Fluorescent labeling of heparins

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

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). 

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... 

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 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.

For energy transfer experiments, fluorescence spectra were obtained by subtracting the fluorescence spectra of the phospholipids, in the absence or in the presence of the energy acceptor, to correct for light scattering and for the fluorescence of the probe. Energy transfer efficiencies (Et) were calculated as previously described (11). The absorbance of samples in the presence of either heparin or labelled lipids never exceeded 0.1, therefore, the inner filter effect was negligible. 

Heparin has been fluorescently labeUed in a manner that does not alter the functional properties of the polysaccharide. The labelled polysaccharide has been used in conjunction with fluorescence polarization spectroscopy to monitor the binding of heparin to the L-serine proteases thrombin, factor XIa, factor Xa, and plasmin. The stoicheiometry and dissociation constants of the interactions have been measured. The kinetics of inactivation of the four proteases by anti-thrombin as a function of heparin concentration have also been measured. Evidence shows that the direct binding of heparin to anti-thrombin is probably responsible for the polysaccharide-dependent acceleration of hemostatic enzyme-inhibitor reactions. 

The test is a whole blood assay and uses fluorescence-labelled latex particles which are added to the heparinized whole blood sample. The PMN phagocytize the opsonised latex particles and the PMNL, having ingested fluorescent beads, are separated in a cell sorter and the size and fluorescence of the PMNL are determined. The mean fluorescence is equal to the number of latex particles ingested per cell. The phagocytosis index and percentage are calculated from the proportion of cells which underwent phagocytosis and that of the control. 

In any event, proteins transferred can be controlled by altering membrane fluidity of either site, liposome or cell. This technique must be very convenient and useful for fractionation of membrane proteins and the more detailed investigation of them in cell and membrane biologies. Specific binding of these glycoproteins directly transferred to liposome is now under investigation by the use of fluorescent probe-labelled and fractionated heparins. 

Fluorescence monitoring provides a high degree of sensitivity and sometimes additional selectivity. Binding of a fluoresceinyl-labelled heparin to albumin was investigated by the fluorescence measurement (ref. 53). The refactive index detector is suited to the... 

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