3.6. Analysis of hemoglobin-binding ability of isolated porcine, cervine, and human Hp
Fig. 9 reveals that isolated Hp possessed the ability to form a complex with
hemoglobin using a 7% native PAGE analysis. Each isolated Hp resulted in a
hemoglobin complex identical to that native Hp present in the plasma. The results
suggest that the isolation Hp retained the hemoglobin binding nature under our
isolation procedures [13,21].
3.7. Summary
In summary, the HPLC gel-filtration used in this study is appropriate for the
isolation of those Hp with a homogeneous structure such as (αβ)2 and (αβ)4.
Contamination of apoA-1 can be eliminated using HDL-depleted plasma. Albumin
and some other proteins coeluted by gel-filtration column can be further removed by a
Cu (II)-IMAC, while the presence of 4 M urea is essential to enhance or differentiate
the binding affinity of Hp from the other contaminants. The purity of each isolates
species is greater than 90% and retains its hemoglobin binding ability. Per our
experience, once it contaminates hemoglobin during the collection of blood, it is
almost impossible to remove using a conventional gel-filtration HPLC. Therefore,
hemolysis should be avoided while preparing the plasma.
References
1. Dobryszycka W. Biological functions of haptoglobin--new pieces to an old
puzzle. Eur J Clin Chem Clin Biochem. 35 (1997) 647-54.
2. Langlois MR, Delanghe JR. Biological and clinical significance of haptoglobin
polymorphism in humans. Clin Chem. 42 (1996) 1589-600.
3. Cheng T-M, et al. Immunochemical property of human haptoglobin phenotypes:
Determination of plasma haptoglobin using type-matched standards. Clin
Biochem (2007)
4. Raijmakers MT, Roes EM, Steegers EA, van der Wildt B, Peters WH.
Umbilical glutathione levels are higher after vaginal birth than after cesarean
section. J Perinat Med. 31 (2003) 520-2.
5. Engstrom G, Hedblad B, Stavenow L, Lind P, Janzon L, Lindgarde F.
Inflammation-sensitive plasma proteins are associated with future weight gain.
Diabetes. 52 (2003) 2097-101.
6. Sadrzadeh SM, Bozorgmehr J. Haptoglobin phenotypes in health and disorders.
Am J Clin Pathol. 121 (2004) S97-104.
7. Arredouani MS, Kasran A, Vanoirbeek JA, Berger FG, Baumann H, Ceuppens JL.
Haptoglobin dampens endotoxin-induced inflammatory effects both in vitro and
in vivo. Immunology. 114 (2005) 263-71.
8. Kristiansen M, Graversen JH, Jacobsen C, Sonne O, Hoffman HJ, Law SK,
Moestrup SK. Identification of the haemoglobin scavenger receptor. Nature. 409
(2001) 198-201.
9. Maeda N, McEvoy SM, Harris HF, Huisman TH, Smithies O. Polymorphisms in
the human haptoglobin gene cluster: chromosomes with multiple
haptoglobin-related (Hpr) genes. Proc Natl Acad Sci U S A. 83 (1986) 7395-9.
10. Patzelt D, Geserick G, Schroder H. The genetic haptoglobin polymorphism:
relevance of paternity assessment. Electrophoresis. 9 (1988) 393-7.
11. Kurosky A, Barnett DR, Lee TH, Touchstone B, Hay RE, Arnott MS, Bowman
BH, Fitch WM. Covalent structure of human haptoglobin: a serine protease
homolog. Proc Natl Acad Sci U S A. 77 (1980) 3388-92.
12. Tseng CF, Lin CC, Huang HY, Liu HC, Mao SJ. Antioxidant role of human
haptoglobin. Proteomics. 4 (2004) 2221-8.
13. Yueh SC, Lai YA, Chen WL, Hsu HH, Mao SJ. An improved method for
haptoglobin 1-1, 2-1, and 2-2 purification using monoclonal antibody affinity
chromatography in the presence of sodium dodecyl sulfate. J Chromatogr B
Analyt Technol Biomed Life Sci. 845 (2007) 210-7.
14. Petersen HH, Nielsen JP, Heegaard PM. Application of acute phase protein
measurements in veterinary clinical chemistry. Vet Res. 35 (2004) 163-87.
15. Katnik I, Pupek M, Stefaniak T. Cross reactivities among some mammalian
haptoglobins studied by a monoclonal antibody. Comp Biochem Physiol B
Biochem Mol Biol. 119 (1998) 335-40.
16. Mominoki K, Nakagawa-Tosa N, Morimatsu M, Syuto B, Saito M. Haptoglobin
in Carnivora: a unique molecular structure in bear, cat and dog haptoglobins.
Comp Biochem Physiol B Biochem Mol Biol. 110 (1995) 785-9.
