Chapter 2 Materials and Methods
2.10. Western blot analysis
Following the separation of proteins by SDS–PAGE, the gel was electrically transfer to a
nitrocellulose-membrane attached with a 3MM filter paper pre-soaked in a transfer buffer
containing 48 mM Tris–HCl, 39 mM glycine, 0.037% SDS (w/v), and 20% methanol (v/v),
pH 8.3. The rest of the procedures for immunoblot was similar to that described previously
[9,10].
RESULTS
In our previous studies, we have described mAb and hemoglobin affinity-column
methods for the purification of human Hp [10,11]. Although these two methods, using PBS
(pH11) as an eluent, were simple; the average Hp recovery was lower than 50% and an
additional chromatographic step was need to remove the contaminated apoA-I. To improve
the recovery and the purity of isolated Hp in the present report, the following steps described
below were conducted.
3.1. Evaluation of dissociation of bound Hp from immobilized mAb
To optimize the elution conditions for the yield of Hp while isolating, we evaluated the
Hp and mAb interaction on an ELISA plate to mimic the Hp binding on mAb affinity column.
Various modified eluting buffers including PBS (pH 11), 4 M urea-PBS (pH 11), and 0.1%
SDS-PBS (pH 11) were studied. Fig. 2 demonstrates that the dissociation of bound Hp from
mAb was not completed using PBS, pH 11. However, the bound Hp was completely
dissociated when using the same PBS buffer containing 0.1% SDS. Buffer containing 4M
urea was only partially effective. The data suggest that SDS with pH 11 could be a useful
reagent for eluting the bound Hp.
3.2 Effect of the concentration of SDS-PBS, pH 11, on the dissociation of bound Hp from
immobilized mAb
To optimize the concentration of SDS capable of dissociating Hp from immobilized mAb,
serial diluted SDS was used for the assay. Fig. 3 shows that the SDS concentrations greater
than 0.08% completely dissociated Hp from the mAb. Since the mAb was immobilized on
the plate as a capture, it is worth mentioning that SDS at 0-2.5% did not cause the dissociation
of immobilized mAb from the plate (data not shown).
3.3. Initial purification of Hp1-1, 2-1, and 2-2 on immunoaffinity chromatography
Fig. 4 shows the optimal conditions for the recovery of Hp phenotypes from the column.
Overall, the Hp yield using SDS-PBS, pH 11 as an eluant was two times higher than that
using PBS, pH 11 (a conventional method) or SDS-PBS, pH 7.4. Interestingly, eluting
buffer of SDS with pH 7.4 delayed the Hp retention time. One of the explanations is that
antibody-antigen interaction is mostly ionic dependent; SDS at neutral pH slowly alters the
conformation of antigen or antibody via a protein-SDS micelle formation [15]. Further
conformational change is induced at high pH resulting in the dissociation of antigen-antibody
complex. Thereby, elution of Hp by SDS combined with pH 11 resulted in high recovery of
Hp 1-1 (71%), 2-1 (68%), and 2-2 (85%) from the column (Table. 1). Nonetheless, the
isolated Hp appeared to contaminate some apoA-I (Fig. 4D).
3.4. Removal of apoA-I contamination
ApoA-I is a major apolipoprotein residued in HDL [16]. It has been known for some
time, apoA-I is a major contaminant in isolated Hp using variety of the methods [9,10]. To
minimize the apoA-I contamination, we evaluated the HDL and Hp interaction on an ELISA
plate immobilized with Hp. Fig. 5A reveals that apoA-I was able to bind Hp, but bound
apoA-I was dissociated from Hp at SDS (pH 7.4) concentrations greater than 0.025%. In the
next experiment, we mimicked the above condition directly on the affinity column. By
SDS-PAGE, concentrations between 0.02 and 0.04% of SDS could wash bound apoA-I away
from the column, but higher concentrations of SDS (≧0.06%) removed both apoA-I and Hp
from the immobilized mAb (Fig. 5B). Likewise, 0.04% SDS-PBS (pH 11) also removed
apoA-I and Hp (data not shown). The data indicate apoA-I can be selectively removed using
0.04% SDS-PBS, pH 7.4, prior to a final elution of Hp.
