Int. J. Peptide Protein Res. 40, 1992, 415-422
Fatty acid and drug binding to a low-affinity component of human serum albumin, purified by affinity chromatography HENRIK VORUM, ANDERS 0. PEDERSEN and BENT HONORE
Institute of Medical Biochemistry, University of Aurhus, Aarhus, Denmark
Received 19 November 1991, accepted for publication 3 May 1992
Binding equilibria for decanoate to a defatted, commercially available human serum albumin preparation were investigated by dialysis exchange rate determinations. The binding isotherm could not be fitted by the general binding equation. It was necessary to assume that the preparation was a mixture of two albumin components about 40% of the albumin having high affinity and about 60% having low affinity. By affinity chromatography we succeeded in purifying the low-affinity component from the mixture. The high-affinity component, however, could not be isolated. We further analyzed the fatty acid and drug binding abilities of the low-affinity component. The fatty acids decanoate, laurate, myristate and palmitate were bound with higher affinity to the mixture than to the low-affinity component. Diazepam was bound with nearly the same affinity to the low-affinity component as to the albumin mixture, whereas warfarin was not bound at all to the low-affinity component. Key words: affinity chromatography; binding analysis; binding isotherm; heterogeneity; human serum albumin; low-affinity albumin
Albumin is synthesized in the liver and circulates in the blood plasma. One of the main functions of albumin is to bind and carry endogenous as well as exogenous ligands. Over decades, several binding studies have been performed to investigate this function. Most binding studies have been performed by using models suggesting that albumin is a homogeneous protein with respect to binding in line with the detection of only one copy of the albumin gene (1). However, several examples of physicochemical heterogeneity of albumin have been described in the literature (for reviews see refs. 2-4). In recent years, thanks to the development of a more precise dialysis exchange rate method (5) some binding studies have revealed that albumin also shows binding heterogeneity for some ligands. We have thus shown that a defatted commercial human serum albumin preparation is heterogeneous with respect to binding of the medium-chain fatty acids, hexanoate, octanote, and decanoate (6), as well as the branched octanoic acid valproate (7), used as a drug in the treatment of epilepsy. The binding of these ligands could only be described by assuming the presence of two albumin components, about two thirds having high affinity and one third having low affinity. The high-affinity albumin component was able to bind one mol decanoate, 1 mol
octanoate, 2 mol hexanoate or 1 mol valproate more than was bound to the low-aflinity component. The chemical basis for this observed binding heterogeneity has not been elucidated. One way to go for further progress in studying the binding heterogeneity of albumin is to separate highand low-affinity components of albumin on a preparative scale. In the present paper we describe an affinity chromatographic procedure used to isolate the lowaffinity component from a commercial albumin preparation. To demonstrate the carrier heterogeneity, binding studies to the low-affinity component and to the original albumin mixture were carried out with the four fatty acids decanoate, laurate, myristate, and palmitate, and with diazepam and warfarin, each demonstrating different drug binding functions of the albumin (8,9). EXPERIMENTAL PROCEDURES
Human serum albumin Human serum albumin was obtained from AB Kabi Vitrum (Stockholm, Sweden; lot no. 88181). It was defatted with charcoal in solution acidified with 0.1 M H2S04, pH 3, at 0" (lo), dialyzed against pure water, and lyophilized. The fatty acid content of the protein 415
H. Vorurn et al. after this procedure, has previously been measured (6) by an enzymatic method (NEFAC, Wako Chemicals GmbH, Neuss, Germany) and was less than 0.1 mol/ mol albumin.
Fatty acids Decanoate, laurate, myristate, and palmitate were obtained from Fluka AG, Buchs, Switzerland. The purity of all four fatty acids was determined as > 99?0 by gas liquid chromatography. [ 'TIDecanoate was obtained from CEA, Cen Saclay - Molecules Marquees, Gifsur-Yvette, France. [ 14C]Laurate, and [ I4C]myristate were received from Amersham International, England. [3H]Palmitatewas purchased from New England Nuclear Research Products, NEN Chemicals GmbH, Dreiech, Germany. According to the manufacturers the radiochemical purity for all four fatty acids was 98"; (by thin-layer chromatography). In all four preparations the content of unbound, dialyzable radioactive impurities (11) accounted for less than 0.15", of the total activity.
acetate-acetic acid buffer, pH 4.0, 1 L 0.02 M sodium bicarbonate-5% COz buffer, pH 8.0, and finally with large amounts of distilled water. For a separate control experiment the sepharose was processed as described above, except that decanoic acid was omitted from the dioxane/water solution.
