Interaction of fibrinogen with n-alkylagaroses and its purification by critical hydrophobicity hydrophobic interaction chromatograpy

Journal of Chromatography A, 1109 (2006) 197–213

Interaction of fibrinogen with n-alkylagaroses and its purification by critical hydrophobicity hydrophobic interaction chromatograpy夽 Herbert P. Jennissen ∗ , Anastasios Demiroglou Institut f¨ur Physiologische Chemie, Universit¨at Duisburg-Essen, Universit¨atsklinikum Essen, Hufelandstrasse 55, D-45122 Essen 1, Germany Received 11 April 2005; received in revised form 3 January 2006; accepted 3 January 2006 Available online 20 February 2006

Abstract A rational application of hydrophobic interaction chromatography (HIC) to the purification of proteins has remained an enigma in spite of over 30 years of research. The critical hydrophobicity parameter, which can be determined from a concentration series of n-alkyl Sepharose 4B (Seph-Cn) offers the possibility of adapting the HIC gel to the needs of purification. To this end a library of HIC gels (Seph-C4 to Seph-C6) of different immobilized alkyl residue concentrations was synthesized and tested with purified bovine fibrinogen. Binding of fibrinogen to such a concentration series resulted in sigmoidal binding curves. Analysis of the Seph-C5 data according to the lattices-site binding model yielded adsorption coefficients (nS ) between 5 and 10 indicating that 5–10 lattice-sites (alkyl residues) interact multivalently with a fibrinogen molecule for adsorption at low ionic strength. The apparent lattice-site half-saturation constant of dissociation lies between 21 and 25 ␮mol/ml packed gel. For each alkyl chain length a critical hydrophobicity could be determined. For fibrinogen purification the critical hydrohobicity gel, Seph-C5 (13 ␮mol/ml packed gel), was selected. With the help of the cosolvents NaCl or glycine a fully reversible adsorption of fibrinogen could be facilitated on the critical hydrophobicity gel. Application of the method to human and bovine blood plasma resulted in a single step purification of fibrinogen in high yields. A comparison of the classical purification of fibrinogen with the critical hydrophobicity HIC (CHIC) method demonstrates a reduction in preparation time from several days to ca. 1 h. The subunit structure of HIC-purified human fibrinogen is identical to the classically purified protein. In the case of bovine fibrinogen however HIC-purified fibrinogen displayed a different subunit structure in that the A␣ chain of fibrinogen had a ca. 5 kDa higher molecular mass. This may be due to the rapidity of the new one-step method and an avoidance of proteolysis. © 2006 Elsevier B.V. All rights reserved. Keywords: Hydrophobic interaction chromatography; Critical hydrophobicity; Alkyl Sepharose; Fibrinogen; One-step purification of fibrinogen

1. Introduction Hydrophobic interaction chromatography (HIC), a form of multivalent interaction chromatography [1], of proteins has remained as one of the most difficult chromatographic procedures, since the rational adjustment of critical parameters such as selectivity, capacity and yield has eluded in-depth analysis and understanding. In addition proteins are often unfolded or “irreversibly” adsorbed during such a purification step. Critical hydrophobicity HIC (CHIC) which has its roots in a paper from our laboratory 30 years ago [2], is a recent development [3–5] resulting from a comprehension of certain basic elements governing HIC and offers a rational optimization scheme for the 夽 Dedicated to Professor Dr. Ludwig M.G. Heilmeyer on the occasion of his 65th birthday and emeritation. ∗ Corresponding author. Tel.: +49 201 723 4125; fax: +49 201 723 5944. E-mail address: [email protected] (H.P. Jennissen).

0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.01.043

HIC process as a whole. Recently, a model for the prediction of protein binding to alkylagaroses has been suggested on the basis of critical hydrophobicity HIC [6]. It is a general method which can be applied to very hydrophilic as well as to hydrophobic proteins, since the HIC gel can be adapted to the special hydrophobicity needs of each protein. Besides enhancing the selectivity of HIC for proteins a further important characteristic of this method is the reversibility of protein binding, allowing a gentle, high-yield, negative cosolvent-gradient elution without addition of alcohols, chaotropics [7] or detergents. Fibrinogen, a 340 kDa Ca2+ -binding glycoprotein [8], named by Virchow in 1847 [9], consists of three heterologous subunits (molecular masses for human form): A␣ (m = 73 kDa), B␤ (m = 60 kDa) and ␥ (M = 53 kDa) in a stoichiometry of (A␣, B␤, ␥)2 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [10]. The rod-like protein is 45 nm long and consists of seven domains (heptanodular model) with a maximal diameter of 4.8 nm [11]. Recently, the crystal structure

198

H.P. Jennissen, A. Demiroglou / J. Chromatogr. A 1109 (2006) 197–213

of a modified form of bovine fibrinogen was reported [12]. Fibrinogen has been the subject of purification procedures for over 100 years (see [13]). About 40–50% pure fibrinogen [14] can be obtained in fraction 1 of Cohn’s method 6 [15] from 1946. The method of freezing-out the fibrinogen of plasma in 1947 [15] led to preparations 90–94% pure [14]. Fibrinogen preparations of 93–98% purity were reported from 1948 onward [13,14,16]. Of these the method of Blomback and Blomback [13], in which the precipitation of fibrinogen with glycine was described, developed into one of the standard preparation methods. All of these methods rely on precipitation procedures by salts and organic solvents and if highly pure fibrinogen is wanted, the preparation involves at least five steps. Affinity chromatographic methods for the purification of fibrinogen on fibrin monomer agarose [17] and ristocetin agarose [18] have been described. However, these methods did not develop into easily applicable standard preparation methods. Although fibrinogen does not bind to lysine-Sepharose [19], consisting of butylamine end groups, we have found that it strongly adsorbs to certain uncharged n-alkyl Sepharoses [20]. In this paper, the first systematic experimental analysis of critical hydrophobicity HIC is described and in the following applications it will be shown that human and bovine fibrinogen can be purified to homogeneity (96–98% clottability) from blood plasma in a single step. 2. Materials and methods 2.1. Materials 2.1.1. Chemicals Sepharose 4B was obtained from Pharmacia (Freiburg, Germany). n-Alkylamines (butyl-, pentyl- and hexylamine for synthesis, purity >99%) were obtained from Merck (Darmstadt, Germany). Carbonyl diimidazole (CDI) was obtained from Sigma (Munich, Germany). Dry acetone (H2 O < 0.3%) was obtained from Merck. [1-14 C]ethylamine hydrochloride and [1-14 C]butylamine hydrochloride were from New England Nuclear (NEN-Life Science; Cologne, Germany) and [14 C]hexylamine from Amersham (Braunschweig, Germany). Commercial purified human fibrinogen (Haemocomplettan HS powder; clottability 70–85%) was from Behringwerke (Marburg, Germany). Aprotinin (Trasylol) was a kind gift from Dr. E. Truscheit (Bayer, Leverkusen, Germany) and was used in a stock solution of 10,000 KIU/ml. The plasmin inhibitor ␧-aminocaproic acid was from Sigma and thrombin (Topostasin, 150 NIH units/ml) from Hoffmann-La Roche (Basle, Switzerland). Aluminum hydroxide moist gel was from British Drug House (BDH, Poole, UK). Water was prepared by distillation of deinonized water and then passing it through a Milli-Q system (Millipore, Eschborn, Germany). All other chemicals were of the highest available or analytical grade. 2.2. Methods 2.2.1. Preparative methods 2.2.1.1. Preparation of bovine fibrinogen. Fibrinogen was purified according to the modifications of the methods described by

Blomback and Blomback [13] Mahn and Muller-Berghaus [21] as follows. Citrate plasma (final concentration of citrate 20 mM) was prepared from bovine blood (step 1) by standard methods and stored frozen. Frozen bovine plasma was slowly thawed at 0 ◦ C. The prothrombin complex was removed by adsorption on aluminum hydroxide (step 2) by mixing 30 g of solid aluminum hydroxide gel with 1000 ml of plasma for 45 min. After centrifugation at 5000 × g for 15 min, 160 g of solid glycine was added per 1000 ml of the supernatant plasma, which corresponds to 65% saturation at room temperature. The precipitate was collected by centrifugation at 6300 × g for 15 min at 5 ◦ C and dissolved in buffer A (50 mM sodium citrate, 5 mM EDTA, 5 mM ␧-aminocaproic acid, 100 KIU aprotinin, pH 7.4). Precipitation with glycine at room temperature was repeated twice (steps 4–5). After the third addition of glycine, the precipitate was dissolved in buffer B (50 mM Tris–HCl, 150 mM NaCl, 0.1 M EDTA, 5 mM ␧-aminocaproic acid, 100 KIU aprotinin, pH 7.4). The solution was cooled to −2 ◦ C while ethanol 30% (v/v) was added (step 6) drop by drop to yield a final ethanol concentration of 6% (v/v). After stirring the precipitate was collected by centrifugation at 8600 × g for 10 min at 5 ◦ C and dissolved in buffer B yielding 3.0 g of pure fibrinogen. Classical fibrinogen preparations end at this step with clottabilities of 95–96%. Under certain conditions it may be desirable to have higher clottabilities. Thus, clottabilities of 98–100% may be obtained by a purification step according to Laki [16,22] which is as follows (step 7): to a fibrinogen solution was added 1.0 M phosphate buffer, pH 6.4 to give a solution of 0.1 M phosphate final concentration and this was allowed to stand for 1 h at 5 ◦ C. This solution was diluted with the same volume of water, pH 6.5, and left standing over night in the cold room at 5 ◦ C. The formed precipitate was filtered at room temperature and discarded. To this clear solution 1/3 volume of saturated ammonium sulfate solution was added at room temperature in small portions. The resulting precipitate was centrifuged at 6300 × g for 15 min, dissolved in buffer B and dialyzed against buffer B over night (=purified step 7 fibrinogen). The step 7 fibrinogen solution (620 mg) was stored in small aliquots at −80 ◦ C. In the present case a clottability of ∼100% is obtained, however at the high cost of losing 75% of the fibrinogen. Bovine fibrinogen preparations with clottabilities of 96–100% are homogenous when examined by SDS-PAGE. All column experiments in this paper were run with this highly purified Laki-fibrinogen. Table 1 shows a purification table of fibrinogen from bovine plasma by the stated classical method (differential precipitation procedures). In the shown preparation, fibrinogen only has to be enriched ca. eight-fold. Since fibrinogen plasma levels can vary by a factor of 2 (150–350 mg/dl) the enrichment factors can lie between 8 and 20-fold. Nevertheless, Table 1 clearly demonstrates how hard it sometime is to purify a protein only ca. 10-fold. The yield for fibrinogen lies between 8 and 2% depending on whether the Laki procedure (step 7) is employed or not. Thus, fibrinogen can be prepared by this method in amounts of 0.5–3 g having a clottability between 96 and 100%. The pure bovine fibrinogen obtained displayed the following subunit molecular masses (Fig. 1, lanes 3–5) as determined by SDS-PAGE: A␣ 65.4 kDa, B␤ 61.9 kDa, γ 53.3 kDa. After addi-