3.5. Final affinity purification of bound Hp via pre-wash with 0.04% SDS-PBS, pH 7.4
Finally, we used 0.04% SDS as a pre-wash for the removal of bound apoA-I from Hp
over the affinity column. Fig. 6 shows a typical chromatographic profile for the plasma of
Hp 1-1, 2-1, or 2-2. Following a flow through of plasma and washes with a PBS, pH 7.4, the
column was then pre-washed with 0.04% SDS-PBS, pH 7.4 to remove bound apoA-I as well
as other non-specifically bound proteins as fraction 1 (F1). Next, 0.1% SDS–PBS, pH 11
was used to elute bound Hp from mAb as fraction 2 (F2). Fig. 6 exhibits that pre-wash
fraction (F1) had most of apoA-I contaminant as identified by a SDS-PAGE and confirmed by
a Western blot analysis. The homogeneity of final isolated Hp was about 97% (Fig. 6, F2
fraction). The final recovery of isolated Hp 1-1, 2-1, and 2-2 was approximately of 56, 55,
and 71%, respectively (Table 1).
3.6. Analysis of hemoglobin-binding property of isolated Hp 1-1, 2-1, and 2-2
Fig. 7 reveals that final isolated Hp possessed the ability to form complex with Hb using
a 7% native PAGE analysis. Each phenotype formed complex with Hb as a monomer (Hp
1-1) and polymers (Hp 2-1 and 2-2), while Hp 2-1 shared one completely identical monomer
to Hp 1-1.
DISCUSSION
Clinically, Hp phenotypes are found to be related to several inflammatory diseases. For
examples, polymeric form of Hp 2-1 or 2-2 is associated with the complications of myocardial
infarction [17], kidney failure [18], and diabetics [19]. In a prospective study, human
subjects with Hp 2-2 are at a 5-fold increased risk for the development of CAD as compared
to those with Hp 1-1 [20]. The risk in heterozygous Hp 2-1 participates is intermediate [20].
Low levels of Hp are also found among HIV-1 seropositive patients with Hp 2-2 [21]. Due
to the difficult purification procedures for each Hp phenotype, the relationship between the
Hp levels and its affected diseases are rarely reported. One of the most difficulties is to
isolate Hp 2-1 and 2-2 because of their heterogeneity with polymeric molecular forms (Fig. 1).
On the other hand, it is essential to study the structural and functional relationship among the
Hp phenotypes. Previously, Rademacher et al. [22] utilize the chicken
hemoglobin–Sepharose affinity column to isolate human Hp; the harsh-elution condition (8M
urea) causes the dissociation of a hemoglobin subunit from the Sepharose matrix.
Meanwhile, human apoA-I appears to be another major contaminant. Travis et al. [23]
employ Sephadex G-200 gel filtration, but the purified Hp is accompanied with large amounts
of IgM and a-2 macroglobulin. Morimatsu et al. [24] 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.
In our previous studies, we utilized hemoglobin or Hp mAb (prepared against Hp
β-chain;clone 8B1-3A) affinity column to isolate three phenotypes of Hp [10,11]. Both
methods showed that there was a contamination of apoA-I, when eluting Hp by PBS, pH 11.
To remove contaminated apoA-I, a re-chromatography on an HPLC Sephorose-12
gel-filtration was required [11]. For this reason, one major focus of this report was attempt
to minimize the contamination of apoA-I. To test the possibility that plasma HDL (apoA-I
containing lipoprotein) may directly bind to Hp, we immobilized purified Hp on an ELISA
plate followed by the addition of excess amount of HDL to saturate its interaction with Hp, if
any. Most interestingly, we found that HDL could bind Hp (Fig. 5), although the binding
affinity between HDL and Hp was not readily known. Such binding is not non-specific,
since Hp can inhibit the apoA-I-dependent lecithin:cholesterol acyl transferase (LCAT)
activity in vitro [25], which plays a role in the reverse cholesterol transport [26]. The data
suggest that HDL is associated with Hp in plasma, at least in part. Fortunately, the binding
affinity seemed to be differentially lower than that of Hp-mAb. It was why 0.04% SDS-PBS
(pH 7.4) only removed apoA-I from Hp, but not Hp from immobilized mAb (Fig. 5). But,
SDS concentrations ≧ 0.06% or 0.04% SDS-PBS at pH 11 should be avoid for pre-wash as
Hp could come off the column. In an early study [9], we proposed to use HDL depleted
plasma (a bottom fraction obtained by ultracentrifugation at KBr d.1.21 g/ml) for Hp
purification, assuming the contamination of plasma apoA-I could be eliminated. This
experiment was conducted in the present study. The isolated Hp still contained some apoA-I,
but not as much as that of whole plasma (data not shown). Therefore, the use of
HDL-deficient plasma for Hp purification may not be considered in the future.
