Dextran adipic acid dihydrazide

The following protocols make use of the compounds adipic acid dihydrazide and carbohy-drazide to derivatize molecules containing aldehydes, carboxylates, and alkylphosphates. The protocols are applicable for the modification of proteins, including enzymes, soluble polymers such as dextrans and poly-amino acids, and insoluble polymers used as micro-carriers or chromatographic supports. 

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. 

To make an amine derivative of dextran, dissolve ethylene diamine (or another suitable diamine) in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2, at a concentration of 3 M. Note Use of the hydrochloride form of ethylene diamine is more convenient, since it avoids having to adjust the pH of the highly alkaline free-base form of the molecule. Alternatively, to prepare a hydrazide-dextran derivative, dissolve adipic acid dihydrazide (Chapter 4, Section 8.1) in the coupling buffer at a concentration of 30 mg/ml (heating under a hot water tap may be necessary to completely dissolve the hydrazide compound). Adjust the pH to 7.2 with HC1 and cool to room temperature. 

Spacer molecules used in this experiment were ( ) 1,2 Diaminoethane ( + ) 1,2 Diaminoethane ( ) Adipic Acid Dihydrazide ( ) Polyamine Dextran ( x ) Pectinamine. 

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See also in sourсe #XX -- [ ]

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