Colloids and Surfaces B: Biointerfaces 14 (1999) 99 – 104

www.elsevier.nl/locate/colsurfb

Influence of macroscopic and microscopic interactions on

kinetic rate constants
I. Role of the extended DLVO theory in determining the
kinetic adsorption constant of proteins in aqueous media,
using von Smoluchowski’s approach
C.J. van Oss a,b,c,*, A. Docoslis b, W. Wu d, R.F. Giese c
a
Department of Microbiology, State Uni6ersity of New York at Buffalo, Buffalo, NY 14214, USA
b
Department of Chemical Engineering, State Uni6ersity of New York at Buffalo, Buffalo, NY 14260, USA
c
Department of Geology, State Uni6ersity of New York at Buffalo, Buffalo, NY 14260, USA
d
Department of Chemistry, State Uni6ersity of New York at Buffalo, Buffalo, NY 14260, USA

Abstract

In the case of adsorption in an aqueous medium of a hydrophilic protein (e.g. human serum albumin (HSA)) onto
a hydrophilic solid substratum such as a clean glass surface, one has to deal with a macroscopic-level repulsion
between HSA and glass at (generally) the majority of orientations of the protein molecules, and also a microscopic-
level attraction between HSA and glass at (generally) the minority of orientations of the protein molecules. The first
phenomenon represents von Smoluchowski’s improbability of adhesion or adsorption and the second represents the
probability of adhesion or adsorption [1]. Both contingencies have to be taken into account in determining von
Smoluchowski’s net probability factor, f of the kinetic association constant, ka, pertaining to protein adsorption. In
the exceptional case where both the protein and the solid substratum are hydrophobically/hydrophilically and
electrostatically neutral, f=1, and the ka-value is only proportional to the diffusion coefficient of the protein [2]. In
order to determine the contributions of both the macroscopic repulsion and the microscopic attraction pertaining to
the kinetics of protein adsorption, an extended DLVO analysis (XDLVO) needs to be done on these interactions at
all distances and at all protein orientations. The XDLVO analysis comprises the Lewis acid – base interaction energies
as a function of distance, in addition to the Lifshitz – van der Waals and the electrokinetic interaction energies [2–5].
© 1999 Elsevier Science B.V. All rights reserved.

Keywords: DLVO theory; Human serum albumin; Protein

1. Introduction

Von Smoluchowski’s mathematical description

* Corresponding author. of the flocculation kinetics of suspended particles

0927-7765/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 9 2 7 - 7 7 6 5 ( 9 9 ) 0 0 0 2 8 - 4
100 C.J. 6an Oss et al. / Colloids and Surfaces B: Biointerfaces 14 (1999) 99–104
[1] can be adapted to express the kinetic constant
(ka) of the adsorption of protein molecules from
an aqueous solution onto a flat plate [2];
ka =4pl0DfA
where l0 is the minimum equilibrium (Born–Ki-
hara) distance between the outer electron shell
boundaries (van der Waals boundaries) of adjoin-
ing non-covalently interacting molecules (l0 =
0.157 nm [3,4]); D is the diffusion coefficient of
the protein, f is a factor proposed by von Smolu-
chowski to comprise both the ‘probability’ and
negative–positive physisorption and the micro-
scopic scale interactions also fall into the category
of physisorption. This is because both macro-
scopic and microscopic interactions of this type
(1)
only depend on strictly noncovalent forces, which
rules out chemisorption [6].
2. The f-factor of Eq. (1)
Factor f (Eq. (1)) comprises all interaction ener-
gies (repulsive and attractive), at all distances, l,
the ‘improbability’ [1] of particles (or molecules) from l=l0 to , and at all orientations (f of the
approaching each other sufficiently closely for potentially adsorbing protein or other macro-

