Langmuir 2004, 20, 169-174
169
Binding of Amino Acids to “Smart” Sorbents: Where Does
Hydrophobicity Come into Play?
Havazelet Bianco-Peled* and Shlomit Gryc
Department of Chemical Engineering, Technion-Israel Institute of Technology,
Haifa 32000, Israel
Received September 14, 2003. In Final Form: October 21, 2003
Poly(N-isopropylacrylamide) (PNIPA)-based sorbents have been successfully used as sorbents in
temperature-sensitive chromatography. Yet, the mechanisms controlling the binding of biochemicals to
these sorbents and, therefore, the separation process are not fully understood. In the current work, the
role of hydrophobic interactions in the binding of amino acids of different hydrophobicities to PNIPA
microgels was studied. Binding experiments were conducted both below (25 °C) and above (37 °C) the
volume-phase transition temperature of the gel. At 25 °C, no straightforward correlation between the
partition coefficient and the hydrophobicity could be suggested for low hydrophobicity values. Contrary,
at higher hydrophobicities the partition coefficient increases with increasing hydrophobicity. This correlation
holds for the whole hydrophobicity range at 37 °C; however, the binding data suggests two different binding
mechanisms of the hydrophilic amino acids and the hydrophobic ones. Isothermal titration calorimetry
measurements confirmed this suggestion: The binding of hydrophobic amino acids seems to be driven by
hydrophobic interactions, as evident from the positive binding enthalpy and the clear correlation between
the amino acid’s hydrophobicity and the binding entropy. Contrary, the binding of the hydrophilic amino
acids was exothermic, implying a binding mechanism based on specific interactions, most probably hydrogen
bonding.
Introduction
Stimuli-responsive polymers, often termed “smart materials”, are attracting a great deal of attention because
of the dependence of their physical properties on the
conditions of the surrounding environment. A well-known
example is the temperature-responsive polymer poly(Nisopropylacrylamide) (PNIPA), which is soluble in water
below its “lower critical solution temperature” (LCST) of
about 32 °C but precipitates at higher temperatures.1,2
Cross-linked PNIPA hydrogels exhibit a similar hydrophilic-hydrophobic transition, associated with a dramatic
decrease in the gel volume, when heated above the critical
temperature. This unusual phase behavior has led to the
suggestion of several types of bioseparation processes
utilizing PNIPA, such as temperature-modulated extraction 3,4 and affinity precipitation.5,6 PNIPA-based materials
were also exploited as “smart” sorbents in temperaturemodulated chromatography,7-15 in which temperature
swings around the LCST affect the affinity of the bio* Corresponding author. Tel.: 972-4-8293588. Fax: 972-48295672. E-mail: [email protected].
(1) Okano, T. Biorelated Polymers and Gels: Controlled Release and
Applications in Biomedical Engineering; Academic Press: San Diego,
1998; p 257.
(2) Schild, H. G. Prog. Polym. Sci. 1992, 17, 163.
(3) Vasheghani-Farahani, E.; Cooper, D. G.; Vera, J. H.; Weber, M.
E. Chem. Eng. Sci. 1992, 47, 31.
(4) Freitas, R. F. S.; Cussler, E. L. Chem. Eng. Sci. 1987, 42, 97.
(5) Balan, S.; Murphy, J.; Galaev, I.; Kumar, A.; Fox, G. E.;
Mattiasson, B.; Willson, R. C. Biotechnol. Lett. 2003, 25, 1111.
(6) Kumar, A.; Khalil, A. A. M.; Galaev, I. Y.; Mattiasson, B. Enzyme
Microb. Technol. 2003, 33, 113.
(7) Teal, H. E.; Hu, Z.; Root, D. D. Anal. Biochem. 2000, 283, 159.
(8) Yoshioka, H.; Mikami, M.; Nakai, T.; Mori, Y. Polym. Adv. Technol.
1995, 6, 418.
(9) Hosoya, K.; Kimata, K.; Araki, T.; Tanaka, N.; Frechet, J. M. J.
Anal. Chem. 1995, 67, 1907.
(10) Ivanov, A. E.; Zhigis, L. S.; Kurganova, E. V.; Zubov, V. P. J.