H.P. Jennissen, A. Demiroglou / J. Chromatogr. A 1109 (2006) 197–213

199

Table 1 Purification table for the classical preparation of fibrinogen from bovine plasma Step

Procedure

Volume (ml)

Fibrinogen (280 nm) Clottability (%)

1

Blood plasma (coagulation process) Aluminum hydroxide precipitation Glycine precipitation 1 Glycine precipitation 2 Glycine precipitation 3 Ethanol precipitation Sodium phosphate and ammonium sulfate precipitation

2 3 4 5 6 7

a

Formed fibrin (mg/ml)

Protein (Lowry) Total fibrinogen (g)

Concentration (mg/ml)

Total protein (g)

Purification (n-fold)

Fibrinogen yield (%)

4600

12

5.6

25.7

75

345

1.0

100

4500

13

5.5

24.8

62

279

1.1

96

1150 450 300 250 107

70 92 94 96 100

4.9 7.8 10.6 8.4 5.3a

5.8 7.7 7.8 8.0 8.3

22 14 12 8.2 2.2

5.6 3.5 3.2 2.1 0.57a

10.6 8.5 11.3 12.1 5.8

12 3.8 3.4 3.0 0.62

Separate calculations.

tion of thrombin the fibrinopeptides A and B are split off (Fig. 1, lane 2) and the resulting ␣ chain acquires a mass 63.6 kDa and the ␤ chain a mass of 57.5 kDa, respectively. The absence of high molecular weight bands in lane 2 indicates a factor XIII-

Fig. 1. SDS-PAGE of bovine fibrinogen purified by classical methods. Bovine fibrinogen was purified according to the procedure described in Section 2.2 and Table 1 up through the Laki procedure. The final product was applied to the depicted gel lanes 3–5). In a clottability assay thrombin was added to fibrinogen and a clot was formed (lane 2). No high molecular mass bands are seen demonstrating absence of factor XIII. From the difference between the subunit molecular masses fibrinogen/fibrin the fibrinopeptide molecular masses can be calculated; fibrinopeptide A: 1.78 kDa, fibrinopeptide B: 4.4 kDa. The electrophoretic separation was performed on a 10% polyacrylamide gel in the presence of SDS according to Laemmli [34]. For further details, see Section 2 and Table 1. Lanes: (1) molecular mass standards ∼30 ␮g; (2) fibrin, 6 ␮g (fibrinogen + thrombin), α: 63.6 kDa, β: 57.5 kDa, γ: 53.1 kDa; 2–5 = fibrinogen 6–10 ␮g, A␣: 65.4 kDa, B␤: 61.9 kDa, γ: 53.4 kDa.

free preparation. It should be noted that the seven purification steps in Table 1 correspond to one man-week of work. 2.2.1.2. Activation of Sepharose 4B with carbonyl diimidazole (CDI). Sepharose 4B consists of 4% agarose, a polysaccharide composed of agarobiose units (see [23]). The concentration of agarobiose (m = 306 Da, for anhydroform) in Sepharose 4B is ∼100 ␮mol/ml packed gel [1]. On agarobiose there are one primary hydroxyl and three secondary hydroxyls, which means that there are ∼100 ␮mol primary and ∼300 ␮mol secondary hydroxyls/ml packed gel on the base matrix of Sepharose 4B. The primary hydroxyls probably display a higher reactivity towards CDI than the secondary hydroxyls similar to the CNBr reaction [23]. Uncharged alkyl-N-Sepharoses were prepared according to the carbonyl diimidazole method of Hearn et al. [24]. A solid-phase 13 C-NMR analysis has shown the gel products to be fully charge free [25]. The immobilized butyl residue concentration was determined by addition of the tracer [14 C]ethylamine to the 2 M butylamine solution [2,26,27]. Before activation 25.0 g of wet weight Sepharose 4B was washed on a B¨uchner funnel sequentially with 500 ml water, 500 ml water/acetone (3:1), 500 ml water/acetone (1:3) and 750 ml dry acetone (<0.3% water). The water-free gel was transferred to a spherical flask and 50 ml dry acetone (<0.3% water) containing, e.g. 1.5 g CDI (20 mg/ml mixture) was added and stirred slowly at room temperature for 30 min. After incubating for 30 min the gel suspension was washed on a B¨uchner funnel sequentially with 500 ml water/acetone (1:3), 500 ml water/acetone (3:1) and 750 ml water and sucked to wet weight dryness. 2.2.1.3. Coupling of alkylamines to CDI activated Sepharose 4B. For coupling 50 ml 2 M butylamine (pentyl- or hexylamine), pH 10.0 (14 C-labelled) was added to 25.0 g of CDI activated wet weight Sepharose 4B and incubated at 4◦ for 60 min. Then the gel was washed sequentially on a Buckner funnel with 500 ml 0.01 M NaOH, 500 ml 0.01 M HCl and 500 ml water. The gel was sucked to wet weight dryness and resuspended in water to which spatula tip of NaN3 is added and stored at 4 ◦ C. [1-14 C]ethylamine, [1-14 C]butylamine, and [1-14 C]hexylamine were employed as tracers. No large differences were found using

200

H.P. Jennissen, A. Demiroglou / J. Chromatogr. A 1109 (2006) 197–213

Table 2 Analytical calibration function for the synthesis carbonyl diimadazole (CDI) methoda

14 C-butyl

Sepharoses by the

Carbonyl diimazole (mg/ml)

Degree of substitution (␮mol/ml) packed gel

0.39 0.84 2.2 4.4 6.5 9.0 9.5 14.4 18.0

1.0 2.0 5.0 10.0 15.9 20.0 25.6 30.0 41.2

a

For further details, see ref. [32], Section 2 and the text.

[1-14 C]ethylamine with the higher homologues for coupling. The method can be scaled up by a factor of ten (250 g wet weight). For analytical experiments only fresh unregenerated gel was used in the experiments. In preparative experiments the gels could be reused after regeneration with 5 vol., respectively of 1 M NaCl, 0.1 M NaOH, H2 0, 0.1 M HCl, H2 O and equilibration buffer. The dependence of the surface grafting density of butyl Sepharoses labelled with [1-14 C]butylamine on the CDI concentration in the incubation mixture in an analytical series is shown in Table 2. If the data are plotted a linear function is found. For determination of the immobilized residue density 1 ml of packed substituted or unsubstituted washed Sepharose 4B was added to 1 ml 32% HCl and heated in a water bath to 50 ◦ C until solubilized [2]. The sample was then neutralized with 0.2 ml 10 M NaOH and counted in the scintillator Quickszint 212 (Zinsser Analytik, Frankfurt, Germany). The analyses were done in triplicate. The samples were corrected for quenching by quench calibration curves. The volume of packed gel was determined as previously described [2]. Thus, a selective synthesis of an alkyl Sepharose of specific immobilized residue density is possible within an error of ca. ±5%. 2.2.2. Analytical methods 2.2.2.1. Clotting procedure. The fibrinogen concentration and activity was assayed by a clotting procedure according to the method of Jacobsson [28], using the following modifications [21]: The fibrinogen solution (0.2 ml, 0.4–0.8 mg fibrinogen) was diluted with 1.4 ml buffer A and then thrombin (Topostatin, Hoffmann-La Roche) was added to a final concentration of 100 U/ml. The formed clot was allowed to stand at room temperature for 20 h, collected by centrifugation at 14,000 × g for 5 min and dissolved in alkaline urea [28] for at least 1 h. The optical density of the solution was determined at 280 nm against an alkaline urea blank. The fibrinogen concentration was calculated according to the extinction coefficient of fibrin in alkaline urea of A1% 1 cm = 16.17 [14,28]. Attempts to replace the photometric measurements at 280 nm [28] by protein determinations according to Lowry et al. [29] were unsuccessful. In general, the Lowry clottability values were ca. 20% lower than the Jacobsson clottability. The method of Claus [30] even gave ∼40% lower clottability values than the Jacobson method. The immunologi-

