Atherosclerosis and its complications are the major causes of mortality in industrialized countries [1-3]. Research into the oxidation of lipoprotein has yielded many insights into the process underlying the development of atherosclerosis. Oxidative modification of low density lipoprotein (LDL) has been suggested as an initial step in the pathogenesis of atherosclerosis [4,6]. However, up until now, investigations of antioxidants have focused on three main dietary antioxidant vitamins (β-carotene, vitamin C, and vitamin E) [39-41] and some synthetic compounds [42-44]. Among those antioxidants described above, probucol, a
synthetic compound, has been shown to be an extremely potent and effective antioxidant in preventing against the formation of atherosclerosis in both in vitro and ex vivo studies [42,43].
The present review focuses on commonly used analytical methods for measuring the antioxidant potency and outlines the critical steps as how to evaluate and design a potent antioxidant agent that can be used for the intervention of atherosclerosis. We conclude that an antioxidant should be first targeted and incorporated into human LDL. Second, the candidate compound should possess high bioavailability.
From the atherogenesis process and we evaluated those currently-used and potential antioxidant candidates for preventing the formation of atherosclerosis. The critical consideration in designing a compound that can be effectively used for antioxidant therapy in atherosclerosis are reviewed as 5 sections: 1) The oxidation hypothesis and atherogenesis induced by oxidized LDL; 2) Recent antioxidant therapies for atherosclerosis; 3) potential antioxidants as antiatherosclerotic agents; 4) commonly used analytical methods of antioxidant potency; 5) rational design of a synthetic antioxidant as an antiatherosclerotic agent.
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Section 1: A novel approach for Hp purification
Abstract
Similar to blood type, human plasma haptoglobin (Hp) is classified as 3 phenotypes: Hp 1-1, 2-1, or 2-2. The structural and functional relationship between the phenotypes, however, has not been studied in detail due to the complicated and difficult isolation procedures. This report provides a simple protocol that can be used to purify each Hp phenotype. Plasma was first passed through an affinity column coupled with a high affinity Hp monoclonal antibody. The bound material was washed with a buffer containing 0.2 M NaCl and 0.02 M phosphate, pH 7.4, eluted at pH 11, and collected in tubes containing 1 M Tris-HCl, pH 6.8. The crude Hp fraction was then chromatographed on a HPLC Superose 12 column in 0.05 M ammonium bicarbonate at a flow rate of 0.5 ml/min. The homogeneity of purified Hp 1-1, 2-1, or 2-2 was greater than 95% as judged by SDS polyacrylamide gel electrophoresis. Essentially, each Hp isolated was not contaminated with hemoglobin and apolipoprotein A-I as that reported from the other methods, and was able to bind hemoglobin. Neuraminidase treatment demonstrated that the purified Hp possessed a carbohydrate moiety, while Western blot analysis confirmed α and β chains corresponding to each Hp 1-1, 2-1, and 2-2 phenotype. The procedures described here represent a significant improvement in current purification methods for the isolation of Hp phenotypes. Circular dichroic spectra showed that the α-helical content of Hp 1-1 (29%) was higher than that of Hp 2-1 (22%) and 2-2 (21%). The structural difference with respect to its clinical relevance is discussed.
Keywords: Human haptoglobin 1-1, 2-1, 2-2; Affinity purification; α-helix; Monoclonal
antibodies
Introduction
Hp is known as an acute phase protein, and its plasma level elevates in response to inflection or inflammation. For this reason, Hp is a useful indicator for some infectious diseases [1-3]. It is also a hemoglobin-binding protein present in the plasma of all vertebrates and believed to participate in hemoglobin transport [4].
Human Hp is a tetrameric structure joined by disulfide linkages among the 2 α and 2 β chains [4-6]. Based on the length of α chain, there are three phenotypes of Hp in the population, Hp 1-1, 2-1, and 2-2 (Fig. 1). All the phenotypes share the same 2 β chains (each with about Mr 40,000 dalton containing 245 amino acids and approximately 30% carbohydrate). A typical structure of homozygous Hp 1-1 is composed of two identical α1 chains (each with about Mr 9,000 dalton containing 83 amino acids). Homozygous Hp 2-2 is composed of two identical α2 chains (each with about Mr 16,500 dalton containing 142 amino acids) as compared to that of heterozygous Hp 2-1 containing one each of α2 and α1 (Fig. 1). Likewise, the tetrameric arrangement is also found in other animal species such as rat, rabbit, and pig [7-12]. However the 2 identical αβ units (Hp 1-1), joined by a non-covalent interaction rather than a disulfide
bridge, are found in dog, cat, and bear [13-14].