Packing, ioading and eluting procedures. The decanoatesepharose column was packed and allowed to equilibrate for 24 h with 66 mM sodium phosphate buffer, pH 7.4 (the starting buffer). One hundred mg of a defatted human serum albumin preparation from Kabi Vitrum@ was dissolved in 7 mL of the sodium phosphate buffer and applied to the column. Elution with the starting buffer yielded an albumin component amounting to 60°, of that applied. The elution volume for this component was similar to the total volume of the column indicating that it possessed no or very low affinity for the decanoate-sepharose resin. Attempts to elute the high-affinity component with different solvents failed. The column was also packed with sepharose without coupled decanoate and allowed to equilibrate for 24 h with 66 mM sodium phosphate buffer, pH 7.4. Ninety Diazepam and warfarin Diazepam and [ I4C]diazepam were obtained as a gift one mg of albumin was dissolved in 7 mL of the sodium from Hoffmann-La Roche Ltd., Base1 Switzerland. Ra- phosphate buffer and applied to the column. The albucemic warfarin sodium salt was a gift from Nyco MED, min recovered amounted to 98.4% of that applied h d o v r e , Denmark. [ 14C]Warfarin was obtained from showing that albumin was not bound covalently to some Amersham International plc., Buckinghamshire, En- reactive groups of the modified sepharose. gland. Before use the radioactive preparations of diazepam and warfarin were purified by chromatography on Dialisis eschange rate determinations precoated TLC silica gel plates. Diazepam was eluted All binding experiments were performed in a sodium with hexane/dioxane/acetic acid (85jl5,i 1) (by vol.) and phosphate buffer, final concentration 66 mM, pH 7.4, at warfarin with toluene/ethylacetate/ethanol (90,'5/5) (by 37 '. A volume sufficient for one or a few days work of vol.). The radiochemical purity was checked as previ- the radioactively labeled ligand solution, was evapoously described (1 1). In both preparations the content rated in a stream of nitrogen and the ligand was then ofunbound, dialyzable radioactive impurities accounted dissolved in ethanol and added under stirring to the for less than 0.2% of the total activity. buffered albumin solution to give an ethanol concentration for all ligands less than 1% (by vol.). Separation of serum albumin bj! afinitj. chromatograph!. In one experiment the binding of decanoate to the total albumin was investigated by the dialysis exchange Coupling procedure. Fifty mL of EAH Sepharose 4B rate method using exactly the same rotating dialysis from Pharmacia was washed with 3 L 0.5 M NaCl and chambers which have previously been used for studies 1 L distilled water on a sintered glass filter (G3). De- of decanoate binding (6). This experiment was carried canoic acid 200 mg was dissolved with 100 mL dioxanei out and used as a standard of reference for comparing distilled water ( l / l ) (v/v) and the pH adjusted to 4.5. the decanoate binding abilities of different batch numThis solution was added to the gel suspension. The bers of albumin preparations. In the experiments exmixture was gently stirred. EDC (1-Ethyl-3[3- amining both the low-affinity component and the total dimethylaminopropyl]carbodiimide), 2.5 g, was dis- albumin together, static dialysis chambers, containing solved in 10 mL distilled water and added drop by drop only 25 p L sample volumes ( 5 ) , were used because only to the suspension. The reaction was allowed to proceed small amounts (63.3 mg) of the low-affinity component for 24 h at room temperature. Carbodiimide was used were available. These latter experiments are thus less to promote the condensation between free amino groups precise and we abstain from a detailed binding analyat the end of the 6-carbon spacer arms of AH-Sepharose sis of these results. However, they can be used to comand the free carboxyl group in the decanoic acid mol- pare the binding properties of the mixed albumin relaecule to form an amide link by acid-catalyzed removal tively to the chromatographically isolated component. of water, as illustrated in Fig. 1. The gel was then thor- Cellophane membranes, cut from dialysis tubing (Union oughly washed with 1 L dioxane/distilled water ( l / 1) Carbide Corp) were applied for studies with diazepam (viv) followed by washes with 1 L 0.2 M sodium and warfarin. For the binding experiments with de416
Binding properties of albumin
~
C
O
,
H+
c%-
CH,-
N = C=N-
decanoic acid
carbodiirnide
/
-c-o-c II
- NH
-
Sepharose
CH3
a,CH,-
CH,-
N (cH,),
r” H,N
II
NH- CH,N-
0
C-
CH,-CH,-CH,-N(CH,),
+
0
0
% Sepharose
=
C
/
NH-
‘
CH,-
NH - CH,-
CH3 CH~-CH,-
N(cH,),
isourea FIGURE 1 Carbodiirnide coupling reaction.
canoate, laurate, myristate, and palmitate we used cellulose membranes (Dianorm type 10.16) from Diachema AG, Switzerland. The dialysis exchange rate method, which has previously been described (6, 12) allows determination of the equilibrium concentration of unbound ligand by measuring the rate of exchange of radiolabeled ligand between compartments with solutions of identical molar composition. Eqns. 1 and 2 were used for calculating the free (c) and the molar ratio of bound ligand to albumin (r):
y=-
c-c
P
where Q I and Qr denote the radioactivity in the left and right solution after dialyzing for t min, C is the total of ligand (bound and unbound), P is the total concentration of albumin, and ko is a rate constant.
Dialysis of the ligands follows strictly first-order kinetics with reproducible rate constants, 0.099 and 0.089 min- for diazepam and warfarin respectively, and 0.114, 0.102, and 0.064 min- for decanoate, laurate, and myristate respectively. The solubility of palmitate in neutral buffers is too low for dialysis-rate determinations in the absence of albumin. Attempts to perform such determinations give irreproducible results (13). However, the availability of palmitate can be found experimentally and expressed as C/p (14). The concentration of bound ligand in this case is the same as the total ligand concentration, C, since free ligand is absent. The concentration of reserve albumin for binding of palmitate is expressed as p , and has previously been defined as the concentration of a standard albumin which in buffered solution binds the ligand as tightly as it is bound in the sample (5). A relative binding isotherm for palmitate can be obtained by picturing C/P versus Clp. For an exact deduction of the thermodynamic content of the availability entity, see (14), eqns. 8-11. 417
H. Vorum et al. RESULTS
Decanoate binding properties of albumin - batch nr. 88181 Fig. 2 shows the binding isotherm for decanoate to the mixed albumin (batch 88181) used in this study. This isotherm is obtained by using the same rotating dialysis chambers and dialysis exchange rate method as previously described (6). A best-fit approximation of the binding constants to the experimental data were obtained by a computer-mediated procedure (15). The criterion for significant acceptability of a best-fit obtained from one binding model compared with a best-fit from another was evaluated by an F-test (16). An attempt was made to analyze the binding isotherm in terms of the stoichiometric binding equation (17, 18).
+ 2c2K,K, + 3c3K,K,K, + . . . + Nc‘ KIK,K,. . . K ,
cK,
r = 1 + c K , + c 2 K , K 2 +c3K,K,K3 + . . . + c“ K,K,K,. . . K ,
perimental data did not result in acceptable fits for decanoate as seen by the dashed lines in Fig. 2. An alternative stoichiometric model with one binding albumin component and another not binding at all, cK,
r = . f 1 i- c K , +
cK,
r=f
Decanoate
-8
* *
+ 2c2K,K2 + 3c3K,K2K3
+ NcNK,K2K3.. . K ,
1
+ c K , + c2K,K2 + c 3 K l K 2 K , + c”K,K2K, K , cK2 + 2c2K,K3 i- 3c3K2K,K4
(’ - f )
+ . . . + ( N - I ) c ( ~ - ’ 2) K3 K4K. . . K
1 + cK,
-7
4
-5
-6
log c
~
...
+ c2K2K3+ c3K2K,K4
+
(5)
c(h’- 1)K2K 3K 4 . . . K N
Fitting of eqn. 5 to the observed points was successful for decanoate as seen in Fig. 2, where the full curves represent the best least-squares fit. Four stoichiometric constants were used: K I = 7.09.106 M - I, K? = 3.35.10’ M - ’ , K3 = 2.40. lo3 M - I and K4= 5.51.105M - I . The best estimate off was 0.40. A model with two albumin components, where one is binding decanoate with one high-affinity step more than the other component, thus gives a good fit to the experimental data. The f value of 0.40 shows that 40% of the albumin possesses high affinity for binding of decanoate whereas the remaining 60% possesses low -4 affinity.