SDS is used more often than any other detergent as an excellent denaturing or unfolding
reagent [15,27]. It breaks mostly the quaternary and tertiary protein-protien interaction
[28,29]. As such, at low SDS concentration (0.04%), pH 7.4, it dissociates the apoA-I from
Hp. While at 0.1% of SDS, pH 11, it elutes bound Hp from the immobilized mAb over the
affinity column. However, one concern is that SDS may alter the conformation of isolated
Hp. Biswas and Das reported that α-Crystallin was able to refold to native structure after
unfolding by SDS [29]. To address whether isolated Hp could refold closely to its native
conformation, we monitored the structure of Hp 1-1 using a circular dichroic
spectrophotometer. We found SDS-eluted Hp being slightly more disordered than that of Hp
eluted without using SDS. Following extensive dialysis in 4 l PBS with three changes, the
disordered structure, however, refolded to the original alpha-helical content (about 30% helix)
as that values previously reported [11] (data not shown). Furthermore, we have shown that
the formation of Hp-Hb complex is dependent on the overall conformation of Hp [8]. In the
present study, the purified Hp following a dialysis could form complex with Hb (Fig. 7),
suggesting that Hp have refolded to native form. Nevertheless, the purified Hp also retained
its immunochemical property as determined by Western blot and ELISA (data not shown).
Taking together, the present method using SDS as an eluant has certain advantages.
First, Hp can be purified in a predictable way by passing it through the immobilized mAb.
Second, the bound apoA-I (or other non-specially bound proteins) is selectively removed prior
to final elution of Hp. Third, the technique allows isolation of the polymeric form of Hp 2-1
and 2-2 (Fig. 7) unlike standard methods which may significantly lose part of them. Some
standard methods which rely on different molecular masses or charges can distribute the
component to different fractions. Fourth, the simple technique can be used for analytical
purpose, for example, for the determination of polymeric forms of Hp that may be of
important for the investigation of metabolism in pathological cases. Because SDS is used in
the elution buffers, it is worth mentioning that a large volume of PBS (300 ml), pH 7.4, is
required for the regeneration of the column, which is time consuming and considered to be
one disadvantage of the present method.
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Fig. 1. Schematic drawing of molecular arrangement in huamn Hp phenotypes. Hp 1-1
possesses only the basic dimer (α1-β)2. While Hp 2-1 is comprised of many structures, a
dimer (α1-β)2, a trimer (α-β)3, and other linear polymers. Here, α represents both α1 and α2
chains. Hp 2-2 is comprised of a trimer (α2-β)3 and other cyclic polymers. Each α1, α2,
or β is 83, 142, or 245 amino acids in length, respectively. α2 is similar to α1, differing only
by an additional insertion of a repeat identical to 3/4 of α1. Due to the extra Cys-74 in α2,
Hp 2-1 and 2-2 form complicated polymers.
Fig. 2. Evaluation of dissociation of bound Hp from immobilized mAb at various elution
conditions. To determine a simple and optional condition that is able to elute the Hp from
immobilized mAb, Hp mAb was first immobilized on an ELISA plate followed by incubating
50 μg of purified Hp 2-1 in 50 μl PBS. After washing, bound Hp was then treated with
various eluting buffers. (A) Total binding of Hp without eluting reagents; (B) PBS, pH 11;
(C) 4 M urea-PBS, pH 11; (D) 0.1% SDS-PBS, pH 11. Binding of Hp was determined using
goat anti-Hp followed by HRP conjugated anti-goat IgG for final development of
chromogenicity. Each value represents a mean of triplicates ± SD.
Fig. 3. Effect of SDS concentrations (pH 11) on the dissociation of bound Hp from
immobilized mAb. The remaining Hp was determined similar to that described in Fig. 2.
Each point represents a mean of triplicates ± SD. Typical deviation is shown in the first
three doses.
Fig. 4. Elution profile of bound Hp from immobilized mAb on immunoaffinity column
(A-C) and contamination of apoA-I in isolated Hp (D). (A-C) One ml of human plasma
was applied onto the immunoaffinity column and washed with a PBS, pH 7.4. The bound
Hp was subsequently eluted with a 0.1% SDS-PBS, pH 11 and with a 0.1% SDS-PBS, pH 7.4.