cal method indicated ca. 30% inactive fibrinogen in plasma. In our hands the Jacobsson method was most reliable, therefore in all cases clottability was determined according to Jacobsson [28] as modified by Mahn and Mueller-Berghaus [21] as stated above. Commercial purified human fibrinogen (Haemocomplettan, Behring) had a clottability of 70–85%. These low values were primarily due to added human serum albumin (HSA) in the fibrinogen. 2.2.2.2. Measurement of fibrinogen binding to alkyl Sepharose. The limited sample-load (LSL) method [5,31] is employed on a quantitative chromatographic basis. In this method a defined amount of protein (e.g. 1 mg) in a defined sample volume is applied to each column under identical conditions and the massbalance is determined. The applied nonsaturating amount is dimensioned so as to be 100% adsorbed (i.e. no protein in run-through) on an adsorbent displaying the expected maximal affinity and capacity. In order to titrate the high affinity sites (i.e. potentially denaturing) on the gel, the measurements are made non-isocratically [3,32] although isocratic chromatographic analysis of low affinity binding sites is also possible on such gels [33]. Limited sample-load chromatography of fibrinogen [32] was performed on a column (0.9 cm × 12 cm) containing 2 ml of packed gel. The gel was washed and equilibrated with 20 vol. buffer C (50 mM Tris–HCl, 150 mM NaCl, 1 mM EGTA, pH 7.4] at 5–10 cm/h. As sample 1 mg purified fibrinogen was applied in a sample volume of 1 ml (in buffer C) and fractions of 1.5 ml were collected. The column was then washed with 15 ml buffer C and then eluted with 7.5 M urea and at high hydrophobicities of the gel with 1% SDS. In all cases of LSL-chromatography the experiments were performed at room temperature, flow was achieved by gravity (at 5–10 cm/h) and only fresh, unregenerated gel was employed. 2.2.3. Critical hydrophobicity HIC of fibrinogen 2.2.3.1. Human fibrinogen. Human blood was collected in 20 mM trisodium citrate and centrifuged at 900 × g for 20 min to remove red cells. The supernatant (plasma) was adjusted to 2 mM EGTA, 5 mM ␧-aminocaproic acid, 100 U/ml aprotinin, pH 7.4 and either used immediately or stored at −80 ◦ C. For fibrinogen purification by critical hydrophobicity HIC 20 ml fresh or frozen human plasma is slowly thawed at 0 ◦ C. Unless otherwise stated solid NaCl was added to the plasma to yield a concentration of 1.5 M. This was applied to a column (13 cm × 1.4 cm) containing 20 ml of packed Seph-C5 (13.6 ␮mol/ml packed gel), washed and equilibrated with buffer D (50 mM Tris–HCl, 1.5 M NaCl, 1 mM EGTA, pH 7.4) [Seph-Cn = alkyl Sepharose 4B of specified alkyl chain length (n)]. The column was then washed with 10 volumes of buffer D and the adsorbed proteins were eluted first with buffer C (50 mM Tris–HCl, 150 mM NaCl, 1 mM EGTA, pH 7.4) then with water and finally with 7.5 M urea. Clottability in the run-through and eluted fractions was tested. The fractions of the second peak (i.e. fibrinogen) were pooled and stored at −80 ◦ C. 2.2.3.2. Bovine fibrinogen. Bovine plasma (see above) to which glycine had been added to a final concentration of 1.5 M

H.P. Jennissen, A. Demiroglou / J. Chromatogr. A 1109 (2006) 197–213

adjusted to pH 7.4 was applied to 20 ml packed gel Seph-C5 (13.6 ␮mol/ml packed gel) in a column (13 cm × 1.4 cm) equilibrated with 50 mM Tris, 1 mM EGTA, 1.5 M glycine pH 7.4 (buffer E). The column was then washed with buffer E and eluted with 50 mM Tris, 1 mM EGTA, 0.150 M glycine, pH 7.4 (buffer F). The fraction volume was ∼ 6.3 ml. 2.2.3.3. Protein. Protein (standard: bovine serum albumin, BSA) was determined after precipitation with 5% trichloroacetic acid on an AutoAnalyzer II (Technicon, Tarrytown, NY, USA) according to Lowry et al. [29]. 2.2.4. SDS-gel electrophoresis Electrophoretic separation of proteins on polyacrylamide gels (10%) in the presence of SDS was performed according to Laemmli [34]. Protein standards were phosphorylase b (97 kDa), BSA (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa) and trypsinogen (24 kDa). The molecular masses of polypeptides were calculated from scans taken with a Pharmacia-LKB Ultroscan laser densitometer. 2.2.5. Conductivity measurements Conductivity measurements were made with a CDM 80 Conductivity Meter (Radiometer, Copenhagen). 2.2.6. Data analysis and curve fitting Evaluation of protein binding was carried out according to ref. [3,31] and is described in detail under Section 3. The sigmoidal binding curves (“sigmoidal dose-response”) were fitted to Eq. (1) (see below) either by non-linear logistic regression of data in semi-logarithmic form [35,36] or by linear regression of data in double logarithmic form [37,38] with the personal computer program GraphPad Prism Version 4. The constants of Eq. (1) (KS,0.5 , nS ) are expressed ± standard error of the mean (SEM). Unless otherwise stated other statistical data is expressed as mean value (¯x) ± standard deviation (SD). 3. Theoretical considerations and terminology Three parameters govern the adsorption of a protein to a homologous series of hydrophobic alkyl Sepharoses: (i) the chain-length, (ii) the surface concentration of immobilized alkyl residues and (iii) the cosolvent concentration (e.g. salt or amino acid). The used terminology is based on IUPAC recommendations [39,40] as adapted in [33]. The alkyl residue density or covalently immobilized residue concentration of alkyl Sepharose 4B can be expressed in two ways [33,41]: (i) on a surface concentration basis in nmol/m2 or (ii) on a volume basis in ␮mol/ml packed gel (CS(res) ) which is employed in this paper. On alkylagaroses two categories of protein adsorption isotherms may be distinguished: (a) a lattice-site binding function and (b) a bulk ligand binding function [33,36,42,43]. For critical hydrophobicity HIC the lattice-site binding function is decisive. The lattice site binding function (isotherm), which can also be applied to immobilized protein ligands [36], governs the binding of an immobilized residue, constituting a surface lattice-site, to

201

a complementary site (patch, pocket) on the protein adsorbed from the bulk solution. Protein adsorption is multivalent [44] and the more lattice sites that simultaneously interact with a protein molecule the higher the affinity of binding will be [45]. Protein adsorption, therefore, increases as a function of the concentration of lattice sites (at a constant equilibrium concentration of free bulk protein) according to the following equation [31,33,36,37,46]: θS nS = KS (CS(res) ) 1 − θS

(1)

where θ S corresponds to the fractional saturation of the involved protein with immobilized alkyl residues [33] as a function of the concentration of lattice-sites (Cs(res) ) in a binding unit. KS is the lattice-site adsorption constant and nS is the lattice-site adsorption coefficient. Both constants can either be obtained from a double logarithmic plot of log θ S /1 − θ S versus log CS(res) [33,37,44] or from a fit to a logistic equation [36]. The saturation of the involved protein with immobilized alkyl residues can in general be equated with the apparent saturation of the gel [33]. As the protein is saturated with immobilized residues the affinity to the surface increases and the binding units fill up. For nS > 1, we obtain a sigmoidal curve displaying positive cooperativity due to multivalence. Typical lattice-site adsorption coefficients are nS ∼ 4–8 [33,44,47]. For the case nS = 1, Eq. (1) reduces to a rectangular hyperbola. Saturation of binding occurs when the complementary binding sites of the protein in a binding unit on the adsorbent surface cannot accommodate further alkyl lattice sites. The corresponding lattice-site half-saturation association S(res) −1

constant KS,0.5 is defined according to: KS,0.5 = (C 0.5 ) = S(res) (KS )1/nS and reflects the affinity of binding. C 0.5 denotes half maximal saturation of the protein with immobilized residues in ␮mol/ml packed gel under non-isocratic (non-equilibrium) conditions (this paper). Since hydrophobic adsorption can be strongly promoted by neutral salts or glycine (i.e. cosolvents, see below) halfS(res) saturation constants can be defined at low C 0.5,LC and high

C 0.5,HC cosolvent concentrations. The surface concentration of alkyl residues for alkyl substituted Sepharose 4B can be calculated according to the relationship that 1 ␮mol/ml packed gel corresponds to 12.8 nmol/m2 for non-shrunken agarose [33,41]. In general, 1 ml packed Sepharose 4B as defined in ref. [2] corresponds to ca. 30 mg (29–32 mg) of agarose mass after drying [41], allowing a calculation in terms of the often used ␮mol/g dry agarose [41]. Hydrophobic interactions – being endothermic by nature – display positive temperature coefficients (for review, see ref. [3]). The experiments in this paper were preformed at room temperature (∼20 ± 2 ◦ C). A lowering of the temperature to cold room conditions (∼5 ◦ C) will reduce the binding affinity of proteins to the alkyl gels altering the critical hydrophobicity and cosolvent dependence. S(res) The critical hydrophobicity point (Ccrit ) was originally defined in saturation binding capacity experiments [2,3] as that immobilized alkyl residue concentration (threshold) at which protein adsorption at low salt concentrations on a defined alkyl S(res)

202

H.P. Jennissen, A. Demiroglou / J. Chromatogr. A 1109 (2006) 197–213

Table 3 Alkyl gel libraries employed in this papera Seph-C4 (␮mol/ml) packed gel

Seph-C5 (␮mol/ml) packed gel

Seph-C6 (mol/ml)packed gel

4.4 15.3 21.7 31.0 36.8 46.8

3.0 8.4 12.1 18.0 22.0 41.6

1.9 4.3 7.7 11.6 17.2 42.0

a For the determination of the critical hydrophobicity on Seph-C5 an additional gel (13.6 ␮mol/ml packed gel was synthesized. For further details, see Section 2, Figs. 5 and 6 and the text.