FIGURE 2 Calculated isotherm for binding of decanoate to the mixed albumin shown in a semilogarithmic plot of the concentration of bound. r. versus free decanoate, c. Inset shows the isotherm in a Scatchard plot, ric versur r . Full lines represent best-fit, obtained nith equation (5) with the values: K I = 7.09. lo6 M l , K - = 3.35.10’ M l . K 3 = 2 . 4 0 . 1 O 3 ~ - ’ and K 4 = 5 . 5 1 . 1 0 5 M - 1 , and /=0.40. Dashed lines show the best-fit with the stoichiometric binding equation (3). The tangent to the isotherm at intercept tvith r,c-axis (dotted line in the Scatchard plot) intersects the r-axis belon 1 indicating that albumin is heterogeneous. 30 p~ albumin in 66 m M sodiuni phosphate buffer, pH 7.4, temperature 37’.
418
. . . + cNK,K2K3’ KN
+
3
(4)
* * *
+
2
+ c2K,K2 + c3K,K,K3
also resulted in unacceptable fits, in good agreement with the binding results shown later, demonstrating that decanoate, to some extent, actually binds to the lowaffinity component. A modified binding equation was then tried. It was assumed that a fraction, f , of the albumin molecules binds decanoate according to the stepwise binding constants, K l , K,, K3; . . ., K N . The first step, with highest affinity. is missing on binding to the remaining fraction, f - 1, which has the binding constants K z , K3; . . ., ( 3 ) K.,. We thus obtain the following binding equation
where r is the average number of bound ligand molecules per albumin molecule, c is the free ligand concentration, Ki is the stoichiometric binding constant for the i’th molecule, and N is the maximum number of ligand molecules bound. Fitting of K-values in eqn. 3 to ex-
-9
+ 2c2K,K, i- 3c3K,K,K3
+ . . . + NcNKlK2K3.*.KN
~
Chromatographic separation of albumin components. One hundred mg of the defatted human serum albumin preparation was applied to the decanoate-sepharose column as previously described. In the first run we obtained 63 “2 of the amount of albumin applied to the column with the total volume, corresponding to the albumin component with low affinity for binding of decanoate. This direct method thus estimates that 37 % of the albumin binds to the decanoate-sepharose resin. This is in very good agreement with the results obtained from the binding analysis described above where it was found that approximately 40% ofthe albumin possesses
Binding properties of albumin high affinity for binding of decanoate whereas the remaining 60% possesses low affinity. The chromatography experiment was then repeated in a second run. This time 39% was attached to the column and 61% was eluted with the total volume. This result was similar to that obtained in the first run, indicating that the column was not saturated in the first run and still was able to bind albumin with high affinity for decanoate. A third and fourth run were then tried but here the binding fractions were reduced to 29% and to 25% respectively, probably as a result of saturation of the column. In these two latter experiments it was assumed
that the low-affinity component was contaminated with the high-affinity component. In consequence of this we only used the low-affinity component obtained from the first chromatographic experiment in the following binding studies. A control experiment was performed with a column packed with sepharose without coupled decanoate to insure that the high-affinity component was not covalently bound to the chemically modified sepharose. The experiment confirmed that the high-affinity component was non-covalently bound to the column. Several attempts were carried out to remove this
Decanoate
Laurate 0
0
-7
-8
-5
-6
-7
-4
-5
-6
-4
log c
Myristate
Palmitate 6
8
r
D
r
'B
-
4
m
OD
8
40
20 I3O
-7
0
2-
0
0 0.
0
0
0 (D
0
(D
0
0
log c
0
I
I
I
I
-6
-5
-4
-3
log c
I 01 -1
0
0 I
I
1
0
1
2
log Clp
FIGURE 3 Binding isotherms for decanoate, Iaurate, myristate, and palmitate to the low-affinity component ( 0 )and to the mixed serum albumin (0). Binding of decanoate (A), laurate (B), and myristate (C)are pictured as Bjerrum plots ( I versus log c). Binding of palmitatc (D) is pictured as a relative Bjerrum plot, where the availability of palmitate, C/p, has taken the place of the free ligand concentration. Albumin concentration is 30 PM for the low-affinity component as well as for the mixed albumin. 66 mM sodium phosphate buffer, pH 7.4, temperature 37".
419
H. Vorum et al. component from the column. We first tried to elute with 25 mM decanoate dissolved in the phosphate buffer solution, then with a 60 mM decanoate phosphate buffer solution containing 10% ethanol and finally with a mixture of ethanol and 0.05 M NaOH ( l / l ) ( v p ) . These attempts to get the high-affinity albumin component out of the column gave only a low yield of albumin (6.5 mg). The best elution was obtained with the 60 mM decanoate phosphate buffer containing looo ethanol. The amount obtained, however, was not sufficient for binding studies. Binding of fatty acids. The results of binding of the four fatty acids to the mixed and to the low-affinity albumin are pictured in Fig. 3 (A-D). Decanoate, laurate, and myristate are presented as r versus log c plots, whereas the analogous r versus log C / p graph is used for palmitate. As expected from the chromatographic separation procedure decanoate binds with lower affinity to the eluted albumin component than to the mixed albumin at r-values below 2. The eluted albumin and the mixed albumin possess almost equal affinity for decanoate at r-values above 2. The medium-chain fatty acids laurate and myristate and the long-chain fatty acid palmitate show a similar pattern with low affinity for the eluted albumin and high affinity for the mixed albumin. With these fatty acids, however, the binding curves do not merge at higher r-values indicating that the low affinity component also binds the fatty acids beyond the first few molecules with weaker affinity than the mixed albumin. Binding of diazepam and wa$arin. Fig. 4A presents the observed results for binding of diazepam to the mixed albumin and to the low-affinity component. The affinity of diazepam to the low-affinity component is practically the same as the affinity for the mixed albumin. The results for warfarin are plotted in Fig. 4B. Warfarin is bound to the mixed albumin, whereas no binding could be detected to the low-affinity component. The low-affinity component seems to have no affinity at all for binding of warfarin. DISCUSSION Afinity chromatography of serum albumin Affinity chromatography occupies a unique place in separation technology since it enables purification of biomolecules on the basis of biological function or individual chemical structure. The molecule to be purified is specifically and reversibly bound to a complementary binding substance, in this case decanoate, immobilized on an insoluble support matrix. A successful separation requires that the ligand can be covalently attached to a chromatographic bed material, the matrix, and at the same time retain a certain degree of affinity for the albumin. Decanoate was chosen as the ligand because it has 420
Diazepam r 31
A
0
-5
-6
-7
-4
log c
-3
Warfarin
B
r
0
80 0
0.5
80
-
8 0
-6
-5
-4
log c FIGURE 4 Binding isotherms for diazepam and warfarin to the low-affinity component ( 0 )and to the mixed serum albumin (0). Binding of diazepam (A) and warfarin (B) are shown as Bjerrum plots (Y versus log c). Albumin concentration is 30 PM for the low-affinity component as \veil as for the mixed albumin. 66 mM sodium phosphate buffer, pH 7.4. temperature 37'.