The overall data suggests that the eluting buffer containing 0.1% SDS at pH 11 results in high
yield for Hp purification. (D) Analysis of isolated Hp by a 15% SDS-PAGE containing 1%
2-mercaptoethanol. It reveals that isolated Hp contaminates apoA-I.
Fig. 5. Binding of HDL with Hp and its dissociation by SDS. (A) Hp was first
immobilized on an ELISA plate followed by incubating 50 μg of purified HDL in 50 μl PBS.
After washing with PBS, the bound HDL dissociated from Hp by treating with SDS in a
dose-dependent fasion. Binding levels of HDL were determined using goat anti-apoA-I
followed by HRP conjugated anti-goat IgG similar to that described in Fig. 2. The data
suggests that Hp associated apoA-I can be eliminated in the presence of SDS. (B) Eluant
from affinity column, eluted with 0.02%, 0.04% and 0.06% SDS-PBS, pH 7.4, was analyzed
by SDS-PAGE in the presence of reducing reagent. It suggests to avoid of using 0.06% SDS
for apoA-I removal.
Fig. 6. Final purification of human Hp 1-1 (A), 2-1 (B), and 2-2 (C) from immunoaffinity column pre-washed with 0.04% SDS-PBS, pH 7.4. (A-C) Initially, 1 ml of human plasma was passed through the column with PBS, pH 7.4 and washed with the same buffer. The bound Hp was then pre-washed with 40 ml of 0.04% SDS-PBS, pH 7.4 to remove Hp associated apoA-I (F1). Finally, Hp was eluted from immobilized mAb by 0.1% SDS-PBS, pH 11 (F2). (D) Analysis of F1 and F2 on 15% SDS-PAGE containing 1%
2-mercaptoethanol and F1 on Western blot (F1'). The α and β subunits corresponding to each Hp phenotype are shown.
Fig. 7. Analysis of hemoglobin-binding property of isolated Hp 1-1, 2-1, and 2-2 (A) and
Hp 1-1, 2-1, and 2-2 from human plasma (B) on 7% native-PAGE. Briefly, each isolated
Hp (5 μg) or each plasma (6 μl) was incubated with hemoglobin (Hb) (5 μg) at room
temperature for 30 min. Following electrophoresis, the gel was stained directly by a freshly
prepared DAB solution containing 0.05% H2O2 as a developer for the endogenous peroxidase
activity of hemoglobin.
INTRODUCTION
Porcine Hp is an acute phase protein with a molecular weight approximate 120 kDa [1-3].
It possesses an electrophoretic mobility and quaternary structure similar to human Hp 1-1
being a homodimer (αβ)2 that is linked by disulfide bridges [3, 4]. Due to its homogeneity,
porcine Hp can be purified by a one-step HPLC gel-filtration chromatography established in
our laboratory [5].
Functions of Hp have been proposed comprehensively; one of the major functions is to
scavenge and complex with plasma free hemoglobin (Hb) that possesses an oxidative toxicity
via iron-containing hemes [6, 7]. The Hp-Hb complex is then metabolized through a
cycteine-rich receptor (CD163) on macrophage [8]. It has the bacteriostasis ability by
attenuating iron necessary for bacteria growth [9, 10]. Hp has also been proved to be an
extremely potent antioxidant which directly inhibits Cu(Ⅱ) induced low density lipoprotein
oxidation and reduces cell oxidative stress [11].
The plasma concentration of porcine Hp increases significantly during infection,
inflammation, and tissue damage [12, 13]. Pigs with lameness, tail biting or diarrhea show a
high level of Hp [14]. Natural infections of pigs with porcine reproductive and respiratory
syndrome virus (PRRS) [15], or with Actinobacillus pleuropneumoniae [13], Mycoplasma
hyorhinis [16] and Toxoplasma gondii [17] bacteria resulted in a significant elevation of their Hp levels. The protein level has therefore been used to identify both clinical and subclinical
diseases [1, 18] as well as to monitor the health status in pig production [14].
Human plasma Hp is classified as three phenotypes: 1-1, 2-1, and 2-2 (Fig. 1) [11, 19].
