Affinity chromatography study of magnesium and

Talanta 53 (2000) 561 – 569 www.elsevier.com/locate/talanta

Affinity chromatography study of magnesium and calcium binding to human serum albumin: pH and temperature variations Yves Claude Guillaume a,*, Christiane Guinchard a, A. Berthelot b a

Laboratoire de Chimie Analytique, Faculte´ de Me´decine et de Pharmacie, Uni6ersite´ de Franche-Comte´, Place Saint-Jacques, F-25030 Basançon, Cedex, France b Laboratoire de Nutrition Pre´6enti6e Expe´rimentale, Pharmacologie, Physiologie, Faculte´ de Me´decine et pharmacie, Place Saint Jacques, 25030 Besançon, Cedex, France Received 28 March 2000; received in revised form 19 July 2000; accepted 25 July 2000

Abstract The magnesium and calcium binding on human serum albumin (HSA) was studied using an affinity chromatography approach. The effects of the mobile phase pH, its ionic strength and column temperature on the transfer equilibrium constants were studied. The thermodynamic data corresponding to the electrostatic interactions occurring during the HSA-ion binding were determined. Enthalpy – entropy compensation revealed that the ion binding mechanism at HSA was independent of the ionic strength, the same at four pH values (6.5, 8, 8.5 and 9), but presented a weak change at physiological pH around 7 – 7.5 due to a HSA phase transition. A theoretical model based on the Gouy–Chapman theory allows to determine the relative charge density of the HSA surface implied in the binding process and the variation of the number of ions bound to one albumin molecule with the pH. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Calcium; Magnesium; Affinity-chromatography; Human serum albumin

1. Introduction Human serum albumin (HSA) is the most abundant protein in blood plasma and possesses a capability of reversible binding of a great number

* Corresponding author. Tel.: +33-3-81665546; fax: + 333-81665655. E-mail address: [email protected] (Y.C. Guillaume).

of substances including bilirubin, hormones, drugs and ions [1]. HSA is a globular protein (molecular mass$ 66 000) consisting of a single chain of 585 amino acid residues, which is formed into subdomains by paired 17 disulfides bonds. Equilibrium dialysis is specially suited to the study of drug–protein interactions [2]. Several high performance liquid chromatographic (HPLC) separation methods have been also introduced. Hummel and Dryer [3] uses a single component added to the mobile phase for the HPLC

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determination of the equilibrium constant of the drug–protein association. This method was used by Soltes and Sebille [4] to study the reversible binding interactions between the tryptophan enantiomers and albumins of different animal species. Only the protein high affinity sites are generally involved as showed by Sebille and his colleagues in a pioneering work [5]. Affinity chromatography with protein immobilized on the chromatographic support is equally used to study the mechanism of this association. The thermodynamic processes involved in the binding and separation of warfarin and thyroxine enantiomers have been characterized by Hage et al. [6]. Peyrin et al. [7] characterized the dansyl amino acids binding at HSA site II and its geometry using a biochromatographic approach. Both chromatographic study and differential scanning calorimetry (DSC) were used to elucidate the effect of the mobile phase pH and temperature on the HSA – dansyl amino acid binding [8]. It was demonstrated that the HSA protein structure balanced between a disordered and an ordered solid like state. Variations of column temperature and mobile phase pH tend to cause this phase transition between these two states explaining the thermodynamic constant changes of the dansyl amino acids – protein association with pH and temperature [9]. The interaction mode between protein and transition metal ions (Cu(II) or Ni(II)) immobilized on a silica support involves ion pair coordination binding. Using this method, Finette et al. [9] have analyzed the effect of buffer ionic strength and temperature on the HSA – immobilized ligand interactions. A novel chromatographic study based on a largezone Hummel and dryer method was applied to the examination of copper – protein (bovine serum albumin) complexes [10]. Toffaletti et al. examined the distribution of calcium among serum proteins by the use of gel filtration [11]. In this paper, the influences of the mobile phase pH, its ionic strength and column temperature on the electrostatic interactions controlling the HSA– magnesium and HSA – calcium bindings were investigated using affinity chromatography.