previously been shown that a commercial human serum albumin preparation from Kabi Vitrum@ was heterogeneous with respect to binding of decanoate (6). The binding isotherm was at that time described by assuming the presence of two albumin components. One component, amounting to 65% of the albumin, possesses high affinity for decanoate and one component, about
Binding properties of albumin 35%, possesses low affinity. For valproate a similar heterogeneous phenomenon has been reported with 67% of the albumin capable of binding valproate (7). These binding studies were carried out with albumin preparations differing from batch 88181 used in the present study. Another proportion between the highand low-affinity components may therefore be found in batch 88181. We evaluated the proportion by using exactly the same dialysis equipment and procedure as previously for decanoate (6). The binding isotherm (Fig. 2) could be described by a model in which approximately 40% of albumin was capable of binding decanoate with one more high-affinity step than the rest. With a successful separation we thus expect to find a high-affinity component yielding approximately 40 % , and a low-affinity component yielding 60% of the albumin preparation. From the albumin added to the column we in fact eluted 63% with the void volume. This fraction represents the low-affinity component, as confirmed later by binding experiments. We then tried to isolate the high-affinity component by supplementing the elution buffer with decanoate, which could bind to the albumin and thereby compete with the decanoate immobilized on the column. When this failed we additionally added ethanol and finally a mixture of ethanol and NaOH. By this procedure we obtained only small amounts of albumin (6.5 mg) not sufficient for binding studies. It should be mentioned, that even if sufficient amounts are obtained we end up with two problems. The presence of denaturing agents in the elution buffer can denature albumin making it unfit for later binding studies and finally the presence of large amounts of fatty acids would require a delipidation of albumin before binding experiments can be performed. According to Chen (10) this defatting procedure requires relatively large amounts of albumin, which is difficult and expensive to obtain with the presently available technique using just a single column. In conclusion, it is a d f i cult task to isolate the high-affinity component in its native state for functional studies.
conformational changes of the albumin molecule are important in the binding process (19). From the practical point of view, however, drugs bound to serum albumin can generally be classified in two groups, those competing primarily with binding of warfarin (Site I) and those competing primarily with diazepam (Site 11) (8, 9). We found that the low-affinity component showed almost as high affinity for diazepam than the mixed albumin. In contrast to this the affinity of the lowaffinity component for warfarin was extremely low to undetectable. In summary the low-affinity component generally seems to possess lower binding affinity for the ligands investigated than the mixed albumin. In order to explain the binding properties of the mixed albumin we thus expect that besides the low-affinity component isolated in the present work the mixed albumin must contain one or more high-affinity components.
Heterogeneity of albumin It is well known that albumin shows physicochemical heterogeneity. Excellent reviews have been made by Janatova (2), Foster (3), and more recently by Peters (4) on this subject. The binding experiments presented here and from two other studies (6,7) show that pooled, defatted human serum albumin as commercially obtained consists of two or more components showing different affinities for ligands. Previously, it has been observed that albumin from different humans possess differences in binding abilities. Albumin from pregnant women shows decreased binding affinity (20), and adult albumin possesses different affinities for bilirubin (21), palmitate (22,23) and the drugs warfarin and sulfamethizole (24) than does albumin from the newborn. The chemical basis for this heterogeneity remains a matter of debate. Recently, binding studies have shown that albumin carrying single point mutations may possess ligand binding affinities differing up to one order of magnitude as compared with the wild-type albumin A (25,26), e.g., albumin with the amino acid substitutions 313 Lys+Asn or 365 Asp+His binds warfarin with lower affinity than the wild type albumin. The frequency of Binding properties of the albumin components The binding properties of the low-affinity component mutations in albumin is unknown at present. We have previously shown that the oxidative state of and of the mixed albumin were investigated by dialysis exchange rate determinations (6, 12). Since decanoate Cys-34, the dimerisation of albumin or protein-towas used to isolate the low-affinity component of al- protein interaction cannot explain the heterogeneous bumin we measured the binding of some other fatty binding of fatty acids (6, 11). To test whether the obacids to see whether this component in general pos- served phenomenon is an artefact originating during the sessed low affinity to fatty acids. Our findings showed preparation of albumin we have previously performed that this indeed was the case. All fatty acids investi- binding experiments on human serum with octanoate gated did bind with lower affinity to the low-affinity (6). This result indicated that albumin also was heterogeneous in serum, bearing in mind, however, that the component than to the mixed albumin. To test the albumin for drug binding we measured the presence of fatty acids in serum complicates the interaffinity of warfarin and diazepam to the component, pretation of the results. In the present report we have even though the description of the albumin molecule as shown a way to purify the low-affinity component of having a number of discrete, preformed drug binding albumin. Binding studies with fatty acids and drugs sites or binding regions is not entirely satisfactory, since have been performed to confirm that the component in 42 1
H. Vorum et al. general possesses low ligand binding affinity. For further progress, an effort should be made to isolate the high-affinity component, although this may be more difficult, as previously mentioned.
ACKNOWLEDGMENT The authors gratefully acknowledge the technical u o r k of Nina Jorgensen.
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'.