Chromatogr., A 1997, 776, 75.
chemicals to the sorbent and, thus, may be used to alter
the resolution and the selectivity. Often, the resolution in
this process improved considerably at a temperature above
the LCST.7,9,11-14 Because the hydrophobicity of the
“smart” sorbent is higher once heated, most authors
interpreted the improved resolution as evidence that the
main biochemical-sorbent interaction mechanism is a
hydrophobic interaction, leading to enhanced binding
above the phase transition temperature. Contrary, other
researchers concluded that the phenomenon of temperature-modulated binding might be due to the changes in
the pore size, resulting from the PNIPA volume transition.10,16 The possibility that the binding might be influenced from specific interactions between PNIPA and the
biochemicals, such as hydrogen bonding, was considered
as well.17
The work presented in this paper is aimed at obtaining
a better understanding of the role of hydrophobic interactions in the binding of chemicals to PNIPA. As a model,
we have focused on adsorption of amino acids to PNIPA
microgels and studied the effect of the amino acid
hydrophobicity on its binding. In addition, as a route to
distinguish between an interaction-based mechanism and
a size-exclusion mechanism, we have measured the
(11) Kanazawa, H.; Kashiwase, Y.; Yamamoto, K.; Matsushima, Y.;
Kikuchi, A.; Sakurai, Y.; Okano, T. Anal. Chem. 1997, 69, 823.
(12) Kanazawa, H.; Sunamoto, T.; Matsushima, Y.; Okano, T. Anal.
Chem. 2000, 72, 5961.
(13) Kanazawa, H.; Yamamoto, K.; Kashiwase, Y.; Matsushima, Y.;
Takai, N.; Kikuchi, A.; Sakurai, Y.; Okano, T. J. Pharm. Biomed. Anal.
1997, 15, 1545.
(14) Kanazawa, H.; Yamamoto, K.; Matsushima, Y.; Takai, N.;
Kikuchi, A.; Sakurai, Y.; Okano, T. Anal. Chem. 1996, 68, 100.
(15) Kikuchi, A.; Okano, T. Prog. Polym. Sci. 2002, 27, 1165.
(16) Lakhiari, H.; Okano, T.; Nurdin, N.; Luthi, C.; Descouts, P.;
Muller, D.; Jozefonvicz, J. Biochim. Biophys. Acta 1998, 1379, 303.
(17) Kimhi, O.; Bianco-Peled, H. Langmuir 2002, 18, 8587.
10.1021/la0357155 CCC: $27.50 © 2004 American Chemical Society
Published on Web 11/26/2003
170
Langmuir, Vol. 20, No. 1, 2004
Bianco-Peled and Gryc
binding enthalpies and entropies,17-21 using isothermal
titration calorimetery (ITC).22
Materials and Methods
Materials. N-Isopropylacrylamide (NIPA; Sigma) was recrystallized from a mixture of toluene and petrol ether (40:60).
Ammonium persulfate(Carlo Erba) was recrystallized from MiliQ
water. All other chemicals were analytical grade and were used
as received. N,N,N′,N′-Tetramethylethylenediamine (TEMED),
glycine, lysine, and valine were purchased from Sigma. Aspartic
acid, glutamic acid, and alanine were purchased from Fluka.
2-Methoxy ethanol, ninhydrin, tin chloride, and leucine were
purchased from Riedel DeHaen. n-Propanol and phosphate buffer
(in a precalibrated ampule) were purchased from Carlo Erba.
Citric acid was purchased from Bio Lab. Pluronic L-61 was
obtained from BASF. All the solutions were prepared using
Milli-Q water. The amino acid solutions for the binding isotherm
and ITC measurements were prepared in the phosphate buffer
at pH 7.
Synthesis. Cross-linked PNIPA microgels were synthesized
using the inverse polymerization technique as described elsewhere.17 Briefly, 50 mL of aqueous solution containing the
monomer (NIPA, 5 g), the cross-linker (N,N′-methylenebisacrylamide, 0.08 g), and the initiator (ammonium persulfate, 0.1 g)
was dispersed in 250 mL of paraffin oil containing 1% pluronic
L-61. Upon formation of aqueous droplets, 5 mL of TEMED
(accelerator) were added to the continuous phase to initiate the
redox polymerization, which is then performed for 3 h at 4 °C.
After polymerization, the beads were separated by excess water,
washed several times with a mixture of acetone and water (1:1)
to remove the monomer, and filtered to obtain PNIPA microgel
beads. The UV adsorption of the wash water was measured to
ensure that all unreacted monomer was removed from the gel.
