Purification of human plasma haptoglobin by hemoglobin-affinity column chromatography

Liau, Chun Yi; Chang, Tsai Mu; Pan, Ju Pin; Chen, Wen Liang; Mao, Simon J.T.

doi:10.1016/s1570-0232(03)00128-4

Cited by 49 publications

(45 citation statements)

References 23 publications

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“…Human serum haptoglobin was prepared by affinity chromatography using freshly prepared human hemoglobin conjugated to Sepharose 4B, by the cyanogen bromide activation method as described previously. 21 Combined sera (2-3 ml) were subjected to hemoglobin-Sepharose 4B column (1.0 3 18 cm 2 , volume 14 ml) prepared in PBS (-) medium, pH 7.2 (see Abbreviations). An LKB peristaltic pump was used, and the flow rate was 0.5 ml/min.…”

Section: Serum Haptoglobin Levelsmentioning

confidence: 99%

“…Abbreviations: aoWR, N a -((aminooxy)acetyl)tryptophanylarginine methyl ester; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; HPLC, high-performance liquid chromatography; mAb, monoclonal antibody; MALDI-TOF MS, matrix assisted laser desorption/ionization time-of-flight mass spectrometry; PBS (-), phosphate-buffered saline (-), i.e., 0.15 M NaCl, 20 mM of a mixture of Na 2 HPO 4 /NaH 2 PO 4 , pH adjusted to 7.2, without addition of Ca 21 or Mg 21 ); PNGase-F, peptide-N4-(acetyl-b-glucosaminyl)-asparagine amidase; RM2 antigen, GalNAcb4-disialyl-Lc4; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SLe …”

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confidence: 99%

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Glycosylation status of haptoglobin in sera of patients with prostate cancer vs. benign prostate disease or normal subjects

Fujimura

Shinohara

Tissot

et al. 2007

Intl Journal of Cancer

114

115

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We studied chemical level and glycosylation status of haptoglobin in sera of patients with prostate cancer, as compared to benign prostate disease and normal subjects, with the following results. (i) Haptoglobin level was enhanced significantly in sera of prostate cancer. (ii) Sialylated bi-antennary glycans were the dominant structures in haptoglobins from all 3 sources, regardless of different site of N-linked glycan. The N-linked glycans at N184 were exclusively bi-antennary, and showed no difference between prostate cancer vs. benign prostate disease. (iii) Tri-antennary, N-linked, fucosylated glycans, carrying at least 1 sialyl-Lewis x/a antenna, were predominantly located on N207 or N211 within the amino acid 203-215 sequence of the b-chain of prostate cancer, and were minimal in benign prostate disease. Fucosylated glycans were not observed in normal subjects. A minor tri-antennary N-linked glycan was observed at N241 of the b-chain in prostate cancer, which was absent in benign prostate disease. (iv) None of these N-linked structures showed the expected presence of disialylated antennae with GalNAcb4(NeuAca3)Galb3(NeuAca6)GlcNAcbGal, or its analogue, despite cross-reactivity of prostate cancer haptoglobin with monoclonal antibody RM2. (v) Minor levels of O-glycosylation were identified in prostate cancer haptoglobin for the first time. Monoand disialyl core Type 1 O-linked structures were identified after reductive b-elimination followed by methylation and mass spectrometric analysis. No evidence was found for the presence of specific RM2 or other tumor-associated glycosyl epitopes linked to this Oglycan core. In summary, levels of haptoglobin are enhanced in sera of prostate cancer patients, and the N-glycans attached to a defined peptide region of its b-chain are characterized by enhanced branching as well as antenna fucosylation. ' 2007 Wiley-Liss, Inc.

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Section: Serum Haptoglobin Levelsmentioning

confidence: 99%

mentioning

confidence: 99%

Glycosylation status of haptoglobin in sera of patients with prostate cancer vs. benign prostate disease or normal subjects

Fujimura

Shinohara

Tissot

et al. 2007

Intl Journal of Cancer

114

115

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“…Conjugation of hemoglobin to Sepharose-4B was performed as described previously (21). Briefly, 5 g cyanogen bromide (CNBr) conjugated to Sepharose-4B (Sigma) was activated by suspending in 1 mM HCl (30 ml) at room temp for 30-60 min and filtered by gravity through glass filter, applying vacuum briefly at the end.…”

Section: Methodsmentioning

confidence: 99%

“…Haptoglobin ß-chain was separated from crude hetero-tetramer haptoglobin, and cross-linked with inter and intra S-S bridges (·ß) 2 , which were cleaved as described below (21). Briefly, S-S linkage of lyophilized haptoglobin fraction from affinity chromatography on hemoglobin-Sepharose-4B column as above was reduced with 150 mM dithioerythritol, alkylated with 300 mM acrylamide, and subjected to Superdex 200 10/30 GL column chromatography (10x300 mm, GE Healthcare, Uppsala, Sweden), following filtration with Millipore Ultra-free-MC (0.45 μm).…”

