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A heparin binding synthetic peptide from human HIP / RPL29 fails to specifically differentiate between anticoagulantly active and inactive species of heparin.

Hoke DE, Carson DD, Höök M - J Negat Results Biomed (2003)

Bottom Line: Herein, we demonstrate that HIP peptide-1 cannot enrich ATIII-dependent anticoagulant activity from a starting pool of porcine intestinal mucosa Hp through a bio-specific interaction.However, a HIP peptide-1 column can be used to enrich for anticoagulantly active Hp from a diverse pool of glycosaminoglycans known as Hp byproducts by a mechanism of nonspecific charge interactions.Thus, HIP peptide-1 cannot recognize Hp via bio-specific interactions but binds glycosaminoglycans by non-specific charge interactions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Extracellular Matrix Biology; The Texas A&M University System Health Science Center, Institute of Biosciences and Technology, Houston, Texas 77030, USA. dehoke@unimelb.edu.au

ABSTRACT
Despite extensive progress in determining structures within heparin and heparan sulfate (Hp/HS) and the discovery of numerous proteinaceous binding partners for Hp/HS so far; the only detailed characterization of a specific protein-glycosaminoglycan interaction is antithrombin III (ATIII) binding to a Hp pentasaccharide containing a unique 3-O-sulfated glucosamine residue. Previously, it was reported from our laboratories that a 16 amino acid synthetic peptide derived from the C-terminus of human HIP/RPL29 (HIP peptide-1) enriched for ATIII-dependent anticoagulant activity, presumably by specifically binding the ATIII pentasaccharide. Herein, we demonstrate that HIP peptide-1 cannot enrich ATIII-dependent anticoagulant activity from a starting pool of porcine intestinal mucosa Hp through a bio-specific interaction. However, a HIP peptide-1 column can be used to enrich for anticoagulantly active Hp from a diverse pool of glycosaminoglycans known as Hp byproducts by a mechanism of nonspecific charge interactions. Thus, HIP peptide-1 cannot recognize Hp via bio-specific interactions but binds glycosaminoglycans by non-specific charge interactions.

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Anion Exchange Chromatography of Unlabelled or Tritiated Hp. Unlabelled Hp (A) or tritiated Hp (B) was added to 1 ml of DEAE anion exchange resin pre-equilibrated in 0.05 M Acetate pH 4.0 and allowed to bind. A LiCl gradient was directly applied to the unlabelled Hp while the tritiated Hp was washed extensively in buffer without LiCl before the start of a LiCl gradient. The X-axis corresponds to fraction number, the left Y-axis corresponds to total μg (A) or DPM (B), and the right Y-axis corresponds to LiCl concentration.
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Figure 2: Anion Exchange Chromatography of Unlabelled or Tritiated Hp. Unlabelled Hp (A) or tritiated Hp (B) was added to 1 ml of DEAE anion exchange resin pre-equilibrated in 0.05 M Acetate pH 4.0 and allowed to bind. A LiCl gradient was directly applied to the unlabelled Hp while the tritiated Hp was washed extensively in buffer without LiCl before the start of a LiCl gradient. The X-axis corresponds to fraction number, the left Y-axis corresponds to total μg (A) or DPM (B), and the right Y-axis corresponds to LiCl concentration.

Mentions: We subsequently explored the possibility that the difference in the HIP peptide-1 binding ability of unlabelled and tritiated Hp was due to a difference in the charge density of these materials. This was tested by anion exchange chromatography of unlabelled (figure 2A) or tritiated Hp (figure 2B) with LiCl gradient elution. All of the unlabelled Hp is found to bind to an anion exchange column and elute as a single broad peak with an average (determined from three separate experiments) peak LiCl concentration of 1.5 M. The tritiated Hp preparation is found to contain a significant amount (38%) of material that does not bind to the anion exchange resin even before the start of the LiCl gradient. The bound fraction consists of 62% of the radioactivity and elutes at a peak LiCl content of 1.0 M.


