Chromatographic Framework to Determinethe Memantine Binding Mechanismon Human Serum Albumin Surface

Firas Ibrahim, Yves-Claude Guillaume&, Claire Andre

Equipe des Sciences Separatives et Biopharmaceutiques (2SB/EA-3924), Laboratoire de Chimie Analytique, Faculte de Medecine Pharmacie,Universite de Franche-Comte, Place Saint Jacques, 25030 Besancon Cedex, France; E-Mail: yves.guillaume@univ-fcomte.fr

Received: 16 January 2008 / Revised: 9 April 2008 / Accepted: 29 April 2008Online publication: 3 June 2008

Abstract

In this work, the interaction of memantine with human serum albumin (HSA) immobilized onporous silica particles was studied using a biochromatographic approach. The determinationof the enthalpy change at different pH values suggested that the protonated group in thememantine–HSA complex exhibits a heat protonation with a magnitude around 65 kJ mol�1.This value agrees with the protonation of a guanidinium group, and confirmed that an argininegroup may become protonated in the memantine–HSA complex formation. The thermody-namic data showed that memantine–HSA binding, for low temperature (<293 K), is dominatedby a positive entropy change. This result suggests that dehydration at the binding interface andcharge–charge interactions contribute to the memantine–HSA complex formation. Above293 K, the thermodynamic data DH and DS became negative due to van der Waals inter-actions and hydrogen bonding which are engaged at the complex interface. The temperaturedependence of the free energy of binding is weak because of the enthalpy–entropy compen-sation caused by a large heat capacity change, DCp = � 3.79 kJ mol�1 K�1 at pH = 7. Theseresults were used to determine the potential binding site of this drug on HSA.

Keywords

Column liquid chromatographyHuman serum albuminMemantine

Introduction

N-Methyl-D-aspartate (NMDA) recep-

tor antagonists have therapeutic poten-

tial in several central nervous system

disorders, including neuroprotective

treatment in chronic neurodegenerative

diseases, and symptomatic treatment in

other neurologic diseases [1].

Memantine, an NMDA antagonist,

has been recently approved for the

treatment of moderate to advanced

Alzheimer’s disease (AD) [2]. Meman-

tine is a low-affinity voltage-dependent

uncompetitive antagonist for glutama-

tergic NMDA receptors [3, 4]. By bind-

ing to the NMDA receptor with a higher

affinity than Mg2+ ions, memantine is

able to inhibit the prolonged influx of

Ca2+ ions which form the basis of neu-

ronal excitotoxicity [5]. The low affinity

and rapid off-rate kinetics of memantine

at the level of the NMDA receptor-

channel, however, preserves the physio-

logical function of the receptor as it can

still be activated by the relatively high

concentrations of glutamate released

following depolarization of the presyn-

aptic neuron [6]. Age-related changes in

physiology and organ function alter

drug pharmacokinetics. In addition,

older persons take more medications in

treating multiple disorders, increasing

the risk of drug–drug and drug–disease

interactions [7]. Thus, the expanded

pharmacokinetics studies, e.g. binding

on plasmatic proteins, are important for

drugs which are taken by aging patients

as the drugs of Alzheimer’s disease.

Memantine binds on plasmatic proteins

to about 45%, it crosses the blood-brain

barrier but its cerebrospinal fluid level is

20% to 50% lower than the serum level

due to albumin binding in serum [8–10].

HSA is the most abundant protein in

blood and can reversibly bind a large

number of pharmacological substances.