Clinically, polymeric form of Hp 2-1 or 2-2 is associated with the complications of myocardial infarction [15], kidney failure [16], and diabetics [17]. Presumably, this was due to the complicated structure of Hp 2-1 and 2-2 as it forms heterogeneous polymers, in which some of the biologically functional groups are not fully expressed on the surface (Fig. 1). The assumption, however, has not been tested because the structural and functional studies are hampered by lack of a straightforward isolation procedure in preparing sufficient Hp phenotypes. The methods currently used for the purification of Hp frequently suffer some drawbacks. For example, Rademacher et al. utilize the chicken hemoglobin-Sepharose affinity column to isolate human Hp; the harsh-elution condition (8 M urea) causes the dissociation of a hemoglobin subunit from the Sepharose [18]. Meanwhile, human apolipoprotein A-I appears to be another major contaminant. Wassdal et al. use rabbit hemoglobin-Sepharose; the hemoglobin is still co-eluted from the column [19]. Travis et al. employ Sephadex G-200 gel filtration, but the purified Hp is accompanied with large amounts of IgM and α-2 macroglobulin [20]. Morimatsu et al. provide a modified method using HPLC with anion-exchange, Sephacryl S-300, TSK Phenyl-5PW, and TSK DEAE-5PW columns together; the procedures however are time-consuming, and the yield is relatively low (2.5 mg per 130 ml acute phase serum) [21]. Although Katnik et al. have shown a single-step isolation for Hp using an antibody-affinity column, the phenotypes, final purity, and the biological properties of Hp are
not fully reported [22]. Presumably, the purpose of their report was to use isolated Hp for raising monoclonal antibodies [22]. The similar antibody affinity-column procedure [22] was employed in our laboratory, but the isolated Hp was not pure. In the present report, we established simple two-step procedures for each Hp 1-1, 2-1, and 2-2 purification using a monoclonal antibody affinity-column followed by a HPLC Superose 12 gel filtration. Finally, some of the biochemical and physical properties with respect to each Hp phenotype were characterized and discussed.
Materials and methods
Materials
Goat polyclonal antibody against human Hp was purchased from Sigma (St. Louis, MO, USA). Rabbit anti-Goat IgG was purchased from Chemicon. CNBr-activated Sepharose 4B was purchased from Pharmacia. All other chemicals were purchased from Sigma (St. Louis, MO, USA) and Merck (Darmstadt, Germany) without any further purification. The buffers used in this report were all filtered through a 0.45 μm filter before using.
Preparation of monoclonal antibody against Hp
Six monoclonal antibodies: 8B1-3A, W1-11G, 2-3H, G2D-7G, 12B-1 and 4A2-4H, against human Hp were produced and characterized according to the standard procedures established in
our laboratory [23]. Monoclonal antibody 8B1-3A, which possessed the highest binding affinity to Hp, was selected for preparation of the affinity column. Briefly, 120 ml of cultured medium from the 8B1-3A hybridoma were first precipitated in 50% saturated ammonium sulfate. The precipitate was dissolved in 12 ml of phosphate buffered saline containing 0.02 M phosphate and 0.15 M NaCl, pH 7.4 (PBS). The solution was then dialyzed exhaustively in PBS to remove the remaining ammonium sulfate, followed by a dialysis in coupling buffer containing 0.1 M NaHCO3 and 0.5 M NaCl, pH 8.3.