A heparin binding synthetic peptide from human HIP / RPL29 fails to specifically differentiate between anticoagulantly active and inactive species of heparin.

Hoke DE, Carson DD, Höök M - J Negat Results Biomed (2003)

Anion Exchange Chromatography of Unlabelled or Tritiated Hp. Unlabelled Hp (A) or tritiated Hp (B) was added to 1 ml of DEAE anion exchange resin pre-equilibrated in 0.05 M Acetate pH 4.0 and allowed to bind. A LiCl gradient was directly applied to the unlabelled Hp while the tritiated Hp was washed extensively in buffer without LiCl before the start of a LiCl gradient. The X-axis corresponds to fraction number, the left Y-axis corresponds to total μg (A) or DPM (B), and the right Y-axis corresponds to LiCl concentration.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC152653&req=5

Figure 2: Anion Exchange Chromatography of Unlabelled or Tritiated Hp. Unlabelled Hp (A) or tritiated Hp (B) was added to 1 ml of DEAE anion exchange resin pre-equilibrated in 0.05 M Acetate pH 4.0 and allowed to bind. A LiCl gradient was directly applied to the unlabelled Hp while the tritiated Hp was washed extensively in buffer without LiCl before the start of a LiCl gradient. The X-axis corresponds to fraction number, the left Y-axis corresponds to total μg (A) or DPM (B), and the right Y-axis corresponds to LiCl concentration.
Mentions: We subsequently explored the possibility that the difference in the HIP peptide-1 binding ability of unlabelled and tritiated Hp was due to a difference in the charge density of these materials. This was tested by anion exchange chromatography of unlabelled (figure 2A) or tritiated Hp (figure 2B) with LiCl gradient elution. All of the unlabelled Hp is found to bind to an anion exchange column and elute as a single broad peak with an average (determined from three separate experiments) peak LiCl concentration of 1.5 M. The tritiated Hp preparation is found to contain a significant amount (38%) of material that does not bind to the anion exchange resin even before the start of the LiCl gradient. The bound fraction consists of 62% of the radioactivity and elutes at a peak LiCl content of 1.0 M.

Bottom Line: Herein, we demonstrate that HIP peptide-1 cannot enrich ATIII-dependent anticoagulant activity from a starting pool of porcine intestinal mucosa Hp through a bio-specific interaction.However, a HIP peptide-1 column can be used to enrich for anticoagulantly active Hp from a diverse pool of glycosaminoglycans known as Hp byproducts by a mechanism of nonspecific charge interactions.Thus, HIP peptide-1 cannot recognize Hp via bio-specific interactions but binds glycosaminoglycans by non-specific charge interactions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Extracellular Matrix Biology; The Texas A&M University System Health Science Center, Institute of Biosciences and Technology, Houston, Texas 77030, USA. dehoke@unimelb.edu.au

ABSTRACT
Despite extensive progress in determining structures within heparin and heparan sulfate (Hp/HS) and the discovery of numerous proteinaceous binding partners for Hp/HS so far; the only detailed characterization of a specific protein-glycosaminoglycan interaction is antithrombin III (ATIII) binding to a Hp pentasaccharide containing a unique 3-O-sulfated glucosamine residue. Previously, it was reported from our laboratories that a 16 amino acid synthetic peptide derived from the C-terminus of human HIP/RPL29 (HIP peptide-1) enriched for ATIII-dependent anticoagulant activity, presumably by specifically binding the ATIII pentasaccharide. Herein, we demonstrate that HIP peptide-1 cannot enrich ATIII-dependent anticoagulant activity from a starting pool of porcine intestinal mucosa Hp through a bio-specific interaction. However, a HIP peptide-1 column can be used to enrich for anticoagulantly active Hp from a diverse pool of glycosaminoglycans known as Hp byproducts by a mechanism of nonspecific charge interactions. Thus, HIP peptide-1 cannot recognize Hp via bio-specific interactions but binds glycosaminoglycans by non-specific charge interactions.

Show MeSH