Sepharose begins. The most exact method of determining the critical hydrophobicity involves its derivation from adsorption isotherms [1,33,47]. Since sigmoidal protein binding curves as a function of the immobilized residue concentration are also found at high salt concentrations [47] an isothermal critical S(res) hydrophobicity point can be defined at low (Ccrit,LC ) as well as at S(res)

high (Ccrit,LC ) cosolvent/salt concentrations, yielding information on the high affinity sites at low salt concentrations and on the low affinity sites and at high cosolvent/salt concentrations, respectively. Both points can be exploited chromatographically S(res) [1,2,33,47] but only Ccrit,LC will be treated in this paper. In this paper fibrinogen binding is measured at low ionic strength by quantitative hydrophobic interaction chromatography [32] employing the “limited sample-load” method [31] under non-isothermic conditions (i.e. not by way of equilibrium binding isotherms). Since in this case the isothermal distribution coefficient is Dai > 100 [33], we may speak of a so-called “irreversible” form of adsorption chromatography (see also, ref. [48]). Since isocratic elution is not possible but moreover elution is facilitated by 7.5 M urea (see Section 2.2), we speak of nonisocratic conditions. The critical hydrophobicity determined S(res) under non-isothermic conditions is denoted as C crit . A comS(res) prehensive definition of critical hydrophobicity (C crit,LC ) under these conditions can be stated as corresponding to a 5% saturation of the involved protein (see [33]) with immobilized alkyl residues at a defined protein concentration (e.g. 0.5–1.0 mg/ml), under non-isocratic conditions, at a coslovent/salt concentration ≤0.15 M. Due to the possibility of adsorption hysteresis these constants have the prefix “apparent” [38]. 4. Results 4.1. Synthesis of a homologous library of uncharged n-alkyl Sepharoses The chain-length parameter and the surface concentration parameter were determined from a homologous library of alkylagaroses. Sepharose 4B was chosen as matrix, because it has no significant commercially implemented chemical modifications or cross-links. Table 3 shows the variation of the chain-length parameter (Seph-C4 to Seph-C6) and of the surface concentration parameter in a range between 2 and 47 ␮mol/ml packed

gel. The concentration series ends at ca. 40 ␮mol/ml packed gel because at concentrations >40 ␮mol/ml packed gel the gels begin to shrink and structurally change. 4.2. Library screening for the chain-length and surface concentration parameters Imperative for screening hydrophobic gel libraries in the present procedure is that protein adsorption is carried out at a low, i.e. a physiological salt concentration of ∼0.15 M NaCl. Fig. 2 shows the chromatograms obtained with highly purified bovine fibrinogen (Table 1) on the Seph-C4 surface concentration series. No adsorption of fibrinogen can be measured up to 31 ␮mol/ml packed gel. At ∼48 ␮mol/mol packed gel 50% of the fibrinogen is bound to the gel. Elongation of the alkyl chain by one carbon atom to Seph-C5 (Fig. 3) changes the chromatographic profiles significantly. No adsorption is found up to 12 ␮mol/ml packed gel. At 18 ␮mol/ml packed gel however, a significant portion of the applied fibrinogen is adsorbed, so that the critical hydrophobicity lies between these two points. At 41 ␮mol/ml packed gel 100% of the applied fibrinogen is bound. The impact of a further increase in the chain length by one carbon atom to Seph-C6 on fibrinogen adsorption is shown in Fig. 4. Fibrinogen adsorption begins ∼5 ␮mol/ml packed gel. Already at 7.7 ␮mol/ml packed gel ca. 70% of the fibrinogen is bound and at 17 ␮mol/ml packed gel 100% is adsorbed. 4.3. Fibrinogen binding curves showing sigmoidicity on immobilized alkyl residues Surface concentration plots of fibrinogen adsorption (derived from Figs. 1–3) to gel libraries Seph-C4 to Seph-C6 are shown in Fig. 5. Characteristically sigmoidal curves are obtained allowS(res) ing a determination of the critical hydrophobicity C crit as the future point-of-operation for hydrophobic interaction chromatography. Sigmoidicity is obtained because at low surface grafting density (1–5 ␮mol/ml packed gel) none of the alkyl gels adsorb fibrinogen. This non-adsorbing range increases as the chain length is reduced from Seph-C6 to Seph-C4. On the butyl series no adsorption is found between 4 and 35 ␮mol/ml S(res) packed gel. At C crit,LC = 31 ␮mol/ml packed gel the critical hydrophobicity of Seph-C4 is obtained, as the run-through peak is broadened and a small amount of fibrinogen is eluted by 7.5 M urea. Correspondingly the sigmoidal curves shift to the left as the chain-length is increased. On Seph-C6 the critical hydrophoS(res) bicity drops to C crit,LC ∼ 4–5 ␮mol/ml packed gel. From the three curves in Fig. 5, the intermediate chain length Seph-C5 was now selected (chain-length parameter) for a detailed determination of the critical hydrophobicity at low salt concentrations. No fibrinogen is adsorbed at 12 ␮mol/ml packed gel. In order to obtain a stringent value for the critical hydrophobicity an additional gel was synthesized with 13.6 ␮mol/ml packed gel. As shown in Fig. 6, this gel adsorbs low amounts of fibrinogen (∼2–3% of the saturation value) S(res) and thus approximates the critical hydrophobicity (C crit,LC ∼ 13.6 ␮mol/ml packed gel). In addition, Fig. 6 quantifies eluted fibrinogen. The amount of urea-resistant fibrinogen (=difference

H.P. Jennissen, A. Demiroglou / J. Chromatogr. A 1109 (2006) 197–213

203

Fig. 2. Quantitative hydrophobic interaction chromatography of fibrinogen on a surface concentration series of Seph-C4 at low salt concentration. Purified bovine fibrinogen (1 mg) was applied in 1 ml according to the “limited sample load” (LSL) method to a column (0.9 cm × 12 cm) containing 2 ml of packed gel in buffer C (50 mM Tris–HCl, 150 mM NaCl, 1 mM EGTA, pH 7.4]. The column was the washed with 15 ml buffer C and then eluted with 7.5 M urea (see arrows). Flow was by gravity. Fractions of 1.5 ml were collected. The following immobilized residue concentrations were employed: (A) 4.4 ␮mol/ml packed gel, (B) 15.3 ␮mol/ml packed gel, (C) 21.7 ␮mol/ml packed gel, (D) 31.0 ␮mol/ml packed gel, (E) 36.8 ␮mol/ml packed gel, (F) 46.8 ␮mol/ml packed gel. Protein was determined in the fractions according to Lowry. The experiments were performed at room temperature. For further details, see Section 2 and Table 3 and the text.

between the two curves) increases as the surface concentration of pentyl residues increases. At 22 ␮mol/m, packed gel ∼15% and at 40 ␮mol/ml packed gel 20% of the adsorbed fibrinogen cannot be eluted by 7.5 M urea. Binding data analysis and fitting of the data of Fig. 6 to Eq. (1) is illustrated in Fig. 7. Fitting of the adsorption data by logistic regression is shown in the semilogarithmic plot in Fig. 7A. The obtained constants are: nS = 10.4 ± 3.1 S(res) and (KS,0.5 )−1 = C0.5,LC = 21.5 ± 1.0 ␮mol/ml packed gel,

(r2 = 0.98). Fitting directly to the double logarithmic form of Eq. (1) by linear regression (Fig. 7B) yields: nS = 5.1 ± 1.1 s(res) and (KS,0.5 )−1 = C0.5,LC = 25.3 ± 1.1 ␮mol/ml packed gel 2 (r = 0.92). The apparent lattice-site adsorption coefficient (nS ) thus lies between 5 and 10 indicating that 5–10 lattice sites (alkyl residues) interact multivalently with a fibrinogen molecule for adsorption at low ionic strength. The apparent lattice-site s(res) half-saturation dissociation constant (KS,0.5 )−1 = C0.5,LC lies between 21 and 25 ␮mol/ml packed gel. A comparison of the

204

H.P. Jennissen, A. Demiroglou / J. Chromatogr. A 1109 (2006) 197–213

Fig. 3. Quantitative hydrophobic interaction chromatography of fibrinogen on a surface concentration series of Seph-C5 at low salt concentration. Purified bovine fibrinogen (1 mg) was applied in 1 ml according to the “limited sample load” (LSL) method to a column (12 cm × 0.9 cm) containing 2 ml of packed gel in buffer C. The column was the washed with 15 ml buffer C and then eluted with 7.5 M urea (see arrows). Flow was by gravity. Fractions of 1.5 ml were collected. The following immobilized residue concentrations were employed: (A) 3.0 ␮mol/ml packed gel, (B) 8.4 ␮mol/ml packed gel, (C) 12.1 ␮mol/ml packed gel, (D) 18.0 ␮mol/ml packed gel, (E) 22.0 ␮mol/ml packed gel, (F) 41.6 ␮mol/ml packed gel. For further details, see Section 2, legend to Fig. 1, Table 3 and the text.

coefficients of determination (r2 ) indicates that the data obtained by logistic regression is somewhat more reliable than the data obtained by linear regression of logarithmic data. The critical hydrophobicities and half-maximal saturation values obtained from the sigmoidal curves are summarized in Table 4. The half-saturation values decrease from 48 ␮mol for Seph-C4 to 8 ␮mol for Seph-C6. Interestingly the ratio s(res) s(res) C crit,LC /C 0.5,LC is ca. 0.6 in the three cases indicating that the estimation of critical hydrophobicity was realistic.