Equilibrium Binding Isotherms. PNIPA microgel particles
were separated from excess water, and a weighed amount of
swollen particles (ca. 0.5 g at 25 °C) was transferred into a glass
tube and heated to the experiment temperature. A total of 3 mL
of amino acid solution was added, and the mixture was shaken
in a temperature-controlled water bath at 150 rpm for 2.5 h. The
solution was then separated from the gel by vacuum filtering
through a glass fiber filtering paper (GF-A, Whatman). Amino
acids in the remaining solution were dyed using the Ninhydrin
procedure.23 Their concentration C′ was determined from UV
adsorption measurements at 570 nm, using a Unico 2100
spectrophotometer. This procedure was repeated three times for
each concentration at 25 °C and at least five times for each
concentration at 37 °C, where the measurements were more
scattered. Instead of averaging the data, all the data points are
presented on the binding isotherms shown in Figures 1 and 2.
We estimated the experimental error to be 15% at 37 °C and 10%
at 25 °C.
The bound amount U, in terms of the weight of bound acid/g
gel, was calculated from a mass balance:
U)
V′0C′0 - V′C′
wg
(1)
where wg is the weight of the wet gel at the experiment
temperature (meaning, the weight of swollen or collapsed gel
introduced into the system), V′0 and V′ are the volume of the
amino acid solution at the beginning of the experiment and the
volume of the filtrate, respectively, and C′0 and C′ are the
(18) Lin, F.-Y.; Chen, W.-Y.; Hearn, M. T. W. Anal. Chem. 2001, 73,
3875.
(19) Lin, F.-Y.; Chen, W.-Y.; Ruaan, R.-C.; Huang, H.-M. J. Chromatogr., A 2000, 872, 37.
(20) Tsai, Y.-S.; Lin, F.-Y.; Chen, W.-Y.; Lin, C.-C. Colloids Surf., A
2002, 197, 111.
(21) Huang, H.-M.; Lin, F.-Y.; Chen, W.-Y.; Ruaan, R.-C. J. Colloid
Interface Sci. 2000, 229, 600.
(22) Blandamer, M. J. Thermodynamic Background to Isothermal
Titration Calorimetry. In Biocalorimetry: Applications of Calorimetry
in the Biological Science; Ladbury, J. E., Chowdhry, B. Z., Eds.; John
Wiley: New York, 1998.
(23) Snell, F. D.; Snell, C. T. Colorimetric methods of analysis; D.
Van Nostrand: New York, 1955; Vol. 4.
Figure 1. Binding isotherms for (]) glycine, (0) alanine, ([)
valine, and (2) leucine at (a) 25 and (b) 37 °C. The solid lines
show the fits to the linear adsorption isotherms.
corresponding amino acid concentrations. At 25 °C, V′0 and V′ are
equal. At 37 °C, V′ also includes water squeezed from the collapsed
gel (90% of the initial water content of the gel is lost during the
heating process from 25 to 37 °C).17
ITC Measurements. ITC measurements were performed
using a VP-ITC apparatus (Microcal). An amino acid solution
was titrated serially (usually 14 injections, 20-µL each, at 12min time intervals) into the calorimetric cell containing the gel
suspension (ca. 0.1 g gel/mL). Measurements involving the gel
were taken in low-gain mode, for enhanced accuracy. Reference
measurements (i.e., amino acid titrated into buffer and buffer
titrated into the gel suspension) were taken in high-gain mode.
All the ITC measurements were conducted after full degassing
of the solution. It should be noticed that the accessible concentration range for aspartic acid and glutamic acid is limited as a
result of their lower solubilities in water.
The enthalpy of binding, ∆h, defined here as the enthalpy
change associated with the transfer of 1 mg of amino acid from
the aqueous phase to the gel phase, is given by17
∆h ) hgs - hw
s
(2)
g
where hw
s and hs are the partial enthalpies of the solute (amino
acid) when found in the solution environment or in the gel
environment, respectively. The enthalpy of binding could be
calculated from the measured enthalpy change ∆H associated
with the injection of volume ∆V and concentration of Csyg into
the titration cell as follows:
∆H ) hw
s
{(
V0 +
)
}
Vg∆V
C - C0V0 + hgs {(wg - ∆wg)U Vc
wgU0} - ∆(wghg) (3)
Binding of Amino Acids
Langmuir, Vol. 20, No. 1, 2004 171
Table 1. Hydrophobicities and Partition Coefficients for
the Studied Amino Acids
partition coefficient
amino acid
hydrophobicity
25 °C
37 °C
Kp,37/Kp,25
leucine
valine
alanine
glycine
lysine
glutamic acid
aspartic acid
0.943
0.825
0.616
0.501
0.283
0.043
0.028
1.7
1.1
1.0
1.1
0.8
2.6
1.3
27.7
8.7
3.4
7.4
4.7
4.3
3.4
16.3
7.7
3.6
6.7
5.9
1.6
2.4
the amino acids from the “most hydrophilic” to the “most
hydrophobic”. Even though many hydrophobicity scales
are available in the literature, most of them predict the
same “hydrophobicity order” for the amino acids selected
for this study. Therefore, and as a matter of convenience,
only one hydrophobicity scale will be referred to in the
following. A list of the amino acids selected for this study,
along with their hydrophobicities according to the Black
and Mould scale,26 is given in Table 1.