& !  & n"

attachment to ensue, and A is Avogadro’s number molecule [2]:
(6.02× 1020) expressed as the number of
molecules in 1 cm3 of a molar solution. When l0
is expressed in cm (i.e. l0 =1.57 ×10 − 8 cm) and
D in cm2 s − 1, ka has the dimension of cm3
mmole − 1 s − 1 =l M − 1 s − 1 [2]. Thus, if it were
not for von Smoluchowski’s f-factor, the value of
the kinetic adsorption constant, ka, of a protein
onto a flat plate (Eq. (1)), would depend only on
the protein’s diffusion constant, D, and is indeed
just proportional to D when f = 1.
The f-factor however is rarely equal to one. For
instance, a protein such as human serum albumin
(HSA) dissolved in water is not attracted to a
clean glass surface at neutral pH, in most orienta-
tions of the HSA molecule. In some of the possi-
ble orientations of HSA, however, it is attracted
to certain sites (usually Ca2 + -containing sites [5]).
There is therefore a balance between a long-range
macroscopic repulsion and a shorter-range micro-
scopic attraction between HSA and glass in water
[5] or, in von Smoluchowski’s terminology, a bal-
ance between the improbability and the probabil-
ity of the HSA molecules being able to adsorb
onto the glass, expressed as the factor, f which is
further elaborated on below.
For protein adsorption from an aqueous solu-
tion, as well as for ligand – receptor (e.g. antigen–
antibody) interactions in aqueous media, the
difference between interactions at a macroscopic
level is not analogous to the difference between
physisorption and chemisorption (see Adamson
and Gast [6]). In cases like the one described here,
the macroscopic scale interactions are akin to
1 l=
− DG
f= exp dl df (2)
f l kT
l = l0
For the determination of factor f, one therefore
needs a complete energy versus distance analysis
of both the macroscopic repulsion and the micro-
scopic attraction energies, i.e. DLVO-type analy-
ses [7,8].
3. Extended DLVO analysis
When applied to aqueous media, as is virtually
always the case with protein adsorption, one
must, in addition to the Lifshitz–van der Waals
(LW) attractive and the electrostatic (EL) repul-
sive interactions of the classical DLVO theory,
include the quantitatively predominant Lewis
acid–base (AB) interactions, resulting in an ex-
tended DLVO (XDLVO) energy versus distance
analysis [4,5,9]. The LW, the electron-donor (š),
the electron-acceptor (› ) components of the sur-
face tension of HSA and of glass are given in
Table 1, as well as the c0-potential of both [5].
The equations governing the values of DG LW,
DG AB and DG EL have been given in [4,5,9].
4. Macroscopic repulsion
Macroscopic-scale interactions are noncovalent
(here, repulsive) interactions whose energies are
averaged over the entire surface of the adsorbate
 C.J. 6an Oss et al. / Colloids and Surfaces B: Biointerfaces 14 (1999) 99–104 101

Table 1
Surface properties of human serum albumin (HSA) and glass used in the HSA-adsorption calculations

g LW (mJ m−2) g š (mJ m−2) g › (mJ m−2) c0 (mV)

HSA (hydrated) [4] 26.8 6.0 51.5 −31.8

Glass [15] 33.7 1.3 62.2 −59.3

molecule and the substratum. Microscopic-scale site of, e.g. an imbedded Ca2 + ion acts as an
interactions are noncovalent (attractive) interac- electron-acceptor, which (locally) attracts a
tions between biopolymer domains situated at the protein molecule (which is a strong overall elec-
solvent interface, with a small radius of curvature, tron-donor), at loci of small radii of curvature
and small discrete sites present on the adsorbing (which loci are the most prone to pierce the
substratum. overall macroscopic repulsion field). Typically, the
The XDLVO diagrams resulting from the data close-range (l= l0) attraction between Ca2 + and
given in Table 1 and representing the net repulsive an ‘elbow’ of an HSA molecule is  −20 kT,
macroscopic interaction between HSA and glass which at first sight is feebler, energetically than
are shown in Fig. 1. The three dimensional mea- the macroscopic repulsion of + 40 kT in the same
surements and shape of the HSA molecule are configuration [5] (compare the lower curve of Fig.
taken from the X-ray diffraction results given by 1 with Fig. 2). Nonetheless, the microscopic single
He and Carter [10] and Carter and He [11]. The site attraction prevails because at lB R, i.e. at
HSA molecule is V-shaped, where each branch of lB 1.3 nm, the macroscopic repulsion is super-
the V is bent twice. As a first approximation, seded by the microscopic attraction. This is be-
HSA can thus be treated as a number of cylinders
of 4.0 nm length and 2.6 nm diameter, interrupted
by ‘elbows’ which can be considered as half-
spheres, with R =1.3 nm (including the ‘point’ of
the V). Then, at neutral pH, with a cylindrical
moiety parallel to a glass plate, there is a net
macroscopic repulsion which, at closer range,
equals +100 kT, while at l = 6 nm, it still equals
+ 1 kT; see the upper curve of Fig. 1. With an
‘elbow’ pointing to the glass plate, there is a
macroscopic repulsion of + 40 kT at close range,
still amounting to + 1 kT at l =4 nm; see the
lower curve of Fig. 1.

