Hydrazine hydrolysis has been found to be effective in the complete release of unreduced O- and N-linked oligosaccharides. Selective and sequential release of oligosaccharides can be accomplished by mild hydrazinolysis of the O-linked oligosaccharides at 60 °C initially, followed by N-linked oligosaccharides at 95 °C. Hydrazine hydrolysis leaves the glycan intact but results in destruction of the protein component (see Figure 1).
Complete details for this procedure are given in Methods in Enzymology.1 Care should be taken during all steps of this procedure as many of the reagents and reactions are extremely hazardous and highly reactive.
Glycan release is accomplished by the addition of fresh anhydrous hydrazine to a salt-free, freshly lyophilized glycoprotein sample. The volume of hydrazine should produce a protein concentration of 5 to 25 mg/ml. The reaction mixture should be capped immediately. For the release of both N- and O-linked glycans, incubation should be for 4 hours at 95 °C. For the selective release of O-linked glycans, incubation at 60 °C for 5 hours is suitable.
Excess unreacted hydrazine can be removed under high vacuum at a temperature not exceeding 25 °C. The vacuum pump should be equipped with an activated charcoal/alumina trap. Addition of small aliquots of anhydrous toluene may be required to bring the sample to complete dryness.
Acetyl groups attached to the carbohydrate through an amine may be removed by hydrazinolysis. These groups must be replaced to stabilize the reducing ends of the released oligosaccharides. The dried sample is re-N-acetylated while on ice by the addition of ice-cold saturated aqueous sodium bicarbonate, followed immediately by the addition of acetic anhydride. The sample is mixed gently and incubated at room temperature for 10 minutes. A second aliquot of acetic anhydride equal in volume to the first is added to the reaction and incubated for an additional 20 minutes. The volume of the two aliquots should result in a 5× molar excess of acetic anhydride over the amine content of the protein. The volume of sodium bicarbonate added should yield a final concentration of 0.5 M acetic anhydride in the reaction mixture.
A small amount of the released glycan pool may exist as the acetohydrazide derivative. These derivatives may be converted to the unreduced glycans by resuspension of the dried glycan pool in 1 mM Cu(II) acetate in 1 mM acetic acid and incubation at room temperature for one hour.
Dowex® 50W×2 can be used to remove the excess cations by washing the sample through the resin with water.
Glycan and degraded protein components can be separated by gel filtration or by paper chromatography.
Note: Use of hydrazinolysis for release and isolation of glycans is covered by U.S. patents 5,539,090 and 6,180,779, and their foreign equivalents.
References: 1. Patel, T.P. and Parekh, R.B., Release of oligosaccharides from glycoproteins by hydrazinolysis. Meth. Enzymol., 230, 57-66 (1994). 2. Patel, T., et al., Use of hydrazine to release in intact and unreduced form both N- and O-linked oligosaccharides from glycoproteins. Biochemistry, 32, 679-693 (1993). 3. Bendiak, B., and Cumming, D.A., Purification of oligosaccharides having a free reducing-end from glycopeptide sources. Carbohydr. Res., 151, 89-103 (1986). 4. Makino, Y., et al., Structural analysis of N-linked sugar chains of human blood clotting factor IX. J. Biochem. (Tokyo), 128, 175-180 (2000). 5. Takasaki, S., et al., Hydrazinolysis of asparagine-linked sugar chains to produce free oligosaccharides. Meth. Enzymol., 83, 263-8 (1982).