17. Wicher KB, Fries E. Haptoglobin, a hemoglobin-binding plasma protein, is
present in bony fish and mammals but not in frog and chicken. Proc Natl Acad
Sci U S A. 103 (2006) 4168-73
18. Rademacher BE, Steele WJ. A general method for the isolation of haptoglobin
1-1, 2-1, and 2-2 from human plasma. Anal Biochem. 1987 Jan;160(1):119-26.
19. Travis JC, Sanders BG. Haptoglobin evolution: polymeric forms of Hp in the
Bovidae and Cervidae families. J Exp Zool. 180 (1972)
20. Morimatsu M, Syuto B, Shimada N, Fujinaga T, Yamamoto S, Saito M, Naiki M.
Isolation and characterization of bovine haptoglobin from acute phase sera. J
Biol Chem. 266(1991) 11833-7.
21. Tseng CF, Huang HY, Yang YT, Mao SJ. Purification of human haptoglobin 1-1,
2-1, and 2-2 using monoclonal antibody affinity chromatography. Protein Expr
Purif. 33 (2004) 265-73.
22. Yamashita G, Secknus R, Chernosky A, Krivacic KA, Holzbach RT. Comparison
of haptoglobin and apolipoprotein A-I on biliary lipid particles involved in
cholesterol crystallization.J Gastroenterol Hepatol. 11 (1996) 738-45.
23. Cardin AD, Price CA, Hirose N, Krivanek MA, Blankenship DT, Chao J, Mao SJ.
Structural organization of apolipoprotein B-100 of human plasma low density
lipoproteins. Comparison to B-48 of chylomicrons and very low density
lipoproteins. J Biol Chem. 261 (1986) 16744-8.
24. Marcovina S, Kottke BA, Mao SJ. Monoclonal antibodies can precipitate
low-density lipoprotein. II. Radioimmunoassays with single and combined
monoclonal antibodies for determining apolipoprotein B in serum of patients
with coronary artery disease. Clin Chem. 31 (1985) 1659-63.
25. Arnold FH. Metal-affinity separations: a new dimension in protein processing.
Biotechnology (N Y). 9 (1991) 151-6.
26. Ueda EK, Gout PW, Morganti L. Current and prospective applications of metal
ion-protein binding. J Chromatogr A. 988 (2003) 1-23.
27. Liau CY, Chang TM, Pan JP, Chen WL, Mao SJ. Purification of human plasma
haptoglobin by hemoglobin-affinity column chromatography. J Chromatogr B
Analyt Technol Biomed Life Sci. 790 (2003) 209-16.
28. Yang SJ, Mao SJ. Simple high-performance liquid chromatographic purification
procedure for porcine plasma haptoglobin. J Chromatogr B Biomed Sci Appl.
731 (1999) 395-402.
29. U.K. Laemmli, Cleavage of structural proteins during the assembly of the head
of bacteriophage T4. Nature 227 (1970), 680–685.
30. Weber K, Osborn M. The reliability of molecular weight determinations by
dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 244 (1969)
4406-12.
31. Song CY, Chen WL, Yang MC, Huang JP, Mao SJ. Epitope mapping of a
monoclonal antibody specific to bovine dry milk: involvement of residues 66-76
of strand D in thermal denatured beta-lactoglobulin. J Biol Chem. 280 (2005)
3574-82.
32. Martins S, Karmali A, Andrade J, Serralheiro ML. Immobilized Metal Affinity
Chromatography of Monoclonal Immunoglobulin M Against Mutant Amidase
From Pseudomonas aeruginosa. Mol Biotechnol. 33 (2006) 103-14.
33. Imam-Sghiouar N, Joubert-Caron R, Caron M. Application of metal-chelate
affinity chromatography to the study of the phosphoproteome. Amino Acids. 28
(2005) 105-9.
34. Sharma S, Agarwal GP. Interactions of proteins with immobilized metal ions: a
comparative analysis using various isotherm models. Anal Biochem. 288 (2001)
126-40.
35. Nagel RL, Gibson QH. The binding of hemoglobin to haptoglobin and its
relation to subunit dissociation of hemoglobin. J Biol Chem. 246(1971) 69-73.
36. Hvidberg V, Maniecki MB, Jacobsen C, Hojrup P, Moller HJ, Moestrup SK.
Identification of the receptor scavenging hemopexin-heme complexes. Blood.
106 (2005) 2572-9.
37. Spagnuolo MS, Cigliano L, D'Andrea LD, Pedone C, Abrescia P. Assignment of
the binding site for haptoglobin on apolipoprotein A-I. J Biol Chem. 280 (2005)
1193-8.