The binding isotherms for the different amino acids, at
two temperatures, are shown in Figures 1 and 2. For
clarity, the isotherms were divided into two groups: Figure
1 shows the binding isotherms of the four amino acids
having higher hydrophobicities: glycine, alanine, valine,
and leucine, at 25 °C (Figure 1a) and at 37 °C (Figure 1b).
Figure 2 shows the binding isotherms of the amino acids
having lower hydrophobicities: glutamic acid, aspartic
acid, and lysine, at 25 °C (Figure 2a) and at 37 °C (Figure
2b). As can be seen, all curves show a linear behavior and
do not reach saturation in the examined concentration
range. Thus, the binding isotherm may be described using
the following relation:
Figure 2. Binding isotherms for (0) glutamic acid, ([) aspartic
acid, and (2) lysine at (a) 25 and (b) 37 °C. The solid lines show
the fits to the linear adsorption isotherms.
where Vg is the gel’s volume, Vc is the volume of the titration cell,
and ∆wg is the change in the gel weight during ejection. U0 and
U are the amino acid concentration in the gel (in terms of weight
of bound acid/g gel) before and after the current injection,
respectively. C0 and C are the solution concentration of the
unbound amino acid before and after the current injection,
respectively. The term ∆(wghg) is related to the enthalpy of
dilution of the gel and was found to be negligible in our
experiments. We need also to consider the mass balance for the
solute
∆VCsyr + C0(V0 - ∆V0) + U0(wg - ∆wg) ) U(wg - ∆wg) +
C(V0 - ∆V0 + ∆V) (4)
and the relations between U and C, obtained from the binding
isotherms.
Results and Discussion
Equilibrium Binding Isotherms. One of the goals of
the current research was to study the effect of the amino
acid hydrophobicity on its binding to the PNIPA microgel.
A common way to quantify amino acid hydrophobicity is
by using “hydrophobicity scales”,24-29 which allow ordering
(24) Hopp, T. P.; Woods, K. R. Proc. Natl. Acad. Sci. U.S.A. 1981, 78,
3824.
(25) Kyte, J.; Doolittle, R. F. J. Mol. Biol. 1982, 157, 105.
(26) Black, S. D.; Mould, D. R. Anal. Biochem. 1991, 193, 72.
(27) Browne, C. A.; Bennett, H. P. J.; Solomon, S. Anal. Biochem.
1982, 124, 201.
(28) Bull, H. B.; Breese, K. Arch. Biochem. Biophys. 1974, 161, 665.
(29) Berggren, K.; Wolf, A.; Asenjo, J. A.; Andrews, B. A.; Tjerneld,
F. Biochim. Biophys. Acta 2002, 1596, 253.
U ) KpC
(5)
where Kp is a constant partition coefficient.
Kp values at 25 °C (Kp,25) and at 37 °C (Kp,37), which
were calculated from the binding isotherms using a linear
regression, as well as the ratio between them (Kp,37/Kp,25),
are summarized in Table 1. Note that both the bound
amount and the solution concentration shown in Figures
1 and 2 in molal units (i.e., mol/kg) and, therefore, the
calculated Kp values, are dimensionless.
The bound amounts U, shown in Figures 1 and 2, reflect
a nominal value as calculated from the total amount of
amino acid that has penetrated into the gel (see eq 1).
Because the gel is initially free of amino acid but contains
a large percentage of water, the solute is expected to diffuse
into the gel as a result of concentration gradients, even
if there is no preferred binding to the polymer backbone.
Therefore, a reference value for the partition coefficient
under such “no binding” conditions can be easily calculated
from the water content of the gel and a mass balance and
was found to be 0.94 at 25 °C and 0.4 at 37 °C. As is
evident from Table 1, the only amino acid that does not
show Kp values larger than the reference value is lysine
at 25 °C. For all the other amino acids, a preferred binding
to the gel at both temperatures was found. Moreover, the
partition coefficient was always higher at 37 °C than at
25 °C.
