Heparin labeling

Figure 2. The pH dependency of heparin labelling. The degree of labelling intensity of the final product was compared with the concomitantly measured solubility of the F-D labelling reagent.

Fluorescent Labeling of Heparins and Related Polysaccharides Old Problems and New Solutions... 

The nondestructive introduction of a fluorescent label would provide the molecule with a nonradioactive fluorophore, yet would preserve the option for direct radiolabelling of the fluorescent moiety with 125Iodine. This approach was pioneered by Nagasawa et al. (5) who reacted native or /V-desulfated heparins with a fluorescein isothiocyanate (FITC). The resulting degree of labelling was low... 

Figure 3. Effects of molecular weight on labelling efficiency. Three heparins were independently labelled at increasing degrees of substitution. Molecular weight ranges of 3,000 (open square), 4,000-6,000 (solid circle), and 12,000-15,000 (open diamond).

Applications of Fluorescent Labelled Heparin in Experimental Biology. To... 

One of the first practical applications for these fluorescent labelled heparins was to examine the heparin binding behavior of different proteins and peptides under study in our laboratories. To this end we used a modification of the dot-blot assay described by Hirose and colleagues (13). F-D labelled heparin (-1 fluorescein/heparin) was radiolabelled with 125Iodine using iodobeads, to a specific activity of approximately 0.5 x 106 cpm/pg. Solutions of proteins with known heparin-binding capacities were dotted on nitrocellulose paper. A series of replicates... 

Figure 4. The efficiency of labelling of N-blocked or nonblocked heparins with FITC versus F-D. See text for details of experiment a, F1TC b, F-D.

Figure 5. Fluorescence anisotropy of F-D labelled heparin-antithrombin interaction. F-D-heparin (0.02 fluoresceins per uronic acid) at 0.1 mg/ml was incubated with different concentrations of antithrombin (open circles) or bovine serum albumin (solid diamonds) in 20 mM sodium phosphate buffer, pH 7.4.

Table L Intensity of Labelling versus Heparin Anticoagulant Activity...

Maximum labelling of heparin with F-D was achieved at 5 hours at 25 °C, pH 8.4. In the case of heparin, the efficiency of labelling was not dependent on molecular weight, but solely a function of the ratio of the concentrations of labelling reagent to monosaccharide subunit in the reaction mixture. Similar results were encountered in the labelling of dextrans of different molecular weight (9). 

The fluorescent labelling of heparin with F-D by this technique did not observably alter the biologic activity of the heparin as regards to its binding to antithrombin and catalysis of antithrombin s neutralization of activated coagulation factors. F-D labelled heparins also bound to other known heparin-binding proteins in a saturable and reversible manner, as demonstrated by the dot-blot assay technique (Figure 6). 

Figure 6. 125I-F-D Heparin dot blot assay. See text for methods and abbreviations. Upper panel incubation with 125I-F-D labelled porcine mucosal heparin alone, Lower panel the same conditions, but a 100-fold excess of unlabelled heparin has been added to the labelled heparin.

Heparin inhibits many enzymes, including all RNases, therefore, it is criticalfor this step. It also inhibits the labeling step therefore it needs to be removed later (by a LiCl precipitation). We did not find any comparable alternative (in efficiency and cost) to heparin. The composition and concentrations of other ingredients can be changed depending on the cell type or experiment. 

The LiClprecipitation is necessary to remove any residual heparin, which may intefere with the labeling reaction. Some vendors (e.g., Ambion and Qiagen) sell RNA purification columns that should remove heparin. We have not tested any of these yet. 

Probably, one of the most valuable advances in this field has dealt with the first chemoenzymatic synthesis of the stable isotope-enriched heparin from a uniformly double labelled 13C, 15N /V-acetylheparosan from E. coli K5. Heteronuclear, multidimensional nuclear magnetic resonance spectroscopy was employed to analyze the chemical composition and solution conformation of N-acety 1 hcparosan, the precursors, and heparin. Isotopic enrichment was found to provide well-resolved 13C spectra with the high sensitivity required for conformational studies of these biomolecules. Stable isotope-labelled heparin was indistinguishable from heparin derived from animal tissues and might be employed as a novel tool for studying the interaction of heparin with different receptors.30... 

Fig. 4. Radioactivity of the I4C-labelled heparin-containing hydrogel as a function of eluent and elution time. 1 distilled water, 2 physiological solution, 3 human blood plasma 101)...

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