5. Microscopic attraction

The microscopic scale attraction of proteins

such as HSA onto glass and other mineral sur-
faces mainly occurs at discrete site cations (in
glass these are mainly imbedded calcium ions [5]).
HSA can be dissociated from the cations occur-
ring on minerals surfaces with complexing agents
such as Na2EDTA [5,12],or sodium hexam-
etaphosphate (unpublished results). A free ionized
Fig. 1. XDLVO energy vs. distance balance. Macroscopic
interaction curves of the interaction between a human serum
albumin (HSA) molecule and a clean (hydrophilic) glass sur-
face (cf. Table 1, ref. [5]). Upper curve: macroscopic repulsive
interaction between a parallel cylindrical HSA domain and the
glass surface. Lower curve: macroscopic repulsive interaction
of an HSA ‘elbow’, pointing at the glass surface.
102 C.J. 6an Oss et al. / Colloids and Surfaces B: Biointerfaces 14 (1999) 99–104

i.e. the macroscopic repulsion at all unfavorable

Fig. 2. XDLVO energy vs. distance balance. Microscopic
interaction curve of the net attractive interaction between an
HSA ‘elbow’ and a glass surface (cf. ref. [5]). The surface area
above the curve is hatched vertically to indicate the area from
which
orientations, averaged over distance (Fig. 3). Part
two represents the probability of interaction [1],
i.e. the microscopic attraction at the favorable
orientations, averaged over distance (Fig. 2).
Then, from the overall three-dimensional struc-
ture of HSA, the relative import of the influence
of part one (Fig. 3), compared to that of part two
(Fig. 2), must be decided. If all ‘elbows’ are
equally likely to be attracted to attractor sites of
the glass, the ratio of the macroscopic (Fig. 3) to
the microscopic influences (Fig. 2) would be 
1.0. If, on the other hand, only the ‘elbow’ at the
point of the V of HSA plays a role, that ratio
would be  3.0. Both cases are worked out in
Table 2.
The above reasoning applies to the occurrence
of a macroscopic-level repulsion in an aqueous
medium between a hydrophilic biopolymer such
as HSA and a hydrophilic solid substratum such
as clean glass, in which case the first part of the

xmicroscopic =
1 &  
l=
DG
dl
l l = l0 kT
is obtained.

cause locally, the macroscopic repulsion which,

elsewhere, is mainly due to electrostatic and elec-
tron-donor– electron-acceptor interactions, is here
replaced by an electrostatic and electron-accep-
tor–electron-donor attraction. Thus, between the
HSA ‘elbow’ and a discrete calcium-containing
site, the microscopic attraction, at closer range,
does not have to overcome the entire macroscopic
repulsion, but locally replaces it with a micro-
scopic attraction. Fig. 2 shows the XDLVO dia-
gram for the combined macroscopic repulsion
(from l=10 to 2 nm) and microscopic (from
l = 2 to 0) attraction energies versus distance, l.
Fig. 3. XDLVO energy vs. distance balance. Macroscopic
interaction curve of the repulsive interaction between a parallel
cylindrical HSA domain and the glass surface (cf. upper curve
6. Role of XDLVO diagrams in the determination of Fig. 1 and ref. [5]). The surface area below the curve is
hatched vertically to indicate the area from which
of kinetic adsorption constants
xmacroscopic =
1 &  
l=
DG
dl
Factor f (Eq. (2)) consists of two parts. Part l l = l0 kT
one comprises the improbability of interaction [1], is obtained.
 C.J. 6an Oss et al. / Colloids and Surfaces B: Biointerfaces 14 (1999) 99–104 103

Table 2
Kinetics of the adsorption of human serum albumin to glassa

Ratio of orientations of macro- Factor Free energy of each mode of re- Total of average f=exp(−x) ka l M−1 s−1c
scopic repulsion to microscopic pulsion, resp. attraction free energies= x b
attraction