As mentioned before, it was previously observed that
the resolution in a temperature-modulated chromatography process of amino acids improved considerably at a
temperature above the LCST.12 The measured binding
isotherms of the hydrophobic amino acids show the same
behavior, and the differences between the partition
coefficient values of the different acids are more pro-
172
Langmuir, Vol. 20, No. 1, 2004
Bianco-Peled and Gryc
Figure 3. Dependence of the partition coefficient on the amino
acid hydrophobicity at (a) 25 and (b) 37 °C. Lines are guides
to the eye.
Figure 4. Enthalpy of binding between the PNIPA gel and (])
glycine, (0) alanine, ([) valine, and (2) leucine at (a) 25 and
(b) 37 °C.
nounced at 37 °C than at 25 °C (Figure 1). Moreover, the
value of Kp increases as the amino acid hydrophobicity
increases (Figure 3). The influence of the hydrophobicity
is more pronounced at a temperature of 37 °C, which is
higher than the phase transition temperature. These
observations seem to support the hypothesis that the
higher binding above the transition temperature is due
to enhanced hydrophobic interactions. However, a different behavior is observed for the hydrophilic amino acids
(Figure 2). At a temperature of 25 °C, in which the gel is
hydrophilic, the partition coefficient differs considerably
from the reference value of 0.94, indicating either enhanced binding to the gel (for aspartic acid and glutamic
acid) or a preference to the aqueous phase (for lysine).
Interestingly, the resolution between the amino acids
seems to be better at a temperature of 25 °C (Figure 2).
The different behaviors of the hydrophobic and the
hydrophilic amino acids is clearly seen in Figure 3, in
which the partition coefficient is plotted as a function of
the amino acid hydrophobicity. At 25 °C, below the phase
transition temperature of the gel, no straightforward
correlation between the partition coefficient and the
hydrophobicity could be suggested for the low hydrophobicity values. Contrary, at higher hydrophobicities the
partition coefficient increases with increasing hydrophobicity. This correlation also exists for the whole hydrophobicity range at 37 °C, that is, above the transition
temperature. However, two different slopes can be observed in Figure 3, suggesting two different binding
mechanisms of the hydrophilic amino acids and the
hydrophobic ones.
In an attempt to gain a better insight into the binding
mechanism, the binding enthalpies and entropies were
determined from ITC measurements. As a measure of the
strength of the interactions, we have calculated the
enthalpy change ∆h associated with the transfer of amino
acid from the aqueous phase to the gel phase. However,
it should be noted that this analysis is approximated
because it is impossible to separate the thermal effect
caused by the binding from the one caused by the water
desorption from the hydrophobic PNIPA surface.30
Figures 4 and 5 display ∆h as a function of the solution
concentration. As with the binding isotherms, the curves
were divided into two groups. Figure 4 shows the enthalpies of the four amino acids having the higher
hydrophobicities: glycine, alanine, valine, and leucine,
at 25 °C (Figure 4a) and at 37 °C (Figure 4b). Figure 5
shows the binding enthalpies of the three amino acids
having the lower hydrophobicities: glutamic acid, aspartic
acid, and lysine, at 25 °C (Figure 5a) and at 37 °C (Figure
5b). These Figures show that for the hydrophobic amino
acids the heat of binding is positive, suggesting an
endothermic binding process, whereas for the hydrophilic
amino acids an exothermic process is observed. However,
apart from this qualitative observation, no particular
relationship between the binding enthalpy and the amino
acid hydrophobicity could be realized.
(30) Wang, G.; Pelton, R.; Zhang, J. Colloids Surf., A 1999, 153, 335.
Binding of Amino Acids
Langmuir, Vol. 20, No. 1, 2004 173
Figure 5. Enthalpy of binding between the PNIPA gel and (0)
glutamic acid, ([) aspartic acid, and (2) lysine at (a) 25 and (b)
37 °C.
Figure 6. Entropy of binding between the PNIPA gel and (])
glycine, (0) alanine, ([) valine, and (2) leucine at (a) 25 and
(b) 37 °C.
On the basis of the binding isotherms and the ITC results
presented so far, mechanisms for the binding of amino
acids to the PNIPA gel can be hypothesized. As already
mentioned, the hydrophilic and the hydrophobic amino
acids seem to be behaving differently, and, therefore, the
binding of each class will be discussed separately.
The ITC measurements imply that the binding of the
hydrophilic amino acids is due to specific interactions,
such as hydrogen bonding or electrostatic interactions.