Table 4 S(res) Apparent critical hydrophobicities (C crit,LC ) and half-maximal saturation values (C 0.5,LC ) for fibrinogen obtained from the sigmoidal immobilized residue concentration curves in Figs. 5–7a S(res)

Alkyl Sepharose

C crit,LC ␮mol/ml packed gel

C 0.5,LC ␮mol/ml packed gel

4.4. Cosolvent promoted fibrinogen binding on critical hydrophobicity supports

Seph-C4 Seph-C5 Seph-C6

31 14 4–5

48 22 8

As demonstrated above it was not the aim to synthesize a gel of high binding capacity for fibrinogen but just the opposite, a

a The values are rounded to the next integer. As described in the text S(res) (KS,0.5 )−1 = C 0.5,LC . The data for Seph-C5 was obtained from Fig. 7A. The critical hydrophobicity for Seph-C6 is estimated.

S(res)

S(res)

H.P. Jennissen, A. Demiroglou / J. Chromatogr. A 1109 (2006) 197–213

205

Fig. 4. Quantitative hydrophobic interaction chromatography of fibrinogen on a surface concentration series of Seph-C6 at low salt concentration. Purified bovine fibrinogen (1 mg) was applied in 1 ml according to the “limited sample load” (LSL) method to a column (12 cm × 0.9 cm) containing 2 ml of packed gel in buffer C. The column was the washed with 15 ml buffer C and then eluted with 7.5 M urea (see arrows). Flow was by gravity. Fractions of 1.5 ml were collected. The following immobilized residue concentrations were employed: (A) 1.9 ␮mol/ml packed gel, (B) 4.3 ␮mol/ml packed gel, (C) 7.7 ␮mol/ml packed gel, (D) 11.6 ␮mol/ml packed gel, (E) 17.2 ␮mol/ml packed gel, (F) 42.0 ␮mol/ml packed gel. For further details, see Section 2, legend to Fig. 1, Table 3 and the text.

non-adsorbing gel. This gel is unusual however since it is just on the brink of adsorbing fibrinogen. An increase in salt or cosolvent concentration at the critical hydrophobicity point should thus lead to a strong increase in protein adsorption. A neutral salt of intermediate salting-in and salting-out property was therefore selected from the Hofmeister series (see refs. [3,4]). An ideal candidate is NaCl, since it does not lead to protein aggregation, as the strongly salting-out (NH4 )2 SO4 , nor does it lead to an unfolding or denaturation of proteins, as the strongly salting-in NaSCN does. As shown in Fig. 8, an enhancement of the NaCl concentration leads to an increase in gel capacity for fibrinogen. At ∼ 705 mM NaCl the 50% value for 1 mg of fibrinogen is reached. 100% adsorption is found at 1.6 M NaCl. In addition to quantitative elution by low salt concentrations (0.15 M NaCl)

fibrinogen can also be eluted with 7.5 M urea with recoveries of 90–100%. Thus, a 1.5–1.6 M NaCl concentration is ideal for hydrophobic interaction chromatography of bovine fibrinogen on critical hydrophobicity-Seph-C5 (CH-Seph-C5, Fig. 8). However, other cosolvents can substitute for the Hofmeister salt in promoting adsorption at the critical hydrophobicity. Such a cosolvent is glycine. As shown in Fig. 9, glycine promotes adsorption to CH-Seph-C5 in a similar fashion as NaCl. In this case adsorption begins at 0.5 M glycine and is complete (100%) at 1.5 M glycine. This example clearly demonstrates that the ionic strength of the solution is not critical in the cosolvent mediated fibrinogen adsorption on the critical hydrophobicity support (CH-support). In agreement with zwitterionic chemistry conductivity measurement of a 1 M solution of glycine shows a

206

H.P. Jennissen, A. Demiroglou / J. Chromatogr. A 1109 (2006) 197–213

Fig. 5. Sigmoidal immobilized residue concentration curves of the adsorption of fibrinogen on homologous alkyl Sepharoses at low salt concentrations. The data was calculated from the experiments shown in Figs. 2–4. One hundred percent equals to 1 mg purified bovine fibrinogen adsorbed to 2 ml of packed gel of the designated chain length. The amount of adsorbed fibrinogen was not corrected for the amount adsorbed to unsubstituted Sepharose 4B (∼ 0.1 mg/ml packed gel). The horizontal line corresponds to half-maximal saturation and the arrows to the approximate critical hydrophobicities. For critical hydrophobicities S(res) s(res) (C crit,LC ) and half-saturation constants (C 0.5,LC ) see Table 4. For further details, see Section 2, Figs. 2–4, Table 3 and the text. (
) Sepharose 4B control, () Seph-C4 series, () Seph-C5 series and (䊉) Seph-C6 series.

325-fold lower conductivity (62 ␮S/cm, pH 7.0) than a 1 M NaCl solution (20.2 mS/cm, pH 6.8). Thus, the absolute concentration of the cosolvent NaCl or glycine is decisive for the promotion of fibrinogen adsorption to CH-Seph-C5 not the ionic strength. As a rule the minimum cosolvent concentration should be employed which leads to 100% adsorption of the protein sample on the critical hydrophobicity support.

Fig. 7. Analysis of the sigmoidal binding data of fibrinogen to Seph-C5 according to Eq. (1). The sigmoidal binding curve of fibrinogen to the Seph-C5 concentration series of Fig. 6 was fitted to Eq. (1) by two methods (A) nonlinear logistic regression of data in semi-logarithmic form [35,36] or (B) linear regression of data in double logarithmic form [37,38]. The constants of Eq. (1) S(res) determined by logistic regression as shown in (A) are: ns = 10.4 ± 3.1, C 0.5,LC = 2 21.5 ± 1.0 ␮mol/ml packed gel, r = 0.98. For the calculation the bottom plateau value was set to 0% and the top plateau value to 100%. The constants of Eq. (1) determined by linear regression as shown in (B) are: nS = 5.1 ± 1.1, S(res) C 0.5,LC = 25.3 ± 1.1 ␮mol/ml packed gel, r2 = 0.92. As described above the reciprocal of (KS,0.5 )−1 corresponds to C 0.5,LC . The double logarithmic spacing in (B) is identical on both axes illustrating the high increment of 5.1. The constants are shown ±SEM. S(res)

4.5. Critical hydrophobicity HIC of fibrinogen on Seph-C5 (13.2 µmol/ml packed gel)

Fig. 6. Determination of the critical hydrophobicity of Seph-C5 for purified bovine fibrinogen and comparison of the adsorption and elution by 7.5 M urea. The amount of adsorbed or eluted fibrinogen was calculated from the experiments shown in Fig. 3 according to mass balance. For determination of the critical hydrophobicity an additional gel was synthesized (13.6 ␮mol/ml packed gel). One hundred percent equals to 1 mg purified bovine fibrinogen adsorbed to 2 ml of packed gel of Seph-C5. At 40 ␮mol/ml packed gel 20% of the fibrinogen cannot be eluted with 7.5 M urea (emergence of very high affinity sites). For further details see Section 2, Fig. 5, Tables 3 and 4 and the text. (䊉) Adsorbed amount of fibrinogen and () eluted amount of fibrinogen (run-through + 7.5 M urea).

4.5.1. Human fibrinogen 4.5.1.1. Adsorption-elution characteristics of purified human fibrinogen. Critical hydrophobicity HIC in this case involves cosolvent-mediated adsorption and negative gradient elution of a protein on a critical hydrophobicity support. Fig. 10A shows the critical hydrophobicity HIC of 55.4 mg of commercial purified human fibrinogen (clottability 85%) on 20 ml packed Seph-C5 of 13.5 ␮mol/ml packed gel. In the peak fractions of the eluate a clottability of 98–100% (Fig. 10B) is obtained with the pool showing 95–96% clottability. Of the 55.4 mg fibrinogen applied to the column 48 mg are eluted by the negative salt gradient corresponding to a 1.12-fold purification, a protein recovery of 86% and a fibrinogen recovery (see clottability) of ca. 97%,

H.P. Jennissen, A. Demiroglou / J. Chromatogr. A 1109 (2006) 197–213

Fig. 8. Determination of the NaCl cosolvent/salt parameter for fibrinogen adsorption on the critical hydrophobicity Seph-C5 support. Purified bovine fibrinogen (1 mg) was applied in 1 ml according to the “limited sample load” (LSL) method to a Seph-C5 (13.6 ␮mol/ml packed gel) column (0.9 cm × 12 cm) containing 2 ml of packed gel in 50 mM Tris–HCl, 1 mM EGTA, pH 7.4 (= buffer G). The concentration of NaCl added to buffer G is indicated in the figure. For quantification elution is facilitated by 7.5 M urea (arrow 1), H2 O (arrow 2) and 1% SDS (arrow 3). (A) Control: buffer G alone, (B) + 150 mM NaCl, yield 106%, (C) + 759 mM NaCl, yield 97.3%, (D) + 1650 mM NaCl, yield 93.3%. The yields are given for elution with 7.5 M urea. For determination of critical hydrophobicity see Fig. 5. For further details, see Section 2 and the text.

demonstrating that no significant loss or denaturation of fibrinogen occurred. The separated protein peak in the run-through corresponds to HSA contained in this commercial fibrinogen preparation. Thus, the yield of ca. 97% demonstrates that the

207

Fig. 9. Determination of the glycine cosolvent/salt parameter for fibrinogen adsorption on the critical hydrophobicity Seph-C5 support. Purified bovine fibrinogen (1 mg) was applied in 1 ml of equilibration buffer according to the “limited sample load” (LSL) method to a to a Seph-C5 (13.6 ␮mol/ml packed gel) column (12 cm × 0.9 cm) containing 2 ml of packed gel equilibrated with buffer containing 50 mM Tris–HCl, 1 mM EGTA, pH 7.4. The concentration of glycine added to the equilibration buffer is indicated in the figure. For quantification elution is facilitated by 7.5 M urea (arrow 1), H2 O (arrow 2) and 1% SDS (arrow 3). (A) + 150 mM glycine, yield 105%, (B) + 500 mM glycine, yield 102%, (C) + 1000 mM glycine, yield 93.4%, (D) + 1500 mM glycine, yield 112%. The yields are given for elution with 7.5 M urea. For determination of critical hydrophobicity, see Fig. 5. For further details, see Section 2 and the text.