2008, 68, 179–186

DOI: 10.1365/s10337-008-0675-60009-5893/08/08 � 2008 Vieweg+Teubner | GWV Fachverlage GmbH

Original Chromatographia 2008, 68, August (No. 3/4) 179

Few specific binding sites are present on

HSA [11, 12]. The most important sites

are benzodiazepine and warfarin binding

sites. He et al. [12] have determined the

three dimensional structure of HSA and

have shown that these two binding sites

are located in hydrophobic cavities in

subdomains IIA and IIIA. Site I is

formed as a pocket in subdomain IIA

and involves the lone tryptophan of the

protein (Trp214). The inside wall of the

pocket is formed by hydrophobic side

chains, whereas the entrance to the

pocket is surrounded by positively

charged residues. Site II corresponds to

the pocket of subdomain IIIA, which has

almost the same size as Site I, the interior

of cavity is constituted of hydrophobic

amino-acid residues and the cavity exte-

rior presents two important amino-acids

residues (Arg410 and Tyr411) [13, 14].

Two common methods that have tradi-

tionally been used in evaluating the

binding of drugs to albumin include

equilibrium dialysis and ultrafiltration

[15–17]. These two methods suffer of

several disadvantages, as the long peri-

ods of time which are required to

establish an equilibrium during the

dialysis process [15, 16]. Furthermore, it

is necessary to correct for the alterations

in free and bound analyte concentrations

that occur during the dialysis procedure

[15]. Ultrafiltration requires less time to

perform, but like dialysis it requires the

use of a labeled drug and/or an addi-

tional analysis step for the actual mea-

surement of the final free drug

concentration. In addition, the effects of

analyte adsorption to the ultrafiltration

membrane must be considered [15, 16].

Other problems include difficulties with

temperature changes during the separa-

tion and problems when working with

highly bound drugs [15]. Because of

these limitations, there has been contin-

uing research to find better, faster and

more convenient approaches for the

analysis of drug–protein binding. One

such approach involves the use of affinity

chromatography (AC) [18]. AC is a li-

quid chromatography (LC)-based meth-

od in which the stationary phase consists

of an immobilized biologically-related

ligand. In the case of solute–albumin

studies, this ligand consists of serum

albumin which has been adsorbed or

covalently linked to a support like silica.

The association constants of many

ligands have been determined by zonal

elution [19] or frontal analysis [20]. The

thermodynamic process involved in the

binding have also been studied [21–23].

One advantage of utilizing AC for

solute–protein studies is the ability of

this method to reuse the same ligand

preparation for multiple experiments

(small amount of protein is needed for a

large number of studies), this helps to

give good precision by minimizing

run-to-run variations. Other advantages

include the ease with which AC methods

can be automated and the relatively

short periods of time that are required in

AC for most solute binding studies. The

fact that the immobilized protein is

continuously washed with an applied

solvent is yet another advantage of AC

[24, 25]. HSA was the model ligand used

in a great number of studies. The main

advantage of using HSA is the data

available for its interaction with a wide

range of organic and inorganic com-

pounds [18].

In this study, AC was used to deter-

mine and quantify the forces driving the

association between memantine and

HSA by studying the energetic changes

of this association as both a function of

temperature and pH. Moreover, the

number of protons linked to this me-

mantine binding reaction of HSA was

calculated. These results were used for

estimating the binding site of memantine

on HSA.

Experimental

Reagents and OperatingConditions

Memantine (Fig. 1) and diazepam were

purchased from Sigma (Paris, France),

water was obtained from an Elgastat

option water purification (Odil Talant,

France) fitted with a reverse osmosis

cartridge. Sodium dihydrogenophos-

phate and di-natriumhydrogenophos-

phate were obtained from Prolabo and

Merck (Paris, France) respectively. The

mobile phase consisted of 0.1 M sodium

phosphate buffer adjusted at different

pH varying between 5.0 and 7.0 (5.0, 5.5,

6.0, 6.5, and 7.0). Experiments were

carried out over the temperature range

278–308 K (278, 283, 293, 298, 303 and

308 K), and the mobile phase flow-rate

was 0.3 mL min�1. The memantine

(20 lL) was injected three times at each

temperature and pH. Once the mea-

surements were completed at the maxi-

mum temperature, the column was

immediately cooled to ambient condi-

tions to minimize the possibility of any

unfolding of the immobilized HSA.