&_
.4ddress:
HenriX- k'orunt Institute of Medical Biochemistry Universit! o f Aarhus Ole Worms, Alle. Bldg. 170 DK-8000 Aarhus C Denmark
Fatty acid and drug binding to a low-affinity component of human serum albumin, purified by affinity chromatography HENRIK VORUM, ANDERS 0. PEDERSEN and BENT HONORE
Institute of Medical Biochemistry, University of Aurhus, Aarhus, Denmark
Received 19 November 1991, accepted for publication 3 May 1992
Binding equilibria for decanoate to a defatted, commercially available human serum albumin preparation were investigated by dialysis exchange rate determinations. The binding isotherm could not be fitted by the general binding equation. It was necessary to assume that the preparation was a mixture of two albumin components about 40% of the albumin having high affinity and about 60% having low affinity. By affinity chromatography we succeeded in purifying the low-affinity component from the mixture. The high-affinity component, however, could not be isolated. We further analyzed the fatty acid and drug binding abilities of the low-affinity component. The fatty acids decanoate, laurate, myristate and palmitate were bound with higher affinity to the mixture than to the low-affinity component. Diazepam was bound with nearly the same affinity to the low-affinity component as to the albumin mixture, whereas warfarin was not bound at all to the low-affinity component. Key words: affinity chromatography; binding analysis; binding isotherm; heterogeneity; human serum albumin; low-affinity albumin
Albumin is synthesized in the liver and circulates in the blood plasma. One of the main functions of albumin is to bind and carry endogenous as well as exogenous ligands. Over decades, several binding studies have been performed to investigate this function. Most binding studies have been performed by using models suggesting that albumin is a homogeneous protein with respect to binding in line with the detection of only one copy of the albumin gene (1). However, several examples of physicochemical heterogeneity of albumin have been described in the literature (for reviews see refs. 2-4). In recent years, thanks to the development of a more precise dialysis exchange rate method (5) some binding studies have revealed that albumin also shows binding heterogeneity for some ligands. We have thus shown that a defatted commercial human serum albumin preparation is heterogeneous with respect to binding of the medium-chain fatty acids, hexanoate, octanote, and decanoate (6), as well as the branched octanoic acid valproate (7), used as a drug in the treatment of epilepsy. The binding of these ligands could only be described by assuming the presence of two albumin components, about two thirds having high affinity and one third having low affinity. The high-affinity albumin component was able to bind one mol decanoate, 1 mol
octanoate, 2 mol hexanoate or 1 mol valproate more than was bound to the low-aflinity component. The chemical basis for this observed binding heterogeneity has not been elucidated. One way to go for further progress in studying the binding heterogeneity of albumin is to separate highand low-affinity components of albumin on a preparative scale. In the present paper we describe an affinity chromatographic procedure used to isolate the lowaffinity component from a commercial albumin preparation. To demonstrate the carrier heterogeneity, binding studies to the low-affinity component and to the original albumin mixture were carried out with the four fatty acids decanoate, laurate, myristate, and palmitate, and with diazepam and warfarin, each demonstrating different drug binding functions of the albumin (8,9). EXPERIMENTAL PROCEDURES
Human serum albumin Human serum albumin was obtained from AB Kabi Vitrum (Stockholm, Sweden; lot no. 88181). It was defatted with charcoal in solution acidified with 0.1 M H2S04, pH 3, at 0" (lo), dialyzed against pure water, and lyophilized. The fatty acid content of the protein 415
H. Vorurn et al. after this procedure, has previously been measured (6) by an enzymatic method (NEFAC, Wako Chemicals GmbH, Neuss, Germany) and was less than 0.1 mol/ mol albumin.
Fatty acids Decanoate, laurate, myristate, and palmitate were obtained from Fluka AG, Buchs, Switzerland. The purity of all four fatty acids was determined as > 99?0 by gas liquid chromatography. [ 'TIDecanoate was obtained from CEA, Cen Saclay - Molecules Marquees, Gifsur-Yvette, France. [ 14C]Laurate, and [ I4C]myristate were received from Amersham International, England. [3H]Palmitatewas purchased from New England Nuclear Research Products, NEN Chemicals GmbH, Dreiech, Germany. According to the manufacturers the radiochemical purity for all four fatty acids was 98"; (by thin-layer chromatography). In all four preparations the content of unbound, dialyzable radioactive impurities (11) accounted for less than 0.15", of the total activity.
acetate-acetic acid buffer, pH 4.0, 1 L 0.02 M sodium bicarbonate-5% COz buffer, pH 8.0, and finally with large amounts of distilled water. For a separate control experiment the sepharose was processed as described above, except that decanoic acid was omitted from the dioxane/water solution.