208

H.P. Jennissen, A. Demiroglou / J. Chromatogr. A 1109 (2006) 197–213

Fig. 10. Critical hydrophobicity HIC of commercial purified human fibrinogen (Haemocomplettan) on Seph-C5 (13.6 ␮mol/ml packed gel). A sample of 2.1 ml (26.4 mg/ml) commercial purified human fibrinogen (clottability 85%), dialyzed against buffer D was applied to a column (1.6 cm I.D. × 10 cm) filled with 20 ml packed pentyl Sepharose (13.6 ␮mol/ml packed gel) in buffer D (1.5 M NaCl). The flow rate was ca. 70 ml/h by peristaltic pump and fractions of 8 ml were collected. A small peak of HSA is eluted in the run-through. The adsorbed fibrinogen (∼3 mg/ml packed gel) was eluted in a single step with buffer C which leads to a steep negative gradient. The peak fractions of the eluted fibrinogen had a clottability of 96–100%. At fraction, 35 step elution was concluded with pure H2 O. No further protein was eluted. (䊉) NaCl gradient, () protein determined at 280 nm, () fibrinogen concentration and (
) clottability.

Fig. 11. Purification of fibrinogen from human plasma equilibrated with 1.5 M NaCl by critical hydrophobicity HIC. A sample of 16 ml (53.8 mg/ml) human plasma to which NaCl (1.35 g) in solid form was added and applied to a column (11.6 cm gel height. ×1.4 cm I.D.) filled with ∼20 ml packed pentyl Sepharose (13.6 ␮mol/ml packed gel) in buffer D (1.5 M NaCl). The flow rate was ca. 52 ml/h by peristaltic pump and fractions of 8.6 ml were collected. A large peak of serum proteins is eluted in the run-through. The adsorbed fibrinogen was eluted by a linear gradient (vol. 155 ml, 2 h) generated from buffer D and buffer C by an Ultrograd (LKB) gradient mixer. Fibrinogen eluted at NaCl concentrations of 1205–380 mM. The peak fractions of the eluted fibrinogen had a clottability of 92–97%. The total yield was 82%. The fractions 29–33 were pooled. At fraction 38 step elution was continued with pure H2 O and then elution was concluded with 7.5 M urea. For further details, see Section 2, Table 5, part A and the text. (䊉) NaCl gradient and () protein determined at 280 nm.

binding of purified fibrinogen to the critical hydrophobicity gel at 1.5 M NaCl is fully reversible. 4.5.1.2. One-step purification of fibrinogen from high-salt human plasma. After the successful chromatography of purified human fibrinogen on CH-Seph-C5 human plasma in 1.5 M NaCl was applied to the gel. As can be seen in Fig. 11A, fibrinogen is selectively adsorbed. A large symmetrical protein peak can be

Table 5 Purification tables for the preparation of fibrinogen from human plasma by critical hydrophobicity HIC at high (1.5 M, A.) and low (0.15 M, B.) initial NaCl concentration in the blood plasmaa Step

Volume (ml)

Fibrinogen (280 nm)

Protein (Lowry)

Clottability (%)

Formed fibrin (mg/ml)

Total fibrinogen (mg)

Concentration (mg/ml)

Total protein (mg)

Purification (n-fold)

Fibrinogen yield (%)

A Plasma (high salt) Runthrough HIC-eluate

16 60 52

8.9 2.0 96.0

4.8 0.28 0.95

76.6 16.6 46.9

53.8 13.8 1.0

861 828 52

1 – 11

100 21 61

Plasma (low salt) Runthrough HIC-eluate

20 37 10.6

4.8 2.3 96.0

3.10 0.61 1.44

62.0 22.6 15.3

65.0 27.1 1.5

1300 1002 16

1 – 20

100 38 25

B

a

The column was equilibrated with 1.5 M NaCl in both cases. For further details, see Section 2, Figs. 11 and 12 and the text.

H.P. Jennissen, A. Demiroglou / J. Chromatogr. A 1109 (2006) 197–213

209

eluted by a negative gradient. Clottablility analysis (Fig. 11B) demonstrates a high activity of fibrinogen in the eluted fractions. The purification table (Table 5, part A) illustrates the quantitative relationships. Pooling of the fractions 29–33 resulted in a fibrinogen pool with a clottability of 95–96% and a yield of 61%. A 10-fold purification is achieved (compare Table 1). Subsequent elution with H2 O and 7.5 urea elutes two additional large protein peaks from the column illustrating the specific elution of fibrinogen through the negative salt gradient. Thus, 40–50 mg of high purity fibrinogen free of factor XIII (not shown) can be easily purified from 20 ml human plasma in one step within ca. 3 h. The reason for the lower yield in comparison to the chromatography of purified fibrinogen in Fig. 10 is probably due to the heterogeneity of fibrinogen with one major species preferentially adsorbed (Table 6). 4.5.1.3. One-step purification of fibrinogen from low-salt human plasma. In a time-saving procedure fibrinogen can also be purified from human plasma in lower yields directly. This is shown in Fig. 12A. Since the NaCl decreases initially due to the low salt concentration in plasma, a significant percentage of the fibrinogen runs through. The fibrinogen eluted by a negative salt step-gradient is electrophoretically pure with a clottability of 96–97% (Fig. 12B). The price for this time-saving method is however the yield which is ca. 25%. Subsequent elution with H2 O and 7.5 M urea again washes two additional large protein peaks from the column. A small amount of fibrinogen is also eluted in the H2 O step. The purity of fibrinogen by SDS-PAGE criteria as shown in the SDS-PAGE of Fig. 13A, the A␣B␤␥ subunits of fibrinogen can be detected in high purity (lane1) on the gel, comparable to that of commercial fibrinogen preparations (lane 4). The clotting experiment with thrombin (lane 2) demonstrates that there is no factor XIII present to crosslink the subunits to higher molecular mass species. Quantitative evaluation is shown in Table 5, part B. In this case a 20-fold purification with a 25% yield is achieved. This described method is ideal for obtaining 10–20 mg of highly pure, fresh fibrinogen from 20 ml of plasma in ca. 1 h. 4.5.2. Bovine fibrinogen 4.5.2.1. One-step purification of fibrinogen from high-glycine bovine plasma. Since the same high degree of purity as obtained for human fibrinogen could not be procured for bovine fibrino-

Fig. 12. Purification of fibrinogen from human plasma without 1.5 M salt addition by critical hydrophobicity HIC. A sample of 20 ml (65.0 mg/ml) human plasma was applied directly to a column (13 cm gel height × 1.4 cm I.D.) filled with ∼ 20 ml packed pentyl Sepharose (13.6 ␮mol/ml packed gel) in buffer D (1.5 M NaCl). The flow rate was ca. 70 ml/h by peristaltic pump and fractions of 6.0 ml were collected. A large peak of serum proteins is eluted in the runthrough. The adsorbed fibrinogen was eluted by a step gradient by changing the buffer from D to C (arrow 1). The peak fractions of the eluted fibrinogen had a clottability of 92–98%. The clottability of fraction 33 (98%) is the mean of two determinations. The total yield was 63%. The fractions 31–33 were pooled. At fraction 38 (arrow 2) step elution was continued with pure H2 O and then (arrow 3) elution was concluded with 7.5 M urea. For further details, see Section 2, Table 5, part B and the text. (䊉) NaCl gradient and () protein determined at 280 nm.

gen with NaCl the amino acid glycine was utilized as cosolvent (see Fig. 8). In a similar operational setup as in the previous runs bovine plasma containing 1.5 M glycine was applied to CH-Seph-C5 and pure fibrinogen (Fig. 14A) displaying a mean clottability of 96% (Fig. 14B) was eluted by a negative glycine

Table 6 Comparison of the subunit molecular masses of human and bovine fibrinogens of various sources as determined by SDS-PAGE on one polyacrylamide gela Subunits

A␣ A␣ B␤ ␥

McKee et al. [10]

Fbg Behring

Fbg. Sigma

Fbg-this paper

Human

Human

Human

Bovine

Human CHIC

Bovine classic

Bovine CHIC

73.0

71.9

66.1

73.8

60.5 51.4

72.7 69.9 60.2 51.5

66.5

60.0 53.0

71.0 68.6 57.8 50.3

62.6 52.9

63.9 53.4

60.7 51.4

a All experimental molecular masses were obtained from one SDS-PAGE gel (10%) according to Laemmli after laser densitometry employing the protein standards described in Section 2.2. The molecular masses correspond to the posttranslationally modified polypeptide chains. The data of McKee et al. [10] are included as literature standard. All other values were determined within the scope of this paper. For further details, see Figs. 1, 13 and 14 and the text.