Apparatus

The LC system consisted of a Shimadzu

LC- 10ATvp pump (Champs sur Marne-

France), a Rheodyne 7125 injection valve

(Cotati, California, USA) fitted with a 20

lL sample loop, and a Shimadzu

UV–Visible detector.AChromTechHSA

column (Interchim, Montlucon, France)

(150 mm · 4 mm I.D., 5 lm particle

size) was used where HSA was covalently

bound onto spherical 5 lm silica parti-

cles. The temperature was controlled with

an Interchim oven TM701 (Monlucon,

France).

Result and Discussion

Bulk Solvent pH Effects

Valuable information about the pro-

cesses driving the memantine–HSA

association mechanism can be further

gained by examining the effect of pH on

memantine retention. The memantine

retention on the albumin stationary

phase can be evaluated using the reten-

tion factor k:

k ¼ t � toð Þ=to ð1Þ

where (t) is the retention time of

memantine and (to) is the column void

time. The void time was determined using

the mobile phase peak. As well, the

memantine retention factor can be related

to the association constant K between

HSA and memantine as follows:

k ¼ UK ð2Þ

where U is equal to the ratio of the active

binding site number in the column over

180 Chromatographia 2008, 68, August (No. 3/4) Original

the void volume of the chromatographic

column. When the pH of the bulk sol-

vent changed, a full description is

essential, which explicitly maintains

conservation of mass of each species

taking into account binding of H+ to

human serum albumin (HSA), meman-

tine (M) and the complex HSA-M:

HSA Hþð ÞAþM Hþð ÞBþ nHþHþ

$ HSA�M Hþð ÞC ð3Þ

where nH+ = C – (A + B) is the num-

ber of protons linked to this memantine

binding reaction of albumin. The asso-

ciation constant of this equilibrium was

given by:

K ¼ HSA�M½ �= HSA½ � M½ � Hþ½ �nHþ ð4Þ

Equation 4 can be written as:

K ¼ K0= Hþ½ �nHþ ð5Þ

where K0 is the K value for [H+]=1 M.

Taking the logarithm of Eq. 5 gives:

logK ¼ logK0 � nHþ log Hþ½ � ð6Þ

As, �log[H+] = pH, Eq. 6 can be

rewritten as:

logK ¼ logK0 þ nHþpH ð7Þ

Derivation of Eq. 7 gives:

@ logK=@pH ¼ nHþ ð8Þ

Combining Eqs. 2 and 8 the follow-

ing is obtained:

@ log k0=@pH ¼ nHþ ð9Þ

The logarithm of the retention factor

k was plotted against pH, when the

bulk solvent pH increased from 5.0 to

7.0, for a wide variation range of tem-

perature (278–308 K) (Fig. 2). These

plots were linear for all temperatures

with correlation coefficients r higher

than 0.96, and showed that the binding

affinity increased linearly with pH. This

increase of the binding affinity came

from two aspects of effects, one from

the albumin and another from the drug.

Although the influence of the buffer pH

on the secondary structure of albumin is

small, the rigidity of the albumin mol-

ecule will be somewhat affect, and the

changes of charge on the entrance of

the binding pocket would influence on

the access of the drug to the binding site

in some extent [26, 27]. On the other

hand the ionization state of the drug

would be different with the variation of

the bulk solvent pH, and thus, affected

the binding affinity of the drug. From

Eq. 9, the slope of the curve log k ver-

sus pH gives the number of protons

(nH+) at the drug–HSA interface im-

plied in the binding process. For

example, at 298 K, the value of nH+

was 0.25. The positive values of nH+

obtained at all the pH values reflected

the uptake of protons when drug bound

to HSA, and mean that one or more

groups of ligand or/and albumin should

increase its pKa as a consequence of

binding and uptake of protons [28].

