ x lo8 (kJ m-*)

Silica

11.0 1.5 5.0 5.0 5.0

0.10 0.10 0.10 001 0.10

28 28 28 28 37

1.85 + 1.69 k 0.92 + 0 67 + 0 85 +

0.10 0.08 0.02 0.09 0.04

13.2 f 0.7 12.1 k 0.6 6.60*0.16 4.80 k 0.13 6.10 + 0.28

-40.1 -40.0 - 40.8 - 39.6 -41.3

- 529 -487 - 269 - 190 -252

Alumma

11.0 1.5 5.0 5.0 5.0

0.10 0.10 0.10 0.01 0.10

28 28 28 28 37

1.17 + 0.95 f 0.20 * 0.45 * 0.35 *

0.10 0.08 0.02 0.02 0.02

8.35 f 0.71 6 80 + 0.57 1.42+014 3.20 f 0.14 2.47 i_ 0.14

- 40.0 -41.1 - 34.0 _

-351 -280 -48.0

-41.4

- 102

Carbon

11.0 75 5.0 5.0 5.0

0.10 0.10 0.10 0.01 0.10

28 28 28 28 31

1.00 * 0.78 0.60 + 0.50 * 0.72 *

0.05 0.04 0.03 0.05

7 14 & 0.36 5.57 4.28 + 0.37 3.51 * 0.21 5.14 + 0.35

- 40.7 -38.1 -41.2 -38.5 - 38.4

-291 -212 -176 - 137 -197

Chrommm

11.0 7.5 5.0 5.0 5.0

0.10 0.10 0.10 001 0.10

28 28 28 28 31

0.51 0 53 i 0.04 0.52 i_ 0 03 -0.21 * 0.01 0.35 * 0.02

3.64 3.78 k 0.28 3.71 +_0.21 - 1.50 * 0.07 2.17 * 0.12

- 38.5 - 38.4 -39.1 39.4 - 37.6

-140 -145 - 145 59.1 - 78.9

Sephadex

11.0 7.5 5.0 5.0

0.10 0 10 0.10 0.10

28 28 ‘8 31

0.19 - 0.20 * 0.02 -0.07 -0.05

1.35 - 1.42 + 0.14 -0.50 -0.35

- 36.0 38 I 37.2

- 44.0 55.0 18.6

affected by change of pH, ionic strength and temperature. Thus, with decrease of pH from 11.0 to 5.0, q@“’ decreases gradually and carbon surfaces as a result dimensional change of protein phase, due to the presence of charge

of the protein.

for silica, alumina of the increase in in the monolayer increased positive

For the chromium

surface

covered with unfolded lysozyme, G(m) remains unaffected by a decrease of pH from 11.0 to 5.0. In the case of Sephadex, with a decrease of pH, charged lysozyme is unable to penetrate highly hydrated pores so that the second term in the right-hand side of Eq. (3) becomes higher than An, so that l-b becomes negative. At pH 5.0, the surface potential of positively charged lysozyme is significantly increased when the ionic strength is decreased from 0.1 to 0.01. As a result of this, due to a decrease

of ionic strength,

c@“) is observed

to decrease for silica and charcoal

surfaces whereas for the chromium surface it becomes negative due to an increase in water binding to the surface (see Eq. (3)). For positively charged alumina particles the reverse effect of ionic strength is observed. From Table 1, we also note that at pH 5.0, the extent of maximum adsorption increases by increasing the temperature from 28 to 37°C. in the case of alumina and charcoal due to the hydrophobic effect. However, for silica, chromium and Sephadex, its magnitude decreases due to the existence of van der Waals, electrostatic and other types of interactions. Although the shapes of all the isotherms in Fig. 1 are “regular” in nature we note with interest that at pH 5.0 and ionic strength 0.01, r; for the alumina surface (Fig. 2, curve 2) increases beyond

416

D. Sarkur

an apparent

saturation

und D.R

value of 0.45 mg me2 with-

C/mttorq/Colloids

tions.

if available

out limit when C, is very high. This may be caused

utilised

by interfacial

particular

the temperature

coagulation

phenomena.

is increased

Also. when

to 37”C, the adsorp-

tion of lysozyme on the alumina surface at pH 5.0 becomes negative at low C, values due to excess hydration