Hp 1-1 is a molecule of homodimer or (αβ)2, whereas 2-1 is comprised of multiple forms
including homodimer, trimer, tetramer, and other linear polymers. Hp 2-2, on the other hand,
consists of trimer, tetramer, and other cyclic polymers. In non-human mammalians, both
dimeric and polymeric forms of Hp exist. Thus far, polymeric forms analogous to human
polymeric Hp are found only in ruminant families of Artiodactyla order [20]. TLY minipigs
classified as Sus scrofa are aborigine in Taiwan with ears smaller than the other breeds. The
body weight of TLY is usually lower than 25 kg during the first six months of age as
compared with 80 kg of other domestic pigs [21]. Due to its low body weight and conserved
population, TLY minipigs have been used as an experimental animal [21]. In the present
study, we phenotyped the Hp of TLY minipigs. While phenotyping, we noticed that many
of them possessed very low plasma levels of Hp. The cDNA nucleotide sequence of TLY
Hp was also conducted to study whether there was a gene defect. It is of interest to observe
that there is no direct relationship between the putative amino-acid sequence of Hp and its
levels. Their amino-acid sequences are almost completely identical to that of domestic pigs
(except Val-65ÆLeu-65). Finally, a noncompetitive ELISA was employed to determine
their plasma Hp levels. In average, the mean Hp levels of TLY (0.21 ± 0.25 mg/ml) were
significantly lower (4X) than that of domestics (0.78 ± 0.45 mg/ml) (p<0.001). Among
which, 25% of TLY revealed a level lower than 0.05 mg/ml. The present study may provide
a reference value for the future use of TLY as an animal model for inflammatory,
cardiovascular, and infectious disease studies.
MATERIALS and METHODS
2.1. Materials
Purified porcine Hb and rat polyclonal antibodies against porcine Hp were prepared
according to the methods previously described [5, 22]. Goat anti-rat IgG was purchased
from Chemicon (Temecula, CA). Sepharose-12 HR column was obtained from Pharmacia
(Uppsala, Sweden). Total RNA extraction kit was purchased from Geneaid (Taipei, Taiwan).
All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) without any further
purification. The buffers used in this report were all filtered through a 0.45-μm filter before
use (Millipore, MA, USA).
2.2. Preparation of plasma samples
Forty-three TLY minipigs (23 sows and 20 boars) aged from 1 to 2 years were raised in
Taitung Animal Propagation. Thirty-five domestics (12 Durac, 15 Landrace, and 8 Yorkshire)
aged 6-8 months used in this study were from Animal Technology Institute, Taiwan. All of
these pigs were reared in a natural lighting environment. Management and medical
treatment were conducted according to the instruction established by National Science
Council of Taiwan. Pigs were vaccinated against swineenzooticpneumonia, hog cholera,
pseudorabies, atrophic rhinitis, foot and mouth disease, and Actinobacilluspleuropneumoniae.
All selected animals were free of adverse signs of disease after a clinical examination in the
herd. Blood samples were collected via jugular puncture using a 25-gauge needle and added
into the tubes containing 0.1% ethylenediaminetetraacetic acid (EDTA). Plasma was
obtained by a centrifugation at 2,000 g for 20 min and stored at -20℃ until analysis.
2.3. Phenotyping of Hp
A 7% native-polyacrylamide gel electrophoresis (native-PAGE) was conducted
according to the Laemmli’s method with the use of 5% polyacrylamide (w/v) as a stacking gel
[23]. Eight μl of plasma was incubated with 16 μg of porcine or human Hb for 30 min at
room temperature and subsequently mixed with a loading buffer [12 mM Tris–HCl, pH 6.8,
5% glycerol (v/v), 2.88 mM of 2-mercaptoethanol, and 0.02% bromphenol blue (w/v)] in a
final volume of 15 μl. Following the electrophoresis, the gel was briefly washed and then
immersed in freshly prepared 3,3’-diaminobendidine (DAB) solution (0.125 g of DAB
dissolved in 0.5 ml DMSO with the addition of 30 ml deionized H2O containing 0.05% H2O2)
with gently shaking to develop the pattern of Hp-Hb complex.
2.4. Western blot
Following the separation of proteins by SDS–PAGE, the gel was electrically transferred
to a nitrocellulose-membrane attached with a 3MM filter paper presoaked in a transfer buffer
containing 48 mMTris–HCl, 39 mM glycine, 0.037% SDS (w/v) and 20% methanol (v/v), pH
8.3. The rest of the procedures for Western blot were similar to that described previously [5,
24]. Rat polyclonal antibody against β chain of porcine Hp was used for the determination
24]. Rat polyclonal antibody against β chain of porcine Hp was used for the determination