2. Theoretical considerations A general phenomenon found in early studies on HSA is its ability to bind divalent inorganic cations. In contrast, with the great majority of cationic ligands such as Cu2 + , Zn2 + , Ni2 + , or Cd2 + , which are bound specifically to albumin with the binding mode involving the formation of multiple chelate ring [12] Ca2 + , and Mg2 + interact preferentially with several sites, which differ only slightly in their affinity towards the ligand [13]. This ionic binding on the protein is influenced strongly by changes in the medium pH. This dependence on pH is the signature of electrostatically driven processes. For example, there is no calcium ion binding on albumin molecule between pH 4.5 and 5.0 whereas approximately two calcium ions are bound per albumin molecule at pH 8.8 [2]. On the basis of this major ‘non-specific’ binding mode of magnesium and calcium to HSA [13], it is proposed that the binding of the cations is dominant by the electrostatic interactions (ei) between its charge and the oppositely charged surface of HSA. The protein is treated as a multivalent spherical particle with its charges uniformly distributed at the surface. The stoichiometric relations, used to study the system behavior when interactions between the ligand and HSA are short ranged, i.e. for Cu2 + or Ni2 + , are inadequate to describe the electrostatic interactions. Thus, the electrostatic Gibbs free energy change of transfer of the cations from the bulk solvent to the HSA surface was noted DG oei.

3. Gibbs free energy changes of electrostatic interactions between cations and HSA DG oei can be related to the HSA surface potential 8, the charge of the anion z and the Faraday constant F by the following equation: DG oei = zF8

(1)

The Gouy Chapman theory has been previously used in a chromatographic system to establish a generally charged solute–HSA binding model and to investigate the relative contribution of different interactions implied in the solute transfer [7,14].


This theory relates the surface potential to the surface charge density s, which has units of charge per area and ionic strength I [15]: s= 8RToo0I sin h

DG oei 2RT

(2)

This relation accounts for a mobile phase of dielectric constant o and ionic strength I (o0 is the permittivity of free space). As sin h x $x under typical chromatographic conditions [16], inversion of Eq. (2) gives: DG = o ei

2RTs

(3)

8RTo0oI It is known that the retention factor at temperature T, for the cation X denoted k%X,T is related to the change in free energy DG oei incurred during the transfer between the mobile and HSA stationary phases. This relationship is expressed by [17]: ln k%X,T = −

o ei T

(DG ) + ln x RT

The free energyDG oei can be broken down into enthalpic and entropic terms to give the equation DG oei = DH oei−TDS oei

(7)

where DH oei and DS oei correspond to enthalpic and entropic changes due to the electrostatic interactions. Combining Eqs. (6) and (7) gives: DH oei +DS oei* RT

(8)

where

(5)

2s

4. Enthalpy and entropy changes of electrostatic interactions between cations and HSA

(4)

where x represents the phase ratio (volume of the stationary phase divided by the volume of the mobile phase). Combining Eqs. (3) and (4) gives: +ln x (6)

8RTo0o I This equation links the variation of ln0 k%X,T i.e. the variation of KX,T with I. A charge density estimation of the HSA stationary phase surface implied in the cation binding process can be obtained from the slope of this ln k%X,T versus 1/ I plot. In this approach, the s value should be constant regardless of the kind of the test ions. However, the surface charge density value does not take into account all the charges on HSA but only the negative charge with which each cation can interact on the basis of their respective affinity for the protein. In other words, our model takes into account only the dominant electrostatic effect by neglecting the weak shortrange specific forces such as those due to steric hindrance or chelate interaction. So the magni-

ln k%X,T = −

tude of the binding affinity can be quantified by the number of cation accessible sites per HSA molecule. This type of treatment is, of course, limited to the case of divalent ions interacting preferentially roughly identical low-affinity sites such as magnesium or calcium.

ln k%X,T = −

The equilibrium constant KX,T of the cation transfer from bulk solvent to HSA surface is k% = xK

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DS oei*=

DS oei + ln x R

(9)

If the cation binds to the HSA surface with a constant enthalpy of association, then a plot of ln k%X,T versus 1/T should be linear with a slope of − DH oei/R and an intercept of DS oei*. DS oei was not provided due to the ambiguity in the calculation of the phase ratio for the HSA column. Thus only the DS oei* values, which has no unit and the same variation as DS oei will be given.