(see Eq. (2)) but at high values

of C, it

approaches a positive saturation value rrcrn), presumably as a result of relative dehydration at the interface (see curve 4). The corresponding isotherm at 28°C exhibits regular behaviour (see curve 3). The isotherms 3, 4 and 5 in Fig. 3 for protein adsorption at carbon surfaces are S shaped, indicating strong intermolecular attraction between adsorbed lysozyme molecules in the monolayer. The isotherms 1 and 4 for adsorption on chromium (Fig. 4) and isotherm 1 for adsorption by Sephadex (Fig. 5) are S shaped, showing the existence of the same type of interaction in the adsorbed layer. One also notes with interest that lysozyme is throughout negatively adsorbed by chromium at pH 5.0 when the ionic strength is decreased from 0.10 to 0.01 (see Fig. 4, curve 5). The same phenomenon has been observed for adsorption of lysozyme on Sephadex at pH 7.5 and 5.0 (Fig. 5, curves 2, 3 and 4). In all these cases. the term An,n,/n, is greater than An, (see Eq. (3)) so that ri?, becomes negative. We also note with interest from curve 2 of Fig. 3 for adsorption at the carbon surface, and from curve 3 of Fig. 4 for adsorption at the chromium surface. that G under certain physicochemical conditions may reach an apparent maximum value followed by a sharp decrease of the extent of adsorption to values close to zero. This phenomenon is the result of an increase in the excess increased contribution of An,n,/rri so that rb in Eq. (3 ) decreases with increase of C,. The relative affinity of lysozyme for different surfaces may be expressed by the magnitude and sign of the maximum adsorption rgcm). Thus at ionic strength 0.1 and at pH 11.0 and 7.5 the affinities in terms of r;‘*) may be expressed in the following order: silica > alumina > charcoal > chromium > Sephadex. These types of observa-

The

Surfuces B Biointerfarrs

2 ( 1994 ) 41 l-41 7

for different

proteins,

for the chromatographic protein

affinities

particular

expressed

and temperature.

in this manner

depend

on pH, ionic

If water competes surface

of rzcrn) may decrease or negative. Recently

be of a

from mixtures.

surface

for occupying

can

separation

it has

for a strength

with lysozyme

sites, the affinities

in terms

and may even become zero

been

shown

[4,12]

that

the

standard free energy (AG” ) of adsorption per square meter of the surface can be calculated using the equation: AG’ = +T;AG;

(5)

where AG,’ is the standard of one mole of protein when the mole fraction altered

free energy

of adsorbate

from zero to unity.

From

in the bulk is the application

of the thermodynamics of adsorption, the derivation of the Gibbs equation, shown [4,10] that

where X,, stands in solution

of transfer

from the bulk to the surface

for the mole fraction

as used for it may be

of protein

(equal to C,/55.5 ), R is the gas constant

and T the absolute

temperature.

Values of AG: for

different systems were obtained from Eq. (6), using a computer (PC-AT) for graphical analysis. These values

are given

in Table 1. AGf; is found

to be

insensitive to changes in the physicochemical conditions. Values of AG”, calculated from Eqs. (5) and (6) and included in Table 1, are found to follow the same order as rrcrn’ so that -AG’ may be a measure of the relative affinity of the proteins for different cal conditions.

surfaces under In fact

varying

- AG ’

c and the average slope AGr$ ) is 40.3 kJ mall ‘.

physicochemi-

varies linearly

of the line (equal

with to

D. Sarkar and D.K. Chattoraj/Colloids Surfaces B: Biointerfaces ? ( 1994

5 S. HaJra and D.K. Chattoraj, 28 ( 1991) 267.

Acknowledgements

6

The financial assistance of CSIR (New Delhi) to one of us (DS) is acknowledged with thanks. We are also grateful to the DSA Programme, for financial help.

UGC

7

8 References T. Yamashita and H.B. Bull. J. Colloid Interface Sci.. 21 (1968) 19. D. Sarkar and D.K. ChattoraJ, J. Colloid Interface Set., 157 (1993) 219. W. Norde, Proteins at interfaces, Doctoral thesis, Agricultural University. Wageningen, The Netherlands, 1976. S. Hajra and D.K. ChattoraJ, Indian J. Biochem. Biophys., 28 (1991) 114.

9 10 11 12

417

J 411-417 Indian

J. Biochem.

Biophys.,

J.D. Andrade, in J.D. Andrade (Ed.), Surface and Interfacial Aspects of Biomedical Polymers, Vol. 2, Plenum Press, New York, 1985. C. Orr and J.M. Dalvelle. Fme Particle Measurement: Size. Surface and Pore Volume, Macmillan, New York, 1959, p. 207. S. Hajra, Interaction of interfaces and water with protem and protein mixtures. Doctoral thesis, Jadavpur University, Calcutta, 1989. O.H. Lowry, N.J. Rosebrough, A.C. Farr and R.J. Randall, J. Blochem.. 193 (1951) 158. D.K. Chattoraj and K.S. Birdi, Adsorption and the Gibbs Surface Excess, Plenum Press, New York, 1984. T. Yamashita and H.B. Bull, J. Colloid Interface Sci., 24 (1967) 310. M. Das and D.K. ChattoraJ, Colloids Surfaces, 61 (1991) 15.