Several residues of HSA are likely can-

didates for proton acceptance, as the

residue histidine, arginine or tyrosine, as

well as the amine group of memantine.

Many studies have demonstrated that

pH-induced alterations in the binding

sites of protein molecule play an

important role in the changes of ligand

binding to protein [29–32]. Xie et al.

[31] have shown that the binding ability

of morin to HSA decreased with the

increase of buffer pH which showed that

the level of protonation played an

important role during the drug–protein

binding process. Also, Bagnost et al.

[32] demonstrated that the affinity of

nor-NOHA to the arginase enzyme was

high and increased slightly with the pH

and this binding mechanism was

accompanied by a protonation of the

histidine residue in the binding site of

the enzyme. Thus, there are several

reasons that can explain these changes

of pKa values. For instance, a variation

in the micropolarity of the environment

surrounding the side chains of certain

active side residues as a result of drug

binding is a possibility. Alternatively, a

protonated form could be stabilized by

forming hydrogen bond with a neigh-

bouring group. Furthermore, the

magnitude of the corresponding heat

protonation DHH+ in the memantine–

HSA complex was determined using the

following relation [32–34]:

DHHþ ¼ �2:3RT 2 @nHþ=@Tð Þ ð10Þ

The plot nH+ versus temperature

showeda constant values forT < 298, and

increased linearly for T � 298 with a cor-

relation coefficient r higher than 0.96

(Fig. 3). Using the slope of the second part

of the plot, the correspondingmagnitudeof

the heat protonation of the protonated

group in the memantine–HSA complex

was determined around 65 kJ mol�1

(Eq. 10). This value agrees with the heat

protonation of a guanidinium group

[35, 36], and confirmed that an arginine

group may become protonated in the

complex. This result was agreed with other

previous studies which demonstrated that

the binding of ligand to protein might be

accompanied with a protonation in one or

more residues in thebinding site [31, 32, 37].

Thermodynamic Analysis

The temperature dependence of the

memantine retention factor is given by the

well known thermodynamic relation

[38, 39]:

@ ln k0=@T ¼ DH=RT 2 ð11Þ

whereDH is the binding enthalpy andR is

the gas constant. The analysis of the

thermodynamics was carried out by

measuring the memantine retention

factor in the temperature range (293–

308 K) with various pH (5 � pH � 7) of

the bulk solvent. The van’t Hoff plots for

the memantine exhibit a significant non-

linear behaviour as shown in (Fig. 4).

Similar non-linear van’t Hoff plots

were obtained for other solute–albumin

binding processes in previous studies

Fig. 1. Memantine structure

Original Chromatographia 2008, 68, August (No. 3/4) 181

[40, 41]. These non-linear plots indicated

changes in the solute retention mecha-

nism with temperature. From these plots

and using Eq. (11) the DH values were

determined (Table 1). DH depends line-

arly on the temperature in the range (278–

308 K) with correlation coefficients r

higher than 0.99 (Fig. 5). At low temper-

atures (<293 K), the binding enthalpy

contributes non-favourably to the free

energy of binding. At about 293 K, the

enthalpy change of association was

nil and above this value became negative

indicating that the complex formation is

enthalpically governed. This means that

van der Waals interactions and hydrogen

bonding (both characterized by negative

enthalpy changes at these temperatures)

are engaged at the complex interface

confirming strong albumin–ligand

hydrogen bond [42–44]. In the tempera-

ture range 278 to 293 K, as temperature

increases, the binding enthalpy becomes

less endothermic (more favourable). As

can be seen in Fig. 5 the enthalpy change

decreases quicklywith temperature due to

a large negative heat capacity changes. At

pH = 7 the DCp value was �3.79 kJ

mol�1.K�1. this value was determined

from the slope of linear temperature

dependence of enthalpy changes (Fig. 5).

