Neuraminidase Digestion

Treat section A with 1% potassium hydroxide in 70% ethanol for 5 min., and wash in water for 5-10 min. (saponification [KOH]). 

Treat sections A and B with V. cholerae neuraminidase— 00 Behringwerke units per ml in 0.05 M acetate buffer at pH 5.5 (containing approximately 0.1% calcium chloride). 

Treat section C with acetate buffer alone. 

Stain all four slides by the Alcian blue at pH 2.5 technique (see above). Results 

Loss of staining in A and/or B , by comparison with C is due to the removal of sialic acid. Loss of staining in slides A, B, and C by comparison with D indicates non-specific removal of a soluble entity. 

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Neuraminidase digestion of transferrin is performed from 6 pi of serum, 2.5 pi 1 M sodium acetate (pH 5.0), 15.5 pi double-distilled water and 1 pi neuraminidase (0.01 U). Incubation is carried out overnight at 37°C followed by dialysis against double-distilled water at 4°C for 12 h and subsequent drying by lyophilisation. Dried samples are resuspended in 1.5 ml 10 mM Tris (pH 7.0)10.9% NaCl and analysed by IEF, followed by silver staining as described above. 

In addition to H(O) substances, Matsumoto and Osawa198 reported hemagglutination inhibition by B and A substances and neuraminidase-digested, porcine submaxillary-mucin. Although Lea substance did not inhibit, a closely related milk oligosaccharide, lacