Dextran periodate

Reproducibility of separation for a Dextran T-500 sample was tested on a semipreparative Sephacryl system S-500/S-1000 (65 + 95x1.6 cm) over a period of 6 months. The elution profiles of Dextran T-500 could be superimposed with deviations in the elution axis of 3 ml ( 1 fraction), and deviations in carbohydrate content within 5% referring to the maximum value at V,e, = 213 ml (Fig. 16.8). 

FIGURE 16.8 Dextran T-SOO ( ) separated on Sephacryl S-SOO/S-IOOO (60 + 9S x 1.6 cm) sampled in 3-ml fractions normalized (area = 1.0) eluogram profiles (ev) constructed from an off-line determined mass of carbohydrates within each of the fractions flow rate 0.42 ml/min Vexd = 126 ml, V,o, = 273 ml eluent 0.005 M NaOH reproducibility of results over a period of 6 months 5% of ev maximum and 3 ml ( 1 fraction). 

This simple strategy can be used to add amine residues to polysaccharide molecules after formation of aldehydes by periodate or enzymatic oxidation (Section 4.4, this chapter). Thus, glycoconjugates or carbohydrate polymers such as dextran may be derivatized to contain amines for further conjugation reactions. 

Aldehyde-containing macromolecules will react spontaneously with hydrazide compounds to form hydrazone linkages. The hydrazone bond is a form of Schiff base that is more stable than the Schiff base formed from the interaction of an aldehyde and an amine. The hydrazone, however, may be reduced and further stabilized by the same reductants utilized for reductive amination purposes (Chapter 3, Section 4.8). The addition of sodium cyanoborohydride to a hydrazide-aldehyde reaction drives the equilibrium toward formation of a stable covalent complex. Mallia (1992) found that adipic acid dihydrazide derivatization of periodate-oxidized dextran (containing multiple formyl functionalities) proceeds with much greater yield when sodium cyanoborohydride is present. 

Bis-hydrazide-containing molecules also can be used to activate soluble polymeric sub-stances-containing aldehyde groups. For instance, dextran may be periodate oxidized to create numerous formyl functionalities on each molecule. Subsequent reaction with a homobifunctional hydrazide in large excess results in a hydrazide-activated polymer having multivalent-binding capability toward aldehydes or ketones (Chapter 25, Section 2.2). Insoluble support matrices suitable for affinity chromatography have been activated in a similar fashion to create the hydrazide derivative (O Shannessy and Wilchek, 1990). 

Quench the reaction by immediate gel filtration on a desalting column. If a dextran-based resin is used for the chromatography, the support itself will react with sodium periodate to quench excess reagent. Alternatively, N-acetylmethionine may be added to quench the reaction, because the thioether of the methionine side chain will react with periodate to form sulfoxide or sulfone products (Geoghegan and Stroh, 1992). In addition, sodium... 

A second method of immunotoxin preparation by reductive amination involves the use a polysaccharide spacer. Soluble dextran may be oxidized with periodate to form a multifunctional crosslinking polymer. Reaction with antibodies and cytotoxic molecules in the presence of a reducing agent forms multivalent immunotoxin conjugates. The following sections discuss these options. 

Figure 21.15 A periodate-oxidized dextran polymer may be reacted with both an antibody and an intact toxin component using reductive amination to form a multivalent immunotoxin complex.

Figure 25.12 Dextran polymers can be oxidized with sodium periodate to create a polyaldehyde derivative. Note that additional oxidation may occur to cleave off another carbon atom and create an aldehyde on the adjacent C—OH group.

Protocol for Oxidizing Dextran with Sodium Periodate... 

Dissolve dextran (Polysciences) of molecular weight between 10,000 and 40,000 in the sodium periodate solution with stirring. 

Hydrazide derivatives also may be prepared from a periodate-oxidized dextran polymer or from a carboxyl-containing dextran derivative by reaction with te-hydrazidc compounds (Chapter 4, Section 8). A hydrazide terminal spacer provides reactivity toward aldehyde- or ketone-containing molecules. Thus, the hydrazide-dextran polymer can be used to conjugate specifically glycoproteins or other polysaccharide-containing molecules after they have been oxidized with periodate to form aldehydes (Chapter 1, Section 4.4). 

With such polysaccharides110 as the dextrans,149-163 which contain primarily a-D-(l — 6) linkages, periodate oxidation has been most useful. Each (1 — 6)-linked D-glucose unit contains three contiguous hydroxyl... 

This C-13 n.m.r. analytical technique has been referenced against 27 dextrans which had been studied by periodate-oxidation and permethylation fragmentation g.l.c.-m.s. techniques (14). 

Figure 3. Gel filtration of alkali-soluble xyloglucan on columns (95 x 1.5 cm) of Sepharose CL-6B. Pea microsomal membranes were incubated for various periods with GDP-[14C]fucose and unlabelled sugar nucleotides. Products were eluted with 0.1 M NaOH in 1 ml fractions. Molecular weights of dextran markers, 1 = 264000 D 2 = 70000 D 3 = 40000 D 4 = 10600 D Glc=glucose. Redrawn from Camirand and Maclachlan (20).

They are most commonly used plasma expanders. It is polysaccharide isolated from beet sugar which is formed by the action of Leuconstec mesenteroides. It is available in mainly two forms depending upon the molecular weight. Dextran 70 (mol. wt. 70,000) available in 6% solution and Dextran 40 (mol. wt. 40,000) available in 10% solution. They are infused intravenously in the treatment of shock. Dextran 40 acts more rapidly than dextran 70. It decreases the blood viscosity and prevents the sludging of RBC s. Dextran 70 remains in circulation for longer period (upto 24 hrs) and is slowly excreted by glomerular filtration. 

On the other hand, polymeric carriers can also be modified to introduce reactive groups. Polysaccharides such as dextran and inulin may be activated [149] by periodate oxidation to create aldehyde groups, by succinic anhydride activation to create carboxylic groups, or by p-nitrophenyl chloroformate activation to create reactive ester groups. 

---