To examine the relative importance of the latter, a series
of binding experiments at various pH values, both at 25
°C and at 37 °C, have been performed (data not shown).
These experiments detected only a small dependence of
the partition coefficient on the pH, that is, on the net
electric charge of the amino acid. Therefore, the main
mechanism controlling the binding of the hydrophilic
amino acids is assumed to be hydrogen-bond formation.
Below the phase transition temperature of the gel, a
straightforward correlation between the partition coefficient and the hydrophobicity could not be suggested. It
is not surprising that a complex process involving breaking
the amino acid’s hydrogen bonds with water and forming
new ones with the gel could not be expressed using a single
empirical parameter. For example, although lysine is
considered to be slightly more hydrophobic than aspartic
acid, its water solubility is much higher. It is likely that
the high water solubility reflects a high tendency toward
hydrogen bonding with water and, therefore, explains the
preference of the lysine for the aqueous phase. Similarly,
the low water solubility of the aspartic acid and the
glutamic acid might induce their binding to the gel. Above
the phase transition temperature of the gel, there is a
sharp decrease in the hydrogen bonding between the
PNIPA and the water.31 As a result, the number of free
amide groups on the polymer gel increases. The enhanced
binding of the hydrophilic amino acids above the transition
temperature may be attributed to the formation of
hydrogen bonds between the amine groups and the free
amide on the PNIPA.
Contrary to the hydrophilic amino acids, the positive
heat of binding measured for the hydrophobic amino acids
suggests that their binding is due to hydrophobic interactions, both at 25 °C and at 37 °C. This mechanism readily
explains the increase in the partition coefficient with
increasing hydrophobicity (Figure 3). Moreover, the
enhanced binding of the amino acids above the phase
transition temperature of the gel could be easily attributed
to the increased gel hydrophobicity. As an additional
verification for the existence of this mechanism, the
entropy change associated with the binding process has
been calculated according to the relation32,33
∆S )
∆h
+ R ln Kp
T
(6)
where ∆S is the binding entropy, T is the solution
temperature (K), and R is the gas constant.
(31) Ramon, O.; Kesselman, E.; Berkovici, R.; Cohen, Y.; Paz, Y. J.
Polym. Sci., Part B: Polym. Phys. 2001, 39, 1665.
(32) Chiou, M. S.; Li, H. Y. Chemosphere 2003, 50, 1095.
(33) Taniguchi, T.; Duracher, D.; Delair, T.; Elaissari, A.; Pichot, C.
Colloids Surf., B 2003, 29, 53.
174
Langmuir, Vol. 20, No. 1, 2004
Adsorption onto surfaces due to hydrophobic interactions is believed to be associated with desorption of water
from the hydrophobic surface and dehydration of the
adsorbed molecules. Because free water molecules have
larger entropy than bound ones, hydrophobic interactions
are thought to be reflected as the positive entropy of
binding. The binding entropies calculated for the hydrophobic amino acids (Figure 6) are indeed positive, supporting the suggested binding mechanism.
Finally, the entropy of binding is plotted as a function
of the amino acid hydrophobicity in Figure 7. For higher
hydrophobicities, the entropy of binding increases as the
hydrophobicity increases (solid line). However, this relationship is no longer valid for the hydrophilic amino
acids (dashed line). Once again, this finding implies that,
although hydrophobic interactions have a significant role
in the binding of hydrophobic amino acids, other mechanisms are involved in the binding of hydrophilic ones.
Conclusions
The adsorption of amino acids onto PNIPA microgels
was studied using binding isotherms and ITC measurements, at 25 °C and at 37 °C. The binding isotherms were
linear, indicating constant adsorption coefficients. Enhanced binding was found for all acids upon temperature
elevation from 25 °C to 37 °C. However, our results indicate
that the mechanisms involved in the binding of amino
acids to PNIPA depend on their hydrophobicities. While
the binding of hydrophobic amino acids is probably driven
Bianco-Peled and Gryc
Figure 7. Entropy of binding at zero concentration at (9) 25
and (O) 37 °C.
by hydrophobic interactions, hydrogen bonding seems to
dominate the binding of the hydrophilic amino acids.
Acknowledgment. This research was supported by
The Israel Science Foundation (grant 1380021). We thank
the Otto Meyerhof Center for Biotechnology established
by Minerva Foundation (Munich, Germany) for the
financial contribution in purchasing the VP-ITC and VPDSC instruments.
LA0357155