5. Experimental methods

5.1. Apparatus The HPLC system consisted of a Merck Hitachi pump L7100 (Nogent-sur Marne, France), an Interchim Rheodyne injection value model 7125 (Montluçon, France) fitted with a 20-ml sample loop and a Waters conductimetric detector (Saint Quentin en Yvelines, France). The chiral column (150× 4.6 mm), which consists of HSA bound to a 7-mm silica matrix, was supplied by Shandon HPLC (Cergy-Pontoise, France) and used at a

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controlled temperature (in an Interchim Crococil oven TM No. 701 (Montluçon, France) for high temperature and an Osi Julabo FT200 cryoimmerser (Elancourt, France) for low temperature. After each utilization, the column was stored at 4°C until further use. To study the effect of the flow-rate on the retention factor, the retention times of the different ions and dead time marker were measured at 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 ml min − 1. The maximum relative difference of the retention factor of these compounds was never greater than 1.5%, meaning that the k% values (corresponding to the equilibrium constants) were independent of the flow-rate in this range. Thus, the flow-rate was maintained constant equal to 1 ml min − 1 throughout the study.

5.2. Sol6ents and samples Sodium hydrogen phosphate and sodium dihydrogen phosphate were supplied by prolabo (Paris, France). Water was obtained from an Elgastat option water purification system (Odil, Talant, France) fitted with a reverse osmosis cartridge. MgCl2, CaCl2, NaCl and KCl were obtained from Sigma Aldrich (Saint Quentin, France) and were made fresh daily at a concentration of 5.25× 10 − 3 M for MgCl2 and CaCl2 and 1.05× 10 − 2 M for NaCl and KCl in water. The mobile phase consisted of a sodium phosphate buffer with salt concentrations varying from 8.25×10 − 4 to 25.25 ×10 − 4 M. The buffer pH were adjusted to values equal to 6.5, 7, 7.5, 8, 8.5 and 9. The buffer, at all pH values, were stocked for 1, 2 and 4 h at ambient room temperature to study the accuracy of their pH values. No fluctuations were observed. The maximum relative difference observed of the pH value of the different mobile phases was always 0.5%. MgCl2, CaCl2, NaCl and KCl samples (20 ml) were injected at different buffer concentrations and pH. It has been known for many years that monovalent cations such as sodium or potassium are not able to bind to HSA [2,18]. Therefore, NaCl and KCl samples were injected in the HSA column to examine the eventual interference of the chloride ion in the detection of the peak of magnesium and calcium. Contrary to the MgCl2 and CaCl2 sam-

ple injection, no detectable peak different from the blank peak corresponding to the injection of a phosphate buffer sample was detected at various mobile phases. This result demonstrated the specificity of the assay using MgCl2 and CaCl2 salts as suppliers of magnesium and calcium, respectively.

5.3. Temperature studies Retention factors of magnesium and calcium were determined over the temperature range 10– 30°C. The chromatographic system was allowed to equilibrate at each temperature for at least 1 h prior to each experiment. To study this equilibration, the retention time of magnesium and calcium was measured every hour for 5 h and again after 23 and 24 h. The maximum relative difference of the retention time of these compounds was always 0.5%, making the chromatographic system sufficiently equilibrated for use after 1 h. The ions were injected three times at each temperature and phosphate buffer concentration. Once the measurements were completed at the maximum temperature, the column was immediately cooled to ambient conditions to minimize the possibility of any unfolding of the immobilized HSA.

6. Results and discussion

6.1. Van’t Hoff plots 6.1.1. For pH 6.5, 8, 8.5 and 9 The Van’t Hoff plots were all linear for the two ions and at all phosphate buffer concentrations. These linear behaviors were thermodynamically what was expected where there was no change in the interaction mechanism in relation to temperature. The correlation coefficients for the linear fits were over 0.989. The typical S.D. of the slope and intercept were, respectively, 0.004 and 0.02. Fig. 1 represents the Van’t Hoff curve for magnesium and calcium at pH 6.5 and I= 0.005. Table 1 contains a complete list of DH oei and DS oei* for the two ions at all ionic strengths I.


6.1.2. For pH 7 and 7.5 The Van’t Hoff plots for the two ions showed a net break at a low temperature T* between 1 and 7°C, which is indicative of a modification of the cation–HSA binding mechanism. The correlation coefficients of the curves in these two regions (region I corresponding to T \T* and region II corresponding to T BT*) were in excess of 0.988. Table 1 contains the values of DH oei and DS oei* for the two ions in the two regions and at all ionic strengths I. 6.2. Enthalpy– entropy compensation The compensation enthalpy – entropy can be expressed by the formula [17]: DG ob = DH o −bDS o

(10)

where DG ob is the Gibbs free energy of a physico chemical interaction at a compensation tempera-