1:1 0.5× +7.945 kTd =+2.783 0.062 4.42×106

0.5× −2.379 kTe =+2.783 0.062 4.42×106
3:1 0.75× +7.945 kTd =+5.364 0.0047 3.35×105
0.25× −2.389 kTe =+5.364 0.0047 3.35×105

a
Computations of the J-factor (Eq. (2)) of von Smoluchowski’s equation (Eq. (1)) at different ratios of macroscopic repulsion to
microscopic attraction orientations (see text and XDLVO diagrams).
b

x=
1 &  
l=
DG
dl, cf. Eq. (2).
l l = l0 kT
c
See Eq. (1) for f =1 and for HSA, D= 6×10−7 cm2 s−1.
d
See XDLVO diagram, Fig. 3.
e
See XDLVO diagram, Fig. 2.
f-factor (Eq. (2)) is smaller than unity. However,
in the case of an attraction between a hydropho-
bic biopolymer and a hydrophilic substratum, or
between a hydrophilic biopolymer and a hydro-
phobic substratum, or between a biopolymer and
a substratum which are both hydrophobic, the
first part of the f-factor, and thus the entire
f-factor, exceeds unity.
7. Results and discussion
As a first approximation, it is reasonable and
feasible to combine together all the net macro-
scopic repulsive energies, over all distances from
l= l0 to 10 nm, cf. Fig. 3. (Beyond l =10 nm,
the repulsive as well as the attractive energies are
too small to take into account). Similarly, one can
combine together all the net macroscopic attrac-
tive energies (cf. Fig. 2). As seen in Table 2,
column three, the long-range macroscopic repul-
sion energy is about three times greater than the
long-range microscopic attraction energy, aver-
aged over all distances. What is unknown is the
proportion of the contribution of each to the total
interaction energies.
Two plausible proportions are shown in Table
2. The first one is an equal distribution of repul-
sive macroscopic and attractive microscopic ori-
entations (ratio= 1:1), which yields a net average
repulsive energy of about + 2.8 kT, and an f-fac-
tor of 0.062, resulting in ka = 4.42× 106 l M − 1
s − 1. The second one is a 3:1 distribution of
repulsion vs. attraction, which would be the ap-
proximate ratio if HSA adsorbs to glass only, or
mainly, via the principal point of the V of its
tertiary configuration. The net total repulsion
then would amount to  + 5.4 kT, yielding a
much smaller f-factor of 0.0047, and a ka =
3.35× 105 l M − 1 s − l.
Unfortunately, to date there are not sufficient
experimental data to decide between these two
possibilities. There are practically no data in the
literature on the real specific kinetic adsorption
constant of serum albumin onto very hydrophilic
surfaces, comparable to clean glass. This is due to
the fact that only very few workers in the field
appear to realize that the only true value for the
specific ka of a given system, is the ka-value ob-
tained by extrapolation to zero protein concentra-
tion, and to zero time. So far, only Cuypers et al.
[13] and van Regenmortel et al. [14] have demon-
strated the extremely strong dependence of the ka
in protein adsorption (aspecific [13], as well as
specific [14]), on the protein concentration [2]. As
an example, the difference between low (e.g. 400
104 C.J. 6an Oss et al. / Colloids and Surfaces B: Biointerfaces 14 (1999) 99–104
nM) and very low (e.g. 4 nM) protein concentra-
tions can yield a difference between ka-values,
from 5.0 ×103 l M − 1 s − 1 to 3.1 × 105 l M − 1 s − 1
respectively, i.e. a factor of 62 [14]. In a case like
the one presented here, only new experimental
results can help decide which proportion of
protein orientations leads to net macroscopic re-
pulsion, and which proportion corresponds to net
microscopic attraction. To that effect one needs to
be able to measure degrees of adsorption down to
very low protein concentrations (e.g. 10 nM, or
less), and at very short times after the onset of
adsorption (e.g. less than 1 s). We are now begin-
ning to obtain results under these conditions, with
serum albumin, and micron-sized mineral parti-
cles [16]. For the adsorption of proteins onto flat
surfaces such as glass plates, however, it is doubt-
ful whether the determination of the degree of
protein adsorption at very low concentrations and
after extremely short times is possible under con-
ditions of adequate mass transport.
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