Preparation of antibody affinity column
Dialyzed monoclonal antibody was first coupled to CNBr-activated Sepharose-4B (Pharmacia, Uppsala, Sweden) according to the manufacturer’s procedures. Briefly, 2.86 g of freeze-dried Sepharose (1 g of freeze-dried powder gave about 3.5 ml final volume of gel) were swollen and suspended in 1 mM HCl and immediately washed with 20x volume of the same solution within 15 min on a sintered glass filter [24-26]. The gel was then washed with coupling buffer containing 0.1 M NaHCO3 and 0.5 M NaCl, pH 8.3, and degassed. About 10 ml (18.7 mg/ml) of ammonium-sulfate fraction of monoclonal antibody in coupling buffer were slowly added into the gel (in 15 ml), while gently stirring by a magnetic bar for 1 h at room temperature. After coupling, the gel was washed 10x volume of PBS to remove unbound materials via a sintered glass filter. The gel was then treated with a blocking solution containing
0.1 M Tris-HCl and 0.5 M NaCl, pH 8.0, for 2 h at room temperature to saturate the remaining reactive-sites. The degassed gel was then washed with 3 cycles of blocking buffer and a 0.15 M NaCl solution of pH 11.0 (adjusted by ammonium) according to the procedures previously described by us [26]. Finally, the gel was equilibrated in PBS and packed onto a 1.5 x 20 cm column.
Purification of human Hp using antibody affinity-column chromatography
Initially, 1 ml of filtered human plasma of each Hp-phenotype batch was loaded onto the antibody affinity-column (10 ml in bed volume) at room temperature. The column was then washed with 50 ml of PBS. The bound materials were further washed with 50 ml of 0.02 M phosphate buffer containing 0.2 M NaCl, pH 7.4, and then eluted with 50 ml of a freshly prepared 0.15 M NaCl solution with pH 11 adjusted by ammonium [26]. Five ml of each fraction was collected in a tube containing 0.25 ml of 1 M Tris-HCl buffer, pH 6.8, to immediately neutralize the pH value. Pooled fractions containing Hp were then concentrated to a final volume of 1 ml using an Amicon centrifugal filter (Millipore).
Further purification of Hp by gel filtration column
Concentrated solution with Hp was filtered with a 0.45 μm nylon fiber prior to HPLC. The HPLC system (Waters) consisted of two pumps, an automatic sample injector, and a
photodiodearray detector. Superose 12 column (1 x 30 cm) (Pharmacia) was used for further Hp purification. The column was pre-equilibrated with 50 mM ammonium bicarbonate.
Partially purified Hp (0.8 ml) was applied to the column at a flow rate of 0.5 ml/min. Fractions containing Hp were pooled and concentrated to a final volume of 1 ml using an Amicon centrifugal filter and then lyophilized. The lyophilized Hp was stored at –80℃ until analyzing.
Gel electrophoresis and densitometry
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the Laemmli’s method [27] with some modification in using 5% polyacrylamide (w/v) on the stacking gel as previously described [7]. Samples (typically 5 μg) for SDS-PAGE were preheated at 100 ℃ for 10 minutes in a loading buffer [12 mM Tris-HCl, pH 6.8, 0.4%
SDS (w/v), 5% glycerol (v/v), 2.88 mM 2-mercaptoethanol, 0.02% bromphenol blue (w/v)].
For molecular weight calibration, a subset of the following standards was included in each gel:
myosin (200 kDa), β-galactosidase (116 kDa), phosphorylase B (97 kDa), serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), lysozyme (14.4 kDa), and aprotinin (6.5 kDa). The samples were run for about 1.5 h at 100 V and stained using Coomassie brilliant blue R-250. Densitometric analysis of SDS-PAGE was performed using a Molecular Dynamics densitometer for data acquisition and Image Quant software for integration and analysis.
Immunoblot analysis
Following the separation of proteins by SDS-PAGE, the gel and nitrocellulose- and 3MM filter- papers were soaked in a transfer buffer containing 48 mM Tris-HCl, 39 mM glycine, 0.037% SDS (w/v), and 20% methanol (v/v) at pH 8.3 for 30 min. The gel was then electrotransferred to a nitrocellulose membrane (Pharmacia) at 90 mA for 1 h in a semi-dry transfer cell (Bio-Rad) containing a transfer buffer. The transferred membrane was then immersed in 5% skim milk (w/v) in PBS for 1 hour at room temperature while shaking gently.
After three times washing with PBS for 5 min, the membrane was incubated with a primary goat polyclonal antibody against human Hp [1:5000 dilution in PBS washing buffer containing 1% (w/v) skim milk and 0.05% Tween-20 (v/v) for 1 hour] at room temperature and washed three times for 5 min. The membrane was then incubated with 1:10,000 diluted rabbit anti-goat IgG conjugated with horseradish peroxidase in washing buffer for 1 h. In addition, the membrane was washed two times with washing buffer and further washed one time with PBS.
