Colorimetric method for the assay of heparin content in immobilized heparin preparations

Smith, Paul K.; Mallia, A. Krishna; Hermanson, Greg T.

doi:10.1016/0003-2697(80)90679-x

Cited by 305 publications

(151 citation statements)

References 26 publications

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“…Heparin/HS Columns-Affinity columns were generated by coupling heparin or HS to Sepharose 4B based on the method of Smith et al (24). Sepharose 4B was washed with 20 bed volumes of water, re-suspended in 1 ml of water, and transferred to a beaker and placed on ice.…”

Section: Methodsmentioning

confidence: 99%

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A Binding Site for Highly Sulfated Heparan Sulfate Is Identified in the N Terminus of the Circumsporozoite Protein

Ancsin

Kisilevsky²

2004

Journal of Biological Chemistry

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Circumsporozoite protein (CSP) coats the malarial sporozoite and functions to target the liver for infection, which is the first step to developing malaria. An important tissue ligand for CSP is the glycosaminoglycan heparan sulfate (HS) found on the surface of hepatocytes and in the basement membrane of the space of Disse. To better understand this efficient targeting process, we set out to identify and characterize the HS binding site(s) of CSP. We synthesized a series of peptides corresponding to five regions of Plasmodium falciparum CSP containing basic residues, a common requirement of HS binding sites, and screened them for heparin and HS binding activity. Only one of these peptides (Pf 2), which contains a motif we have named region I-plus, demonstrated both high affinity heparin/HS binding activity and the ability to block the binding of recombinant CSP to heparin-Sepharose 4B. Analysis by isothermal titration calorimetry revealed that region I-plus has a binding constant of K d ‫؍‬ 5.0 M and a stoichiometry of n ‫؍‬ 7.8 binding sites/heparin chain. Heparin binding was dependent on the amino acid sequence of region I-plus, and the binding sites on heparin/HS are contained within a decasaccharide. Furthermore, HS oligosaccharides rich in sulfate and iduronic acid content (heparin-like) are required for efficient binding. Because liver HS is exceptionally high in both these components relative to the HS of other organs, the HS structural requirements for efficient region I-plus/HS binding are consistent with this peptide sequence functioning to target sporozoites to the liver for attachment to hepatocytes. Finally, the region I-plus heparin/HS binding site was also discovered for two other species that infect humans, Plasmodium malariae and Plasmodium vivax, further supporting the existence of a HS binding domain in the N-terminal portion of CSP.

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Section: Methodsmentioning

confidence: 99%

“…By this procedure, heparin/HS was linked through its ϪNH 2 and ϪOH groups. The amount of heparin coupled to the Sepharose was 0.5-0.75 mg/ml as determined by the toluidine blue assay (24). Affi-Gel heparin was purchased from Bio-Rad.…”

Section: Methodsmentioning

confidence: 99%

A Binding Site for Highly Sulfated Heparan Sulfate Is Identified in the N Terminus of the Circumsporozoite Protein

Ancsin

Kisilevsky²

2004

Journal of Biological Chemistry

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“…The GAG content of complex membranes was determined using a colorimetric method based on binding of toluidine blue [20,21]. To assay bound GAG in complex membranes on a 24-well plate, the overlying solution was first aspirated and the membrane rinsed once with PBS.…”

Section: Gag Quantification Assaysmentioning

confidence: 99%

Maintenance of CD34 Expression During Proliferation of CD34 ⁺ Cord Blood Cells on Glycosaminoglycan Surfaces

1999

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Recent studies have indicated that glycosaminoglycan (GAG) interactions with hematopoietic progenitors play a significant role in the regulation of hematopoiesis. However, the details of these interactions are not clear. In this study, we examined the role of soluble and immobilized GAGs in the proliferation of CD34 + cells. Chitosan, a cationic polysaccharide, was used to immobilize GAGs in ionic complex membranes. The GAGs studied were heparin, hyaluronate, and chondroitin sulfates A, B, and C. CD34-enriched umbilical cord blood cells were seeded onto tissue culture plates coated with the GAG-chitosan complex membranes. Cultures were maintained in medium supplemented with stem cell factor and interleukin 3 for up to six weeks, during which total and CD34 + cell numbers were determined by flow cytometry. Total cell number expansion ranged from 25-fold to 40-fold after six weeks. However, only heparin and chondroitin sulfate B (CSB) surfaces retained a significant CD34 + fraction. All other surfaces exhibited declines in CD34 expression, with negligible CD34 + percentages remaining after four weeks. In contrast, heparin and CSB surfaces exhibited CD34 + fractions as high as 90% after four weeks.GAG desorption studies indicated that the observed effects were partly mediated by desorbed GAGs in a concentration dependent manner. Subsequent studies showed that sustained high (160 g/ml) heparin levels had toxic effects, while the same concentration of CSB exhibited more rapid early proliferation of CD34 + cells. In conclusion, this culture system has demonstrated the ability to produce simultaneous proliferation and CD34 + cell enrichment of a partially purified cord blood population by controlling the nature and levels of GAG moieties to which the cells are exposed. The results indicate that specific GAGs can significantly influence the growth and differentiation characteristics of cultured CD34 + cells.

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“…After stirring overnight, the final suspension was filtered and rinsed with deionized water. In both functionalized resins, the amount of immobilized heparin was determined by the toluidine blue dye assay (Smith et al 1980). HIV-1 (Clade B) virus solution preparation.…”

Section: Methodsmentioning

confidence: 99%

Removing human immunodeficiency virus (HIV) from human blood using immobilized heparin

et al. 2011

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We report herein that heparin covalently attached to a water-insoluble resin suspended in HIV-infected aqueous buffer or whole blood captures the virus; subsequent physical separation of the immobilized heparin reduced the viral titers by over 80% and 50%, respectively. The detoxification concept has been validated by both circulating an HIV-1 solution through a column packed with the heparin-Sepharose beads and successively mixing an HIV-1 solution with fresh beads.

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Colorimetric method for the assay of heparin content in immobilized heparin preparations

Cited by 305 publications

References 26 publications

A Binding Site for Highly Sulfated Heparan Sulfate Is Identified in the N Terminus of the Circumsporozoite Protein

A Binding Site for Highly Sulfated Heparan Sulfate Is Identified in the N Terminus of the Circumsporozoite Protein

Maintenance of CD34 Expression During Proliferation of CD34 ⁺ Cord Blood Cells on Glycosaminoglycan Surfaces

Removing human immunodeficiency virus (HIV) from human blood using immobilized heparin

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Colorimetric method for the assay of heparin content in immobilized heparin preparations

Cited by 305 publications

References 26 publications

A Binding Site for Highly Sulfated Heparan Sulfate Is Identified in the N Terminus of the Circumsporozoite Protein

A Binding Site for Highly Sulfated Heparan Sulfate Is Identified in the N Terminus of the Circumsporozoite Protein

Maintenance of CD34 Expression During Proliferation of CD34 + Cord Blood Cells on Glycosaminoglycan Surfaces

Removing human immunodeficiency virus (HIV) from human blood using immobilized heparin

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Maintenance of CD34 Expression During Proliferation of CD34 ⁺ Cord Blood Cells on Glycosaminoglycan Surfaces