As well, the entropy change DS was

determined from the DH obtained and

using the value of DG calculated from the

relation:

DG ¼ �RT ln k0 � lnUð Þ ð12Þ

The DS value was then calculated

using the following equation:

DS ¼ �DG Tð Þ=T þ DH Tð Þ=T ð13Þ

TH and TS (reference temperatures at

which DH and DS are nil) for the binding

of memantine to HSA were around

293 K at all pH values of bulk solvent.

About this temperature the entropy

change was & nil. Below this tempera-

ture value, the binding process is

therefore accompanied by a positive en-

tropy change (Table 1), which also

strongly depends on temperature, while

DG changes slightly with temperature

(Fig. 6) because of the enthalpy–entropy

compensation [32]. This behaviour has

been found in many ligand–protein

interactions [45–48]. At low temperature,

below 293 K, the positive enthalpy

change and positive entropy change of

binding upon complex formation can be

justified by charge–charge interactions

and hydrophobic forces [49, 50]. This is

in agreement with the findings reported

by several authors who demonstrated

that hydrophobic and electrostatic

interactions play an important role in the

binding of drugs with an acidic or basic

character to HSA [42, 51]. Memantine is

both highly basic (pKa = 10.42) and

lipophilic (log P = 3.28) [52] suggesting

that ionic interactions thanks to its basic

primary amine group, and hydrophobic

interactions due to its hydrophobic rings

will take place when memantine is in-

cluded in the HSA binding cavity. In the

association of a protein to a ligand,

several contacts between non-polar

groups of memantine and HSA are

engaged. Thus, substantial fraction of

polar and non-polar surface is buried in

the complex formation which is thus

accompanied by negative heat-capacity

changes of the system. Many studies

0,1

0,2

0,3

0,4

0,5

0,6

0,7

275 280 285 290 295 300 305 310

T (K)

nH+

Fig. 3. Temperature dependence of the linked protons (per mol albumin), nH+

-0,8

-0,7

-0,6

-0,5

-0,4

-0,3

-0,2

-0,1

0,04,5 5,5 6,5 7,5

pH

logk

'

5 6 7

Fig. 2. log k versus pH for memantine at T = 298 K

-1,0

-0,9

-0,8

-0,7

-0,6

-0,5

-0,40,0032 0,0033 0,0033 0,0034 0,0035 0,0035 0,0036 0,0036 0,0037

lnk`

1/T (K-1)

0,0034

Fig. 4. Temperature dependence of the ln k values of memantine at pH = 6.5

182 Chromatographia 2008, 68, August (No. 3/4) Original

[53, 54] have suggested that DCp may be

described as a phenomenon in hydration

terms, pointing out that changes in

vibrational modes apparently contribute

slightly to DCp. Similarly, Connely and

coworkers have shown that the heat

capacity of ligand binding can be

approximated by contributions arising

from dehydration of solvents exposed

groups [55–57]. The interaction between

apolar groups of memantine and HSA

requires the dehydration of both protein

and the drug and there is an entropic

gain from the transfer of interfacial

water into the bulk solvent. Assuming

that DCp value is due principally to

the hydrophobic effect [58] and

that the decrease in heat capacity

per mol of water lost is, on average,

24 J mol�1.K�1 [59], one can calculate

that, at pH = 7, about 157 water mole-

cules are released. As well, the enthalpy

and heat capacity values provide an esti-

mation of solvent accessibility changes

during the binding. Murphy and Freire

have suggested the following equations

for DCp and DH60 (enthalpy change at 60

NC) [53].

DCp ¼ 1:88DASAap � 1:09DASAp ð14Þ

DH60 ¼ �35:3DASAap þ 131DASAp

ð15Þ

where DCp, DH60 and DASA are in

J.K�1 mol�1, J mol�1 and A2 units,

respectively [53–60]. DASAap and DASAp

represent the changes in non-polar and

polar areas exposed to solvent (accessible

surface area) that take place upon albu-

min-memantine binding. The tempera-

ture of 60 �C in the expression is themean

value of the denaturation temperature of

the model proteins used in the analysis.