Section: Methodsmentioning

confidence: 99%

N-glycosylation status of β-haptoglobin in sera of patients with prostate cancer vs. benign prostate diseases

Hakomori¹

2009

Int J Oncol

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Abstract. N-glycosylation status of purified ß-haptoglobin separated from sera of patients with prostate cancer was studied in comparison to that of sera from patients with benign prostate diseases, or normal subjects. Two different approaches, as summarized below, one based on binding of lectins and antibodies to ß-haptoglobin, the other on mass spectrometry of released N-linked glycans from ß-haptoglobin, were performed. Some of the results were useful for distinction of prostate cancer vs. benign prostate diseases. i) Binding of Phaseolus vulgaris-L lectin (PHA-L), defining the GlcNAcß6Man·6Man side chain present in tri-or tetraantennary N-linked glycans, to ß-haptoglobin was higher for cases of prostate cancer and high-grade prostate intraepithelial neoplasia than for benign diseases. Binding of Aleuria aurantia lectin (AAL) defining Fuc·3-, ·4-, or ·6-GlcNAc, or monoclonal antibody directed to sialyl-Le x , to ß-haptoglobin was also higher for some of the cancer cases than for benign diseases. Many other lectins and antibodies showed no binding to ß-haptoglobin, or showed no significant difference between cancer vs. benign diseases. ii) Mass spectrometric analysis of N-linked glycans of ß-haptoglobin released by Peptide N-glycosidase-F showed enhanced expression of monosialyl tri-antennary structures in prostate cancer cases. Thus, binding of PHA-L to affinity-purified ß-haptoglobin from sera of patients could lead to development of useful tools for differential diagnosis of prostate cancer vs. benign prostate diseases.

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“…The rest of the procedures for Western blot were similar to those described previously [22,39]. Rat polyclonal antibody prepared against theβ-chain of porcine Hp was used for determination of the presence of Hp.…”

Section: Methodsmentioning

confidence: 99%

Low Levels of Haptoglobin and Putative Amino Acid Sequence in Taiwanese Lanyu Miniature Pigs

Yueh

Wang

Lin

et al. 2008

The Journal of Veterinary Medical Science

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ABSTRACT. Porcine haptoglobin (Hp) is an acute phase protein. Its plasma level increases significantly during inflammation and infection. One of the main functions of Hp is to bind free hemoglobin (Hb) and inhibit its oxidative activity. In the present report, we studied the Hp phenotype of Taiwanese Lanyu miniature pigs (TLY minipigs; n=43) and found their Hp structure to be a homodimer (β-α-α-β) similar to human Hp 1-1. Interestingly, Western blot and high performance liquid chromatographic (HPLC) analysis showed that 25% of the TLY minipigs possessed low or no plasma Hp level (<0.05 mg/ml). The Hp cDNA of these TLY minipigs was then cloned, and the translated amino acid sequence was analyzed. No sequences were found to be deficient; they showed a 99.7% identity with domestic pigs (NP_999165). The mean overall Hp level of the TLY minipigs (0.21 ± 0.25 mg/ml; n=43) determined by enzyme-linked immunosorbent assay (ELISA) was markedly lower than that of domestic pigs (0.78 ± 0.45 mg/ml; p<0.001), while 25% of the TLY minipigs had an Hp level that was extremely low (<0.05 mg/ml). In addition, the initial recovery rate (first 40 min) in the circulation of infused fluorescein isothiocyanate (FITC)-Hb was significantly higher in the TLY minipigs with extremely low Hp levels than those with high levels. This data suggests that the low concentration of Hp-Hb complex is responsible for the higher recovery rate of Hb in the circulation. TLY minipigs have been used as an experimental model for cardiovascular diseases; whether they can be used as a model for inflammatory diseases, with Hp as a marker, remains a topic of interest. However, since the Hp level varies significantly among individual TLY minipigs, it is necessary to prescreen the Hp levels of the animals to minimize variation in the experimental baseline. The present study may provide a reference value for future use of the TLY minipig as an animal model for inflammation-associated diseases. KEY WORDS: amino acid sequence, evolution, haptoglobin concentration, hemoglobin, Lanyu minipig.

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Purification of human plasma haptoglobin by hemoglobin-affinity column chromatography

Cited by 49 publications

References 23 publications

Glycosylation status of haptoglobin in sera of patients with prostate cancer vs. benign prostate disease or normal subjects

Glycosylation status of haptoglobin in sera of patients with prostate cancer vs. benign prostate disease or normal subjects

N-glycosylation status of β-haptoglobin in sera of patients with prostate cancer vs. benign prostate diseases

Low Levels of Haptoglobin and Putative Amino Acid Sequence in Taiwanese Lanyu Miniature Pigs

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