Packing, ioading and eluting procedures. The decanoatesepharose column was packed and allowed to equilibrate for 24 h with 66 mM sodium phosphate buffer, pH 7.4 (the starting buffer). One hundred mg of a defatted human serum albumin preparation from Kabi Vitrum@ was dissolved in 7 mL of the sodium phosphate buffer and applied to the column. Elution with the starting buffer yielded an albumin component amounting to 60°, of that applied. The elution volume for this component was similar to the total volume of the column indicating that it possessed no or very low affinity for the decanoate-sepharose resin. Attempts to elute the high-affinity component with different solvents failed. The column was also packed with sepharose without coupled decanoate and allowed to equilibrate for 24 h with 66 mM sodium phosphate buffer, pH 7.4. Ninety Diazepam and warfarin Diazepam and [ I4C]diazepam were obtained as a gift one mg of albumin was dissolved in 7 mL of the sodium from Hoffmann-La Roche Ltd., Base1 Switzerland. Ra- phosphate buffer and applied to the column. The albucemic warfarin sodium salt was a gift from Nyco MED, min recovered amounted to 98.4% of that applied h d o v r e , Denmark. [ 14C]Warfarin was obtained from showing that albumin was not bound covalently to some Amersham International plc., Buckinghamshire, En- reactive groups of the modified sepharose. gland. Before use the radioactive preparations of diazepam and warfarin were purified by chromatography on Dialisis eschange rate determinations precoated TLC silica gel plates. Diazepam was eluted All binding experiments were performed in a sodium with hexane/dioxane/acetic acid (85jl5,i 1) (by vol.) and phosphate buffer, final concentration 66 mM, pH 7.4, at warfarin with toluene/ethylacetate/ethanol (90,'5/5) (by 37 '. A volume sufficient for one or a few days work of vol.). The radiochemical purity was checked as previ- the radioactively labeled ligand solution, was evapoously described (1 1). In both preparations the content rated in a stream of nitrogen and the ligand was then ofunbound, dialyzable radioactive impurities accounted dissolved in ethanol and added under stirring to the for less than 0.2% of the total activity. buffered albumin solution to give an ethanol concentration for all ligands less than 1% (by vol.). Separation of serum albumin bj! afinitj. chromatograph!. In one experiment the binding of decanoate to the total albumin was investigated by the dialysis exchange Coupling procedure. Fifty mL of EAH Sepharose 4B rate method using exactly the same rotating dialysis from Pharmacia was washed with 3 L 0.5 M NaCl and chambers which have previously been used for studies 1 L distilled water on a sintered glass filter (G3). De- of decanoate binding (6). This experiment was carried canoic acid 200 mg was dissolved with 100 mL dioxanei out and used as a standard of reference for comparing distilled water ( l / l ) (v/v) and the pH adjusted to 4.5. the decanoate binding abilities of different batch numThis solution was added to the gel suspension. The bers of albumin preparations. In the experiments exmixture was gently stirred. EDC (1-Ethyl-3[3- amining both the low-affinity component and the total dimethylaminopropyl]carbodiimide), 2.5 g, was dis- albumin together, static dialysis chambers, containing solved in 10 mL distilled water and added drop by drop only 25 p L sample volumes ( 5 ) , were used because only to the suspension. The reaction was allowed to proceed small amounts (63.3 mg) of the low-affinity component for 24 h at room temperature. Carbodiimide was used were available. These latter experiments are thus less to promote the condensation between free amino groups precise and we abstain from a detailed binding analyat the end of the 6-carbon spacer arms of AH-Sepharose sis of these results. However, they can be used to comand the free carboxyl group in the decanoic acid mol- pare the binding properties of the mixed albumin relaecule to form an amide link by acid-catalyzed removal tively to the chromatographically isolated component. of water, as illustrated in Fig. 1. The gel was then thor- Cellophane membranes, cut from dialysis tubing (Union oughly washed with 1 L dioxane/distilled water ( l / 1) Carbide Corp) were applied for studies with diazepam (viv) followed by washes with 1 L 0.2 M sodium and warfarin. For the binding experiments with de416
Binding properties of albumin
~
C
O
,
H+
c%-
CH,-
N = C=N-
decanoic acid
carbodiirnide
/
-c-o-c II
- NH
-
Sepharose
CH3
a,CH,-
CH,-
N (cH,),
r” H,N
II
NH- CH,N-
0
C-
CH,-CH,-CH,-N(CH,),
+
0
0
% Sepharose
=
C
/
NH-
‘
CH,-
NH - CH,-
CH3 CH~-CH,-
N(cH,),
isourea FIGURE 1 Carbodiirnide coupling reaction.
canoate, laurate, myristate, and palmitate we used cellulose membranes (Dianorm type 10.16) from Diachema AG, Switzerland. The dialysis exchange rate method, which has previously been described (6, 12) allows determination of the equilibrium concentration of unbound ligand by measuring the rate of exchange of radiolabeled ligand between compartments with solutions of identical molar composition. Eqns. 1 and 2 were used for calculating the free (c) and the molar ratio of bound ligand to albumin (r):
y=-
c-c
P
where Q I and Qr denote the radioactivity in the left and right solution after dialyzing for t min, C is the total of ligand (bound and unbound), P is the total concentration of albumin, and ko is a rate constant.
Dialysis of the ligands follows strictly first-order kinetics with reproducible rate constants, 0.099 and 0.089 min- for diazepam and warfarin respectively, and 0.114, 0.102, and 0.064 min- for decanoate, laurate, and myristate respectively. The solubility of palmitate in neutral buffers is too low for dialysis-rate determinations in the absence of albumin. Attempts to perform such determinations give irreproducible results (13). However, the availability of palmitate can be found experimentally and expressed as C/p (14). The concentration of bound ligand in this case is the same as the total ligand concentration, C, since free ligand is absent. The concentration of reserve albumin for binding of palmitate is expressed as p , and has previously been defined as the concentration of a standard albumin which in buffered solution binds the ligand as tightly as it is bound in the sample (5). A relative binding isotherm for palmitate can be obtained by picturing C/P versus Clp. For an exact deduction of the thermodynamic content of the availability entity, see (14), eqns. 8-11. 417
H. Vorum et al. RESULTS
Decanoate binding properties of albumin - batch nr. 88181 Fig. 2 shows the binding isotherm for decanoate to the mixed albumin (batch 88181) used in this study. This isotherm is obtained by using the same rotating dialysis chambers and dialysis exchange rate method as previously described (6). A best-fit approximation of the binding constants to the experimental data were obtained by a computer-mediated procedure (15). The criterion for significant acceptability of a best-fit obtained from one binding model compared with a best-fit from another was evaluated by an F-test (16). An attempt was made to analyze the binding isotherm in terms of the stoichiometric binding equation (17, 18).
+ 2c2K,K, + 3c3K,K,K, + . . . + Nc‘ KIK,K,. . . K ,
cK,
r = 1 + c K , + c 2 K , K 2 +c3K,K,K3 + . . . + c“ K,K,K,. . . K ,
perimental data did not result in acceptable fits for decanoate as seen by the dashed lines in Fig. 2. An alternative stoichiometric model with one binding albumin component and another not binding at all, cK,
r = . f 1 i- c K , +
cK,
r=f
Decanoate
-8
* *
+ 2c2K,K2 + 3c3K,K2K3
+ NcNK,K2K3.. . K ,
1
+ c K , + c2K,K2 + c 3 K l K 2 K , + c”K,K2K, K , cK2 + 2c2K,K3 i- 3c3K2K,K4
(’ - f )
+ . . . + ( N - I ) c ( ~ - ’ 2) K3 K4K. . . K
1 + cK,
-7
4
-5
-6
log c
~
...
+ c2K2K3+ c3K2K,K4
+
(5)
c(h’- 1)K2K 3K 4 . . . K N
Fitting of eqn. 5 to the observed points was successful for decanoate as seen in Fig. 2, where the full curves represent the best least-squares fit. Four stoichiometric constants were used: K I = 7.09.106 M - I, K? = 3.35.10’ M - ’ , K3 = 2.40. lo3 M - I and K4= 5.51.105M - I . The best estimate off was 0.40. A model with two albumin components, where one is binding decanoate with one high-affinity step more than the other component, thus gives a good fit to the experimental data. The f value of 0.40 shows that 40% of the albumin possesses high affinity for binding of decanoate whereas the remaining 60% possesses low -4 affinity.