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ture b. Combining Eqs. (8)–(10) the following equations are obtained: ln k%T = ln k%b −

DH oei 1 1 − R T b

(11)

and ln k%b = −

DG ob +ln x Rb

(12)

A plot of ln k%T (for T= 298 K) versus −DH oei was tested when the pH had values of 6.5, 8, 8.5 and 9 for the two ions for each ionic strength I. All the correlation coefficients for the linear fits were at least equal to 0.978. For example, for Mg2 + and Ca2 + at a ionic strength equal to 0.0050, r was, respectively, 0.981 and 0.987. This degree of correlation confirmed that the HSA– cation binding mechanism for the test ions was independent of the pH value chosen among these four previous values but changed for pH 7 and 7.5. Enthalpy–entropy compensation was also used to test the variation of the HSA–cation binding mechanism with the ionic strength. A plot of ln k%T (for T= 298 K) versus − DH oei was tested when the ionic strength had values 0.0033, 0.0050, 0.0067, 0.0084, 0.0101 for the two ions for each studied pH values. The correlation coefficients for the linear fits were in excess of 0.982. For example, for Mg2 + and Ca2 + at pH 9, r was, respectively, 0.991 and 0.989. This degree of correlation confirmed that the HSA–cation binding mechanism was independent of the ionic strength value chosen between these five previous values.

6.3. Ionic strength effect on equilibrium constants

Fig. 1. Van’t Hoff plot, ln k% vs. 1/T, for (A) magnesium, (B) calcium at pH 6.5 and I, 0.005.

From the retention factor, the plot of ln k% in relation to the reciprocal square root of the ionic strength were determined at each temperature for the two species. The plots were all linear. The correlation coefficients for the fits were over 0.978. According to Eq. (6), the HSA surface charge density can be calculated at different pH values from the slopes of the ln k% versus 1/ I plots. For example, the corresponding s/F values at pH 6.5 and at different temperatures were given in Table 2. The relative difference in these values obtained for all T values was inferior to


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Table 1 Standard enthalpy DH oei (kJ mol−1) and entropy DS oei* (no unit) at different pH and ionic strength I values with S.D. (in parentheses) for, (A)+(B), the magnesium binding with HSA; (C)+(D), the calcium binding with HSA I

pH 6.5

(A) DH oei/Mg 0.0033 15.1 (0.02) 0.0050

13.1 (0.04)

0.0067

11.8 (0.02)

0.0084

9.7 (0.03)

0.0101

7.9 (0.03)

(B) DS oei*/Mg 0.0033 8.0 (0.01) 0.0050

6.6 (0.02)

0.0067

5.5 (0.03)

0.0084

4.6 (0.01)

0.0101

3.0 (0.02)

(C) DH oei/Ca 0.0033 17.3 (0.01) 0.0050

15.0 (0.02)

0.0067

12.9 (0.03)

0.0084

11.9 (0.01)

0.0101

9.2 (0.02)

(D) DS oei*/Ca 0.0033 10.5 (0.01) 0.0050

9.0 (0.02)

0.0067

7.2 (0.03)

0.0084

6.1 (0.01)

0.0101

5.2 (0.02)

7

7.5

8

8.5

9

I 16.4 (0.01) II 15.7 (0.03) I 14.2 (0.04) II 13.6 (0.06) I 12.9 (0.04) II 12.3 (0.03) I 10.9 (0.04) II 10.3 (0.05) I 9.2 (0.03) II 8.5 (0.05)


19.8 (0.03)

22.1 (0.05)

24.5 (0.05)

17.7 (0.04)

19.8 (0.06)

21.6 (0.04)

16.3 (0.05)

18.3 (0.04)

20.4 (0.03)

14.0 (0.04)

15.8 (0.03)

17.5 (0.06)

12.8 (0.03)

14.7 (0.06)

16.5 (0.04)



11.3 (0.02)

12.8 (0.03)

14.4 (0.03)

9.5 (0.01)

11.0 (0.04)

12.7 (0.02)

8.8 (0.03)

9.7 (0.02)

10.6 (0.02)

7.5 (0.02)

9.00 (0.01)

9.9 (0.01)

5.6 (0.01)

6.5 (0.02)

7.4 (0.03)



21.9 (0.06)

23.8 (0.05)

26.2 (0.06)

19.7 (0.07)

22.0 (0.05)

24.1 (0.07)

17.0 (0.04)

19.2 (0.04)

21.6 (0.04)