At pH = 7 using DH60 = �147.62 kJ

mol�1, assuming a DCp = �3.79 kJ

mol�1K�1, the changes in accessible sur-

face areas areDASAap = � 3,168 A2 and

DASAP = � 1981 A2. Therefore, the re-

sults of Murphy’s approach indicated

that the surface area buried on complex

formation comprises 62% nonpolar sur-

face and 38% polar surface. The amount

of non-polar surface involved appeared

too large to be accounted for in ‘‘rigid

body’’ association. That could justify the

accessible surface area value calculated.

At low temperature below &293 K, DHandDS values remained positive since the

contributions of the desorption of the

solvent molecules overweight that of the

memantine adsorption on the albumin

surface. At 293 K DS & 0, it appears so

that there should be some source of neg-

ative entropy compensating the positive

entropy of dehydration. The overall en-

tropy change (DS) at 293 Kcanbe split up

in the following way [32, 54]:

DS ¼ DShydr þ DStrans þ DSspecific ð16Þ

where DShydr is the contribution by the

hydrophobic effect. DStrans accounts for

the reduction in the overall rotational

and translational degrees of freedom,

as well as the immobilization of amino

acid side chain at the complex inter-

face. DSspecific describes system-specific

contributions such as reduction of

main chain mobility and entropic con-

tributions from polar interactions.

DShydr can be estimated from

DShydr ¼ 1:35DCp: ln T=386ð Þ ð17Þ

where DCp (in J mol�1 K�1) is the mea-

sured heat capacity change,T the absolute

temperature and 386 the reference tem-

perature at which the entropy of transfer

of non-polar liquids to water vanishes

[54]. For memantine-albumin com-

plex at 293 K and pH = 7 we obtained

DShydr = + 1.41 kJ mol�1 K�1. From

T DS& 0 kJ mol�1.K�1 at 293 K, we

calculatead that DStrans +DSspecific

=� 1.41 kJ mol�1 K�1. For a great

number of bimolecular association reac-

tions, DStrans has been thought to con-

tribute �0.21 kJ mol�1 K�1 of rotational

and translational entropy [54]. Hence, the

remaining entropic loss of �1.20 kJ

mol�1 K�1mustbe contributed by the loss

in the conformational restrictions of me-

mantine and HSA. This unfavourable

conformational change entropy could

proceed from fixation of side chains at the

interface and structural changes in the

interacting molecules upon complex. Our

Table 1. Thermodynamic parameters DH (kJ mol�1) and DS (J mol�1 K�1) for the memantinebinding to HSA at pH = 7 and for six temperatures

T (K) DH kJ mol�1 DS J mol�1 K�1

278 45.2 174.9283 24.6 101.3293 �14.6 �34.6298 �33.2 �97.5303 �51.1 �157.3308 �68.5 �214.2

-80

-60

-40

-20

0

20

40

60

80

275 285 290 295 305 310

pH=5

pH=5,5

pH=6

pH=6,5

pH=7

H°

(kJ.

mo

l-1)

T (K)

300280

Fig. 5. Temperature dependence of DH for the binding of memantine on HSA at various pHvalues of bulk solvent