FIGURE 2 Calculated isotherm for binding of decanoate to the mixed albumin shown in a semilogarithmic plot of the concentration of bound. r. versus free decanoate, c. Inset shows the isotherm in a Scatchard plot, ric versur r . Full lines represent best-fit, obtained nith equation (5) with the values: K I = 7.09. lo6 M l , K - = 3.35.10’ M l . K 3 = 2 . 4 0 . 1 O 3 ~ - ’ and K 4 = 5 . 5 1 . 1 0 5 M - 1 , and /=0.40. Dashed lines show the best-fit with the stoichiometric binding equation (3). The tangent to the isotherm at intercept tvith r,c-axis (dotted line in the Scatchard plot) intersects the r-axis belon 1 indicating that albumin is heterogeneous. 30 p~ albumin in 66 m M sodiuni phosphate buffer, pH 7.4, temperature 37’.
418
. . . + cNK,K2K3’ KN
+
3
(4)
* * *
+
2
+ c2K,K2 + c3K,K,K3
also resulted in unacceptable fits, in good agreement with the binding results shown later, demonstrating that decanoate, to some extent, actually binds to the lowaffinity component. A modified binding equation was then tried. It was assumed that a fraction, f , of the albumin molecules binds decanoate according to the stepwise binding constants, K l , K,, K3; . . ., K N . The first step, with highest affinity. is missing on binding to the remaining fraction, f - 1, which has the binding constants K z , K3; . . ., ( 3 ) K.,. We thus obtain the following binding equation
where r is the average number of bound ligand molecules per albumin molecule, c is the free ligand concentration, Ki is the stoichiometric binding constant for the i’th molecule, and N is the maximum number of ligand molecules bound. Fitting of K-values in eqn. 3 to ex-
-9
+ 2c2K,K, i- 3c3K,K,K3
+ . . . + NcNKlK2K3.*.KN
~
Chromatographic separation of albumin components. One hundred mg of the defatted human serum albumin preparation was applied to the decanoate-sepharose column as previously described. In the first run we obtained 63 “2 of the amount of albumin applied to the column with the total volume, corresponding to the albumin component with low affinity for binding of decanoate. This direct method thus estimates that 37 % of the albumin binds to the decanoate-sepharose resin. This is in very good agreement with the results obtained from the binding analysis described above where it was found that approximately 40% ofthe albumin possesses
Binding properties of albumin high affinity for binding of decanoate whereas the remaining 60% possesses low affinity. The chromatography experiment was then repeated in a second run. This time 39% was attached to the column and 61% was eluted with the total volume. This result was similar to that obtained in the first run, indicating that the column was not saturated in the first run and still was able to bind albumin with high affinity for decanoate. A third and fourth run were then tried but here the binding fractions were reduced to 29% and to 25% respectively, probably as a result of saturation of the column. In these two latter experiments it was assumed
that the low-affinity component was contaminated with the high-affinity component. In consequence of this we only used the low-affinity component obtained from the first chromatographic experiment in the following binding studies. A control experiment was performed with a column packed with sepharose without coupled decanoate to insure that the high-affinity component was not covalently bound to the chemically modified sepharose. The experiment confirmed that the high-affinity component was non-covalently bound to the column. Several attempts were carried out to remove this
Decanoate
Laurate 0
0
-7
-8
-5
-6
-7
-4
-5
-6
-4
log c
Myristate
Palmitate 6
8
r
D
r
'B
-
4
m
OD
8
40
20 I3O
-7
0
2-
0
0 0.
0
0
0 (D
0
(D
0
0
log c
0
I
I
I
I
-6
-5
-4
-3
log c
I 01 -1
0
0 I
I
1
0
1
2
log Clp
FIGURE 3 Binding isotherms for decanoate, Iaurate, myristate, and palmitate to the low-affinity component ( 0 )and to the mixed serum albumin (0). Binding of decanoate (A), laurate (B), and myristate (C)are pictured as Bjerrum plots ( I versus log c). Binding of palmitatc (D) is pictured as a relative Bjerrum plot, where the availability of palmitate, C/p, has taken the place of the free ligand concentration. Albumin concentration is 30 PM for the low-affinity component as well as for the mixed albumin. 66 mM sodium phosphate buffer, pH 7.4, temperature 37".
419
H. Vorum et al. component from the column. We first tried to elute with 25 mM decanoate dissolved in the phosphate buffer solution, then with a 60 mM decanoate phosphate buffer solution containing 10% ethanol and finally with a mixture of ethanol and 0.05 M NaOH ( l / l ) ( v p ) . These attempts to get the high-affinity albumin component out of the column gave only a low yield of albumin (6.5 mg). The best elution was obtained with the 60 mM decanoate phosphate buffer containing looo ethanol. The amount obtained, however, was not sufficient for binding studies. Binding of fatty acids. The results of binding of the four fatty acids to the mixed and to the low-affinity albumin are pictured in Fig. 3 (A-D). Decanoate, laurate, and myristate are presented as r versus log c plots, whereas the analogous r versus log C / p graph is used for palmitate. As expected from the chromatographic separation procedure decanoate binds with lower affinity to the eluted albumin component than to the mixed albumin at r-values below 2. The eluted albumin and the mixed albumin possess almost equal affinity for decanoate at r-values above 2. The medium-chain fatty acids laurate and myristate and the long-chain fatty acid palmitate show a similar pattern with low affinity for the eluted albumin and high affinity for the mixed albumin. With these fatty acids, however, the binding curves do not merge at higher r-values indicating that the low affinity component also binds the fatty acids beyond the first few molecules with weaker affinity than the mixed albumin. Binding of diazepam and wa$arin. Fig. 4A presents the observed results for binding of diazepam to the mixed albumin and to the low-affinity component. The affinity of diazepam to the low-affinity component is practically the same as the affinity for the mixed albumin. The results for warfarin are plotted in Fig. 4B. Warfarin is bound to the mixed albumin, whereas no binding could be detected to the low-affinity component. The low-affinity component seems to have no affinity at all for binding of warfarin. DISCUSSION Afinity chromatography of serum albumin Affinity chromatography occupies a unique place in separation technology since it enables purification of biomolecules on the basis of biological function or individual chemical structure. The molecule to be purified is specifically and reversibly bound to a complementary binding substance, in this case decanoate, immobilized on an insoluble support matrix. A successful separation requires that the ligand can be covalently attached to a chromatographic bed material, the matrix, and at the same time retain a certain degree of affinity for the albumin. Decanoate was chosen as the ligand because it has 420
Diazepam r 31
A
0
-5
-6
-7
-4
log c
-3
Warfarin
B
r
0
80 0
0.5
80
-
8 0
-6
-5
-4
log c FIGURE 4 Binding isotherms for diazepam and warfarin to the low-affinity component ( 0 )and to the mixed serum albumin (0). Binding of diazepam (A) and warfarin (B) are shown as Bjerrum plots (Y versus log c). Albumin concentration is 30 PM for the low-affinity component as \veil as for the mixed albumin. 66 mM sodium phosphate buffer, pH 7.4. temperature 37'.