210

H.P. Jennissen, A. Demiroglou / J. Chromatogr. A 1109 (2006) 197–213

Fig. 13. SDS-PAGE of CHIC-purified fibrinogen from human (A) and bovine (B) blood plasma. (A) Fibrinogen purified by critical hydrophobicity HIC (pooled fractions 31–33, clottability ∼96% from Fig. 10) was analyzed by SDS-PAGE on a 10% polyacrylamide gel according to [34]. Furthermore, this fibrinogen was clotted by addition of thrombin. In case there is a contamination by factor 13 high molecular mass bands should occur. No such bands were detectible indicating a highly pure fibrinogen. For further details, see Section 2, Fig. 10 and the text. Lanes: 1 = HIC-Elution Pool (12 ␮g); 2 = HIC-Elution Pool (12 ␮g) + thrombin (lane 1); 3 = MW protein-standards; 4 = human fibrinogen (Behring, 12 ␮g); 5 = human fibrinogen (Behring, 12 ␮g) + thrombin. (B) Bovine fibrinogen was purified by critical hydrophobicity HIC (see Fig. 14) and is compared to fibrinogen purified by classical means (see Table 1). Lanes: 1 = bovine fibrinogen purified according to Laki (Table 1) 12 ␮g, subunit molecular masses: A␣ 65.8 kDa, B␤ 62.5 kDa, γ 54.8 kDa; 2 = molecular weight standards 40 ␮g; 3 = bovine fibrinogen purified by critical hydrophobicity HIC (Fig. 13) 14 ␮g, subunit molecular masses: A␣ 70.5 kDa, B␤ 63.3 kDa, γ 55.0 kDa.

gradient. Pooling of the peak fractions 24–27 yields a clottability of 96% with a yield of 40% (41% in the run-through). The overall recovery was 81%. However, as shown by SDS-PAGE in the insert to Fig. 14B the A␣-subunit obtained by the HIC procedure has a higher molecular mass (70.5 kDa) than expected from the classically purified fibrinogen (Table 1, Fig. 1) with an A␣-subunit molecular mass of 65–66 kDa. Fig. 13B shows a comparison of classical and HIC purified fibrinogen. It can be

Fig. 14. Purification of bovine fibrinogen by critical hydrophobicity HIC with a negative glycine gradient. Bovine plasma (see above) to which glycine had been added to a final concentration of 1.5 M adjusted to pH 7.4 was applied to 20 ml packed gel Seph-C5 (13.6 ␮mol/ml packed gel) in a column (1.4 cm × 13 cm) equilibrated with buffer E. The column was then washed with buffer E (1.5 M glycine, k = run-through, Fractions 2–12) and eluted with a negative salt gradient to the concentrations of buffer F (150 mM glycine). The fraction volume was ∼6.3 ml. An amount of ∼12 mg purified fibrinogen was eluted by a negative salt gradient. The total yield was 81%. For further details see Fig. 8, Section 2.2 and the text. (A) Protein by optical density measurement at 280 nm, glycine gradient. (B) Fibrinogen clottability. Insert: SDS-PAGE (10%) of fractions 23–27. The fibrinogen eluted in the experiment of Fig. 13 was analyzed by SDS-PAGE (10% gel) according to Laemmli. Lanes: M = protein standards, 40 ␮g; 25 = fraction 25, 9 ␮g; 23 = fraction 23, 10 ␮g; 26 = fraction 26, 10 ␮g; 24 = fraction 24, 9 ␮g; 27 = fraction 27, 9 ␮g. The mean molecular masses (±SD) calculated from the five fractions are: A␣ 71 ± 0.2 kDa, B␤ 63 ± 0.9 kDa, γ 55 ± 0.6 kDa, respectively.

clearly seen that the difference in molecular mass is outside the range of molecular mass variance. 5. Discussion In this paper with highly purified bovine fibrinogen on uncharged alkylagaroses we show (Figs. 5 and 6) by the limited sample load method that fibrinogen binding is a sigmoidal function of the immobilized residue concentration for SephC4, Seph-C5 and Seph-C6. The sigmoidal curves demonstrate the multivalent molecular mechanism of binding for fibrinogen which involves a threshold—the critical hydrophobicity. The critical hydrophobicity corresponds to the critical surface concentration of immobilized alkyl residues constituting a geometric and sterical configuration facilitating the adsorption of

H.P. Jennissen, A. Demiroglou / J. Chromatogr. A 1109 (2006) 197–213

fibrinogen. The enhancement of fibrinogen binding is due to an increase in binding affinity as a result of the increase in the number of alkyl residues interacting with hydrophobic patches on the surface of the protein molecule. This increase in affinity as a function of the immobilized residue concentration has been called a positive cooperativity [37,44,47] of the latticesite binding function. The analysis of fibrinogen binding to Seph-C5 (Fig. 7) is in full agreement with this model [5,31,33] yielding lattice-site adsorption coefficients of 5–10 as a minimum number of lattice-sites interacting cooperatively with the fibrinogen molecule above the critical hydrophobicity in the adsorption process at low ionic strength. The alkyl residue lattice on the agarose surface therefore constitutes a molecular recognition structure forming the basis for the selectivity of the binding interaction (see [5]). It is therefore very plausible that if one takes the critical hydrophobicity as the point-of-operation for hydrophobic interaction chromatography an increase in the cosolvent concentration (Figs. 8 and 9) pushes adsorption of fibrinogen over the threshold of binding, which is observed as an increase in the gel capacity (Figs. 2–6). It cannot be excluded that the increase in salt concentration also leads to a limited conformational change of fibrinogen. Such changes however appear small since the partial specific volumes of proteins generally remain constant in the employed cosolvents [49,50]. The critical hydrophobicity gel determined from the sigmoidal curves (e.g. Seph-C5, 13.6 ␮mol/ml packed gel, Fig. 6) could then be employed for the purification of human and bovine fibrinogen from blood plasma. In a positive control experiment with purified fibrinogen (Fig. 10) ∼53 mg was applied and adsorbed to 20 ml packed gel giving it a capacity for fibrinogen of ∼3 mg/ml packed gel. This capacity seems low in the light of capacities for ion exchangers [51] or those published for commercial HIC gels which deem acceptable only if values of 20–30 mg/ml packed gel or above are obtained. However, it should be considered that such high capacities for HIC gels only apply to special cases. The working capacity of HIC gels is determined by their negative cooperative properties. For example, from the adsorption isotherms of phosphorylase b on Seph-C4 saturation capacities of 35–45 mg/ml packed gel at low and high salt concentrations have been determined [37]. Thus, Seph-C4 has a very high capacity for phosphorylase b—so it seems. To get near this saturation point of the gel however, protein concentrations in the range of 5–10 mg/ml are required, since the affinity of the gels decreases as it is saturated. As the concentration of phosphorylase b in a crude extract is only ca. 0.05 mg/ml the working capacity is never more than a couple of mg/ml packed gel. A similar reasoning can be applied to fibrinogen as shown here. Other parts of the gel capacity are taken up by the contaminating proteins in the extract or plasma, as shown in Figs. 10, 11 and 14 by elution with pure water or 7.5 M urea. Often proteins such as antibodies are adsorbed from ammonium sulphate solutions just below the precipitation concentration (e.g. 1.0–2.0 M) to obtain high capacities, e.g. on agarose based or polystyrene based HIC gels [52]. One should keep in mind however, that under such salting-out conditions proteins can be non-specifically adsorbed even to unsubstituted,

211

i.e. hydrophilic Sepharose 4B in amounts up to 40–45 mg/ml packed gel [53] (for review, see [4]). In other words, at such high ammonium sulphate concentrations, many proteins will adsorb to any gel. Therefore, such “mixed capacities” cannot readily be compared with capacities obtained on classical HIC gels in the presence of NaCl or glycine as employed in this paper. On the other hand, dynamic binding capacities of HIC gels at critical hydrophobicities are of high interest and are presently under study in our group. One of the major results of the described experiments is that the critical hydrophobicity properties of an alkyl Sepharose towards a protein can be exploited as the point-of-operation for purification procedures. As demonstrated in this paper fibrinogen can be purified in a single step from bovine and human blood plasma to homogeneity (96–98% clottability). It might be said that the purification of a protein from plasma 10–20fold is trivial. However, as demonstrated in Table 1 the classical purification of a protein 10–20-fold in substantial amounts can take one man-week. The purification of fibrinogen by critical hydrophobicity HIC (Table 5) on the other hand takes only a few hours and in a simplified procedure, where the yield is not decisive, only 1 h. A 10-fold upscale of the procedure in Fig. 11 (Table 5, part A) will lead to the same amount of fibrinogen (∼500 mg) as the classical procedure. Another major advantage of the rapidity of critical hydrophobicity HIC is the preservation of native protein structure. In the course of a week proteolytic changes can take place altering the protein primary structure or possibly also posttranslational modifications. Thus, the protein with is isolated may not correspond to the original protein in the extract. In the case of human fibrinogen it appears that the danger of structural changes is not very great. However, in the case of bovine fibrinogen we could show that the A␣ subunit of fibrinogen purified by critical hydrophobicity HIC is ca. 4–5 kDa larger than the A␣ subunit found in fibrinogen purified by the classical procedure (see Fig. 13B). At present, the cause of these differences in molecular mass is unclear. In this connection it is of interest that the genetic molecular mass of the A␣ subunit of fibrinogen has not been unequivocally determined. In the Swiss-Prot Database [54], the C-terminal amino acid of the fibrinogen ␣-chain (FIBA bovin P02672) is stated as unknown. Thus, it may very well be that the actual length of the bovine fibrinogen ␣-chain is larger than the presently known sequence (596 AA, 65,006 Da). Alternatively the fibrinogen ␣-chains might differ in their carbohydrate content lending them a different electrophoretic mobility in SDS-PAGE. It is well known that physiological plasma fibrinogen is heterogenous [55,56] with molecular species containing various amounts of phosphate [57], sulfate [58], sialic acid [59] and glycosyl residues [60]. Thus, if a certain species of the heterogenous fibrinogen plasma pool would be preferably adsorbed to the critical hydrophobicity support then an alteration of the apparent subunit molecular mass would be plausible. These possibilities cannot be excluded at present, since ca. 40% of the applied fibrinogen in Fig. 14 is in the run-through (not shown), due to a slight overloading of the gel. It has been shown that purified human fibrinogen is eluted from the CH-Seph-C5 gel with a yield of 97%. However, in the