16.3 (0.05)

18.1 (0.04)

20.1 (0.08)

14.3 (0.06)

16.6 (0.06)

18.5 (0.07)



14.4 (0.03)

16.7 (0.04)

19.1 (0.04)

13.1 (0.04)

15.1 (0.03)

17.1 (0.05)

11.4 (0.05)

13.4 (0.03)

15.1 (0.06)

10.0 (0.04)

11.8 (0.02)

13.5 (0.07)

8.4 (0.03)

10.2 (0.04)

12.1 (0.04)


10%, thus indicating that the surface charge densities implied in the binding process were independent of the temperature. By determining the surface area of the HSA molecule from its molecular data, it was possible to calculate the number of calcium and magnesium ions bound to one Table 2 Relative charge densities of the HSA stationary phase surface s/F (mmol m−2) related to magnesium (Mg) and calcium (Ca) binding and calculated from Eq. (6) at pH 6.5 and at different temperatures T (°C)

15 20 25 30 35

s/F (×10−3) Mg binding

Ca binding

3.5 3.5 3.6 3.5 3.7

7.4 7.3 7.6 7.4 7.4

Fig. 2. The number of calcium ions, n, per albumin molecule as a function of pH.

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albumin molecule. At pH 8, for example, this number was equaled to 1.3 and 0.6 for calcium and magnesium, respectively. This value was the weakest for magnesium due to the fact that the number of binding sites per protein, which interacted with the calcium ion was higher than with magnesium [2,19]. For example, the calcium binding to albumin is shown in Fig. 2. This approached the curve found in an early study by Fogh-Andersen et al. [2]. The divergence can be explained by the fact that the charge distribution over the protein surface is inhomogeneous, resulting in domains of higher charge density and thus, in stronger interactions than assumed in the model.

6.4. Thermodynamic constant 6ariations with pH and temperature At all pH values, DH oei and DS oei* values of the cation–HSA binding process were always positive. These results indicate that the binding process is entropically driven for all eluent compositions. The interactions between ionic species in aqueous solution are characterized by small positive enthalpy changes and positive entropy changes [20,21]. Accordingly, the present thermodynamic behavior corresponded to the model describing the electrostatic attraction that occurs between the negatively charged non-specific regions of HSA and the positively charged test ions. When the ionic strength increased, both DH oei and DS oei decreased becoming less positive (Fig. 3). In this ionic strength range, the ionic double layer length of charged species was thick with a high Debye length. The phosphate buffer concentration increase was responsible for a Debye length reduction by affecting the electrostatic shielding, which governed an ionic attraction decrease and, thus, DH oei and DS oei values became progressively less positive corresponding to a weaker binding process. For pH 7 and 7.5, it had been demonstrated by DSC that the HSA structure was in equilibrium between an ordered and disordered state [8]. At temperature T\ T* (region I), the protein surface was in high sinuosity (ordered solid like state) but at temperature TB T* (region II), the protein surface was in low sinuosity (disordered solid like state) [8]. The affinity decrease in the test ions for the protein surface in region II can

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tions implied in this process. The experimental values of transfer equilibrium constants obtained by varying the pH of the bulk solvent and its ionic strength provided access to the HSA surface charge density implied in the binding process and the number of ions bound to one albumin molecule. It can also be noted that the thermodynamic data corresponding to this molecular association process supported the fact that the binding was controlled by an entropically driven mechanism corresponding to its electrostatic attraction for different ‘non specific’ areas of serum albumin. Enthalpy–entropy compensation revealed that the HSA-ion binding mechanism is independent of the ionic strength, identical at pH 6.5, 8, 8.5 and 9, but changed at pH 7 and 7.5 due to a phase transition of the albumin molecule.

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Fig. 3. Plot of (A) DH oei (kJ mol − 1) and (B) DS oei* (no unit) vs. the ionic strength I at pH 8.

be explained by a decrease in the surface heterogeneity. This decrease in the test cation binding was accompanied by a reduction in enthalpy and entropy changes for the transfer of magnesium and calcium from the mobile to the stationary phase. Thus the DH oei and DS oei values were less positive in region II than in region I (Table 1) corresponding to a weaker binding process. This behavior, i.e. a lower affinity of ligand for a smooth surface than for an irregular surface, has been suggested by some authors over the past few years [22,23].

7. Conclusion A HSA-ion (Ca and Mg) binding model was established to investigate the electrostatic interac-

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