previously been shown that a commercial human serum albumin preparation from Kabi Vitrum@ was heterogeneous with respect to binding of decanoate (6). The binding isotherm was at that time described by assuming the presence of two albumin components. One component, amounting to 65% of the albumin, possesses high affinity for decanoate and one component, about
Binding properties of albumin 35%, possesses low affinity. For valproate a similar heterogeneous phenomenon has been reported with 67% of the albumin capable of binding valproate (7). These binding studies were carried out with albumin preparations differing from batch 88181 used in the present study. Another proportion between the highand low-affinity components may therefore be found in batch 88181. We evaluated the proportion by using exactly the same dialysis equipment and procedure as previously for decanoate (6). The binding isotherm (Fig. 2) could be described by a model in which approximately 40% of albumin was capable of binding decanoate with one more high-affinity step than the rest. With a successful separation we thus expect to find a high-affinity component yielding approximately 40 % , and a low-affinity component yielding 60% of the albumin preparation. From the albumin added to the column we in fact eluted 63% with the void volume. This fraction represents the low-affinity component, as confirmed later by binding experiments. We then tried to isolate the high-affinity component by supplementing the elution buffer with decanoate, which could bind to the albumin and thereby compete with the decanoate immobilized on the column. When this failed we additionally added ethanol and finally a mixture of ethanol and NaOH. By this procedure we obtained only small amounts of albumin (6.5 mg) not sufficient for binding studies. It should be mentioned, that even if sufficient amounts are obtained we end up with two problems. The presence of denaturing agents in the elution buffer can denature albumin making it unfit for later binding studies and finally the presence of large amounts of fatty acids would require a delipidation of albumin before binding experiments can be performed. According to Chen (10) this defatting procedure requires relatively large amounts of albumin, which is difficult and expensive to obtain with the presently available technique using just a single column. In conclusion, it is a d f i cult task to isolate the high-affinity component in its native state for functional studies.
conformational changes of the albumin molecule are important in the binding process (19). From the practical point of view, however, drugs bound to serum albumin can generally be classified in two groups, those competing primarily with binding of warfarin (Site I) and those competing primarily with diazepam (Site 11) (8, 9). We found that the low-affinity component showed almost as high affinity for diazepam than the mixed albumin. In contrast to this the affinity of the lowaffinity component for warfarin was extremely low to undetectable. In summary the low-affinity component generally seems to possess lower binding affinity for the ligands investigated than the mixed albumin. In order to explain the binding properties of the mixed albumin we thus expect that besides the low-affinity component isolated in the present work the mixed albumin must contain one or more high-affinity components.
Heterogeneity of albumin It is well known that albumin shows physicochemical heterogeneity. Excellent reviews have been made by Janatova (2), Foster (3), and more recently by Peters (4) on this subject. The binding experiments presented here and from two other studies (6,7) show that pooled, defatted human serum albumin as commercially obtained consists of two or more components showing different affinities for ligands. Previously, it has been observed that albumin from different humans possess differences in binding abilities. Albumin from pregnant women shows decreased binding affinity (20), and adult albumin possesses different affinities for bilirubin (21), palmitate (22,23) and the drugs warfarin and sulfamethizole (24) than does albumin from the newborn. The chemical basis for this heterogeneity remains a matter of debate. Recently, binding studies have shown that albumin carrying single point mutations may possess ligand binding affinities differing up to one order of magnitude as compared with the wild-type albumin A (25,26), e.g., albumin with the amino acid substitutions 313 Lys+Asn or 365 Asp+His binds warfarin with lower affinity than the wild type albumin. The frequency of Binding properties of the albumin components The binding properties of the low-affinity component mutations in albumin is unknown at present. We have previously shown that the oxidative state of and of the mixed albumin were investigated by dialysis exchange rate determinations (6, 12). Since decanoate Cys-34, the dimerisation of albumin or protein-towas used to isolate the low-affinity component of al- protein interaction cannot explain the heterogeneous bumin we measured the binding of some other fatty binding of fatty acids (6, 11). To test whether the obacids to see whether this component in general pos- served phenomenon is an artefact originating during the sessed low affinity to fatty acids. Our findings showed preparation of albumin we have previously performed that this indeed was the case. All fatty acids investi- binding experiments on human serum with octanoate gated did bind with lower affinity to the low-affinity (6). This result indicated that albumin also was heterogeneous in serum, bearing in mind, however, that the component than to the mixed albumin. To test the albumin for drug binding we measured the presence of fatty acids in serum complicates the interaffinity of warfarin and diazepam to the component, pretation of the results. In the present report we have even though the description of the albumin molecule as shown a way to purify the low-affinity component of having a number of discrete, preformed drug binding albumin. Binding studies with fatty acids and drugs sites or binding regions is not entirely satisfactory, since have been performed to confirm that the component in 42 1
H. Vorum et al. general possesses low ligand binding affinity. For further progress, an effort should be made to isolate the high-affinity component, although this may be more difficult, as previously mentioned.
ACKNOWLEDGMENT The authors gratefully acknowledge the technical u o r k of Nina Jorgensen.
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'.
&_
.4ddress:
HenriX- k'orunt Institute of Medical Biochemistry Universit! o f Aarhus Ole Worms, Alle. Bldg. 170 DK-8000 Aarhus C Denmark