212

H.P. Jennissen, A. Demiroglou / J. Chromatogr. A 1109 (2006) 197–213

purification of human fibrinogen from plasma only yields of ca. 60% were obtained. This discrepancy, which we generally found with fibrinogen, may also be explained by the above mentioned heterogeneity of fibrinogen in plasma. It may thus be that certain species of fibrinogen are preferentially adsorbed to the CH-gel, whereas other species run through. As shown in Figs. 8 and 14, glycine can substitute as cosolvent for NaCl within the same concentration range. These cosolvents have similar but not identical properties, since bovine fibrinogen purified with glycine buffer showed a higher purity than the NaCl purified protein. Glycine has been suggested as an adjuvant to HIC [61] in the purification of monoclonal antibodies, since it could not promote retention independently. In this paper we show that glycine is just as effective as NaCl in independently promoting the adsorption of fibrinogen to a CHSeph-C5 support. In other work it has been shown [62] that glycine stabilizes protein structure and that the major effect of the salt NaCl [49,63] and the cosolvent glycine [50] is a preferential hydration of proteins. This cosolvent mediated increase in the protein hydration shell would offer an explanation for the increase in the affinity (i.e. free enthalpy of interaction of hydrophobic bonding) of the fibrinogen-CH-support interaction, since more molecules of the ordered water shell of the protein could be shifted to unordered water (>S) upon hydrophobic binding. Acknowledgements This work was supported by a grant of the Bundesministerium f¨ur Forschung und Technologie to H.P.J. (F¨orderkennzeichen: 07024610). We thank Professor Dr. G. M¨uller-Berghaus (MPI Forschungsgruppe f¨ur H¨amostasiologie, Kerckhoff Klinik, Bad Nauheim) for instructing us in the classical method of fibrinogen preparation and analysis. We thank Professor Dr. D. Paar (Klinische Chemie & Laboratoriumsdiagnostik, Universit¨atsklinikum Essen) for immunological determinations of fibrinogen and determinations according to Claus [30]. For stimulating discussions on CHIC applications we thank Associate Professors Lars Hagel and James van Alstine (both GE Healthcare Bio-Sciences AB). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12]

H.P. Jennissen, J. Chromatogr. 159 (1978) 71. H.P. Jennissen, L.M.G. Heilmeyer Jr., Biochemistry 14 (1975) 754. H.P. Jennissen, Int. J. Bio-Chromatogr. 5 (2000) 131. H.P. Jennissen, in: M.A. Vijayalakshmi (Ed.), Biochromatography, Taylor and Francis, London, New York, 2002, pp. 47–71. H.P. Jennissen, Methods Mol. Biol. 305 (2005) 81. M. Laub, H.P. Jennissen, Biophys. J. 88 (2005) 46a. W.G. Hanstein, J. Solid Phase Biochem. 4 (1979) 189. R.F. Doolittle, Ann. Rev. Biochem. 53 (1984) 195. R. Virchow, Arch. Pathol. Anat. Physiol. (Berlin) 1 (1847) 547. P.A. McKee, P. Mattock, R.L. Hill, Proc. Natl. Acad. Sci. USA 66 (1970) 738. C. Cohen, J.W. Weisel, G.N. Phillips Jr., C.V. Stauffacher, J.P. Fillers, E. Daub, Ann. NY Acad. Sci. 408 (1983) 194. J.H. Brown, N. Volkmann, G. Jun, A.H. Henschen-Edman, C. Cohen, Proc. Natl. Acad. Sci. USA 97 (2000) 85.

[13] B. Blombaeck, M. Blombaeck, Ark. Kemi 10 (1956) 415. [14] R.A. Kekwick, M.E. Mackay, M.H. Nance, B.R. Record, Biochem. J. 60 (1955) 671. [15] E.J. Cohn, L.E. Strong, W.L. Hugles Jr., D.J. Mulford, J.N. Ashworth, M. Melin, H.L. Taylor, J. Am. Chem. Soc. 68 (1946) 459. [16] K. Laki, Arch. Biochem. Biophys. 32 (1951) 317. [17] F.R. Matthias, G. Hocke, H.G. Lasch, Thromb. Res. 7 (1975) 861. [18] K. Suzuki, J. Nishioka, S. Hashimoto, Thromb. Res. 18 (1980) 707. [19] M. Matsuda, S. Iwanaga, S. Nakamura, Thromb. Res. 1 (1972) 619. [20] A. Demiroglou, H.P. Jennissen, Biol. Chem. (formerly: Hoppe-Seyler’s Z. Physiol. Chem.) 371 (1990) 778. [21] I. Mahn, G. Mueller-Berghaus, Haemostasis 4 (1975) 40. [22] W.E. Collins, S.L. Cooper, in: Proceedings of the 13th Annual Meeting of the Society of Biomaterials, New York, 2–6 June 1987, 1988, p. 61 (abstract communications). [23] H.P. Jennissen, J. Mol. Recognit. 8 (1995) 116. [24] G.S. Bethell, S. Ayers, W.S. Hancock, M.T.W. Hearn, J. Biol. Chem. 254 (1979) 2572. [25] T. Zumbrink, A. Demiroglou, H.P. Jennissen, J. Mol. Recognit. 8 (1995) 363. [26] H.P. Jennissen, A. Demiroglou, E. Logemann, in: T.C.J. Gribnau, J. Visser, R.J.F. Nivard (Eds.), Studies on the Mechanism of Protein Adsorption on Hydrophobic Agaroses, Elsevier, Amsterdam, 1982, pp. 39–49. [27] H.P. Jennissen, J. Colloid Interface Sci. 111 (1986) 570. [28] K. Jacobsson, Scand. J. Clin. Lab. Invest. 7 (Suppl. 14–17) (1955) 8. [29] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem. 193 (1951) 265. [30] A. Clauss, Acta Haemat. 17 (1957) 237. [31] V. Hlady, J. Buijs, H.P. Jennissen, Methods Enzymol. 309 (1999) 402. [32] H.P. Jennissen, A. Demirolgou, J. Chromatogr. 597 (1992) 93. [33] H.P. Jennissen, J. Chromatogr. 215 (1981) 73. [34] U.K. Laemmli, Nature 227 (1970) 680. [35] H.J. Motulsky, Intuitive Biostatistics, Oxford University Press, New York, Oxford, 1995. [36] H.P. Jennissen, G. Botzet, J. Mol. Recognit. 6 (1993) 117. [37] H.P. Jennissen, Biochemistry 15 (1976) 5683. [38] H.P. Jennissen, G. Botzet, Int. J. Biol. Macromol. 1 (1979) 171. [39] D.H. Everett, Pure Appl. Chem. 31 (1972) 579. [40] I. Mills, T. Cvtas, K. Homann, N. Kallay, K. Kuchitsu, Quantities, Units and Symbols in Physical Chemistry, second ed., Blackwell, Oxford, 1993. [41] A. Demiroglou, H.P. Jennissen, J. Chromatogr. 521 (1990) 1. [42] H.P. Jennissen, Makromol Chem., Macromol. Sympos. 17 (1988) 111. [43] H.P. Jennissen, Ber. Bunsenges. Phys. Chem. (1989) 949. [44] H.P. Jennissen, Biol. Chem. (formerly: Hoppe-Seyler’s Z. Physiol Chem) 357 (1976) 1201. [45] H.P. Jennissen, Adv. Enzyme Regul. 19 (1981) 377. [46] H.P. Jennissen, Biol. Chem. (formerly: Hoppe-Seyler’s Z. Physiol. Chem.) 358 (1977) 255. [47] H.P. Jennissen, Biol. Chem. (formerly: Hoppe-Seyler’s Z. Physiol. Chem.) 357 (1976) 1727. [48] M. Millitzer, E. Wenzig, W. Peukert, Chem. Eng. Technol. 28 (2005) 756. [49] T. Arakawa, S.N. Timasheff, Biochemistry 21 (1982) 6545. [50] T. Arakawa, S.N. Timasheff, Arch. Biochem. Biophys. 224 (1983) 169. [51] E. Karlsson, L. Ryd´en, J. Brewer, in: J.-C. Janson, L. Ryd´en (Eds.), Protein Purification: Principles, High Resolution Methods and Applications, VCH, Weinheim, New York, 1989, pp. 107–148. [52] P. Gagnon, E. Grund, Biopharm 9 (1996) 34. [53] F. von der Haar, J. Solid Phase Biochem. 4 (1979) 207. [54] A. Bairoch, R. Apweiler, C.H. Wu, W.C. Barker, B. Boeckmann, S. Ferro, E. Gasteiger, H. Huang, R. Lopez, M. Magrane, M.J. Martin, D.A. Natale, C. O’Donovan, N. Redaschi, L.S. Yeh, Nucleic Acids Res. 33 (2005) D154.

H.P. Jennissen, A. Demiroglou / J. Chromatogr. A 1109 (2006) 197–213 [55] M.W. Mosesson, Ann. NY Acad. Sci. 408 (1983) 97. [56] A.H. Henschen-Edmann, Ann. NY Acad. Sci. 936 (2001) 580. [57] S.C. Martin, P. Ekman, P.O. Forsberg, H. Ersmark, Thromb. Res. 68 (1992) 467. [58] D.L. Christie, R.M. Hill, K. Isakow, P.M. Barling, Anal. Biochem. 154 (1986) 92. [59] W.P. Gati, P.W. Straub, J. Biol. Chem 253 (1978) 1315.

213

[60] E. Kottgen, B. Hell, C. Muller, R. Tauber, Biol. Chem Hoppe Seyler 369 (1988) 1157. [61] P. Gagnon, http://www.validated.com, 2000, pp. 1–9. [62] B. Schobert, H.P. Jennissen, Biol. Chem. (formerly: Hoppe-Seyler’s Z. Physiol. Chem.) 361 (1980) 329. [63] T. Arakawa, S.N. Timasheff, Biochemistry 23 (1984) 5912.