38. Miller YI, Altamentova SM, Shaklai N. Oxidation of low-density lipoprotein by
hemoglobin stems from a heme-initiated globin radical: antioxidant role of
haptoglobin. Biochemistry. 36 (1997) 12189-98.
Fig. 1. Schematic drawing of molecular arrangement in human Hp phenotypes. Hp
1-1 possesses only the basic dimer (α1β)2 whereas Hp 2-1 is comprised of polymeric
structures: starting with a dimer (α1β)2, trimer (αβ)3, and other linear polymers.
Here, α represents both α1 and α2 chains. Hp 2-2 is comprised of a trimer(α2β)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. Owing to the extra Cys-74 in α2, Hp 2-1 and 2-2 form
complicated polymers.
Fig. 2. Identification of the homogeneity of Hp in porcine, cervine, and human
plasma using hemoglobin binding assay. Briefly, plasma (6 μl) was pre-incubated
with hemoglobin (18 μg) at room temperature for 30 min before conducting the
native-PAGE. The gel (7%) was run at 20 mA for 1.5 h and further developed with
DAB in PBS containing 0.05% H2O2 based on the peroxidase activity of hempglobin.
Fig. 3. Typical chromatographic profile of Hp isolation using HPLC Superose-12
column. Dialyzed supernatant of 50% saturated ammonium-sulfate fraction was
applied onto a HPLC system (see Materials and Methods). The chromatography was
conducted at a flow-rate of 0.3 ml/min with a pressure of 200 psi and run for 60 min
at room temperature using PBS containing 0.01% NaN3 as a mobile phase. The
filled bar represented the pooled fractions corresponding to isolated Hp of each
species.
Fig. 4. Analysis of isolated Hp from HPLC using 15% SDS–PAGE in the presence
of reducing reagent. Following HPLC chromatography, each fraction was subjected
to SDS-PAGE and run at 20 mA for 1.5 h. Lane M: molecular weight markers.
Lane W: Western blot using porcine β chain specific, bovine Hp, and human Hp
polyclonal antibodies for (A), (B), and (C), respectively, were used for Western blot.
Fig. 5. SDS-PAGE of human Hp fractions elution from immobilized metal affinity
chromatography (IMAC) coupled with Cu (II). Following the binding of human
crude Hp preparation (Fig. 3) to IMAC, the bound materials were stepwisely eluted
with imidazole at the concentration indicated on the top of each lane. (A) Cu
(II)-coupled column equilibrated with binding buffer (300 mM NaCl, 50 mM
NaH2PO4, pH 8) and eluted with binding buffer containing imidazole from 0.5 to 10
mM. (B) Cu (II)-coupled column equilibrated with binding buffer containing 4 M
urea and eluted with the binding buffer containing imidazole from 2.5 to 20 mM.
(C) Cu (II)-coupled column equilibrated with binding buffer containing 4 M ureaand
eluted with 4 M urea containing 5 mM imidazole followed by a d 20 mM imidazole.
Finally, the column was “striped off” by EDTA to confirm that no protein was
retained in column. Lane M: molecular weight markers.
Fig. 6. Analysis of purified human, cervine, and porcine Hp by SDS-PAGE and
Western-Blot in reducing condition. The purified human (1-1), cervine, and porcine
Hp was analyzed by a 15% SDS-PAGE (A) and Western blot (B). Lane M:
molecular weight markers (in kDa)
Fig. 7. Characterization of molecular form of human, corvine, and porcine Hp by
4% SDS-PAGE in non-reducing condition. The purified human (1-1), cervine, and
porcine Hp was analyzed by a 4% SDS-PAGE (A) and Western blot (B)
Lane M: molecular weight markers (in kDa). The molecular weight of human (1-1),
cervine, and porcine Hp were 120, 220, and 120 kDa, respectively, as estimated by an
image analysis system Quantity One (Bio-Rad).
Fig. 8. Hemoglobin binding ability on Hp. Human, cervine, and porcine plasma
were incubated with hemoglobin at room temperature for 30 min before conducting a
7% native-PAGE with a procedure similar to that described in Fig. 2.
Fig. 1
Fig. 2
Hb Hp-Hb complex
Pig Deer Human Hp 1-1 Human Hp 2-1 Human Hp 2-2 Hemoglobin
Pig Deer Human Hp 1-1 Human Hp 2-1 Human Hp 2-2 Hemoglobin
(αβ)2
(αβ)3
(αβ)4
Absorbance at 280 nm
Fig. 3
1.5 pHp 1.0
0.5
0
(A) Porcine Hp
1.5
1.0
0.5
0
cHp (B) Cervine Hp
Retention time (min) hHp
1.5
1.0
0.5
0
25 30 35 40 50 45 55
(C) Human 1-1 Hp
Fig. 4
Fig. 5
Fig. 6
(B)
10