Finally, the membrane was developed using 3,3’-diaminobenzidine (DAB) as a substrate for horseradish peroxidase [7, 25].
Circular dichroic spectra
The lyophilized Hp was dissolved in 10 mM phosphate buffer at pH 7.4 with a final
concentration of 0.2 mg/ml. About 300 μl of Hp solution was used to analyze within a cuvette of 1-mm path length. Circular dichroic spectrum was conducted between 190 and 300 nm in a Jasco J-715 spectropolarimetry. The obtained spectrum of each type of Hp was accumulated for 20 times at a scanning rate of 50 nm/min and the % α-helical content was estimated from the mean residue molar elliplicity (θ222). % α-helix = [(θ222 + 3000)/(36000+3000)] X 100 [28].
Results
Preparation of monoclonal antibody against Hp
Six monoclonal antibodies prepared against Hp were characterized, in which 8B1-3A possessed the highest binding affinity (Ka=5.6 x 109 M-1) and was chosen to prepare an affinity column. The binding capacity estimated was greater than 100 μg of Hp per ml of coupled Sepharose (data not shown).
Purification of human Hp using antibody affinity column chromatography
Fig. 2 shows a typical chromatographic profile for Hp 1-1, 2-1, and 2-2 purification on the affinity column. Human plasma was applied to the column followed by an extensive wash with a phosphate buffer containing 0.2 M NaCl. It is worth mentioning, this pre-wash step differed from the conventional method in which 0.12 M NaCl was used. Using 0.2 M NaCl, most of the low-affinity binding proteins were eliminated (Fig. 3). Hp was then eluted at pH 11 and
collected in tubes containing 0.25 ml of 1 M Tris-HCl, pH 6.8, to immediately neutralize the pH.
The purity of each Hp phenotype was approximately 60-80% in homogeneity as analyzed on SDS-PAGE. Apolipoprotein A-I appeared to be a major contaminant. All the phenotypes of Hp converted to α (α1 or α2 or both) and β subunits in the presence of the reducing reagent (Fig.
3). A typical Western blot analysis showing 3 isolated phenotypes is depicted in Fig. 4. The recovery of Hp at this step accounted for 75-94% of the Hp from the plasma with a final of 51-54 fold purification (Table 1).
Further purification of Hp on HPLC gel-filtration column
The obtained Hp 1-1, 2-1, or 2-2 fraction was concentrated and applied onto a gel-filtration Superose 12 column pre-equilibrated with 0.05 M of ammonium bicarbonate, pH 8.0.
Chromatographic profiles (Fig. 5) revealed that the solution property of each Hp phenotype was consistent with its molecular form, in which Hp 1-1 was more homogeneous in size with longer elution time than that of Hp 2-1 and 2-2. Purity of each phenotype was then analyzed on SDS-PAGE containing reducing reagent 2-mercaptoethanol. Homogeneity of each phenotype was greater than 95% (Fig. 6). Thus, HPLC Superose column was markedly effective to remove apoA-I contaminant.
Western blot analysis in the absence of a reducing reagent demonstrated that Hp 2-1 and 2-2 were all polymeric (Fig. 7), in which Hp 2-2 was devoid of monomer and dimer consistent with
the proposed structure of Hp (Fig. 1). Thus, our purification procedures did not apparently alter the structural characteristics of Hp phenotypes.
The polymeric structure of isolated Hp and its binding to hemoglobin
We further studied the ionic property of isolated Hp 1-1, 2-1, and 2-2 on a native-PAGE; the distinct polymorphism of each phenotype was also observed (Fig. 8). Hp 2-2 was the most basic among the Hp phenotypes. Since hemoglobin (Hb) is able to bind Hp and to form a Hb-Hp complex [10], Fig. 8 demonstrates that the Hb could form Hb-Hp complex with each Hp phenotype we isolated.
Circular dichroic spectra
To characterize the secondary structure of each Hp phenotype, we determined the conformation of Hp by CD (Fig. 9). The estimated α-helical content was about 29, 22, and 21%
To characterize the secondary structure of each Hp phenotype, we determined the conformation of Hp by CD (Fig. 9). The estimated α-helical content was about 29, 22, and 21%