Original Chromatographia 2008, 68, August (No. 3/4) 183

results showed that adaptive conforma-

tional transitions are associated with the

memantine–HSA complex formation

where both components are able to ad-

just their recognition surfaces in order to

maximize complementarities through

tightly packed contacts involving Cou-

lomb interactions and hydrogen bonding

[60, 61]. Following previous studies from

Guillaume’s group concerning pH and

temperatureeffectsondansyl aminoacids–

HSA binding mechanism [40, 44, 62], our

present results can be explained by sug-

gesting that memantine binds to albumin

Site II, where these hydrophobic groups

occupy the nonpolar interior of the

cavity and its primary amine group

interacts with charged (Arg410) and

polar (Tyr411) groups on the cavity rim

forming electrostatic and hydrogen-

bond. In order to confirm that meman-

tine mainly bound on HSA Site II, a

zonal elution approach [25, 63] was

carried out. For this, the binding of a

well known ligand on HSA Site II, i.e.,

diazepam, was examined by injecting

small amounts of diazepam into the

HSA column while a known concentra-

tion of memantine was applied to the

column in the mobile phase. By exam-

ining how the mobile phase concentra-

tion of the memantine additive affects

the retention of the diazepam injected

solute, information will be gained on the

type of competition (i.e., allosteric

interaction or direct competition) which

is occurring between the two solute

molecules. Eleven memantine concen-

tration values were included in the con-

centration range 0–10 lM. The retention

factor of diazepam declined with

increasing the concentration of the

competitive agent, i.e., memantine, as

follows [25, 63–65]:

1=kdia ¼ KM M½ �= Kdia Stot½ �ð Þþ 1= Kdia Stot½ �ð Þ ð18Þ

where kdia, Kdia and KM are respec-

tively the diazepam retention factor

and the equilibrium affinity constants

with HSA for diazepam and meman-

tine, [Stot] is the effective concentration

of common binding sites, and [M] is

the memantine concentration in the

mobile phase. (Fig. 7) presents the plot

1/kdia versus the memantine concentra-

tions [M] at 293 K (Eq. 18). As can be

seen in Fig. 7, a decrease in retention

of diazepam was observed as increasing

amounts of memantine were added to

the mobile phase. It appears that the

plot 1/kdia versus the memantine con-

centration is linear (r2 > 0.998). This

plot gave a good agreement between

the experimental intercept (i.e. kdiawhen no competing agent (memantine)

was present) and the intercepts which

were determined by linear regression

of the entire data set (kexperimental

=11.42 & kpredicted & 11.51). The

agreement between these values is

significant since it indicates the dis-

placement of memantine by diazepam

was though a direct, rather than

an allosteric mechanism of competi-

tion and confirmed that memantine

interacts reversibly with a single type

of equivalent binding site, i.e., Site II

[25, 64].

Conclusion

In this paper the memantine binding

mechanism to human serum albumin

was analyzed. This binding was accom-

panied with a proton uptake which can

be attributed to an increase in the pKa of

one or more groups of the memantine

and/or HSA in the complex at the entire

range of pH studied. The protonation

heat showed that an arginine group of

albumin binding site may become pro-

tonated in the complex. The binding was

temperature dependent. In a low tem-

perature domain (<293 K) it was

entropically driven, indicating a contri-

bution from hydrophobic effect due to

0,08

0,085

0,09

0,095

0,1

0,105

0,11

0,115

0 1 2 3 4 5 6 7 8 9 10

1/kdia

[M](µM)

Fig. 7. Plot of 1/kdia vs [M] (lM) in the mobile phase (0.1 M sodium phosphate buffer,pH = 7.0)

-80

-60

-40

-20

0

20

40

60

80

275 280 285 290 295 300 305 310

-T S

H

G

T (K)

kJ.m

ol -1

Fig. 6. Temperature dependence of the thermodynamic parameters for the binding ofmemantine-albumin at pH = 7

184 Chromatographia 2008, 68, August (No. 3/4) Original

the release of water molecules when

memantine and HSA associated. Above

293 K, the thermodynamic data DH and

DS became negative due to van der

Waals interactions and hydrogen bond-

ing which are engaged at the complex

interface. By the use of known correla-

tions between the heat capacity change

and the burial of non-polar surface area,

the surface area that is burried in the

memantine–HSA complex was esti-

mated. These results associated with the

use of a zonal elution approach demon-

strated that memantine seemed to be

good candidate as ligand for the HSA

Site II (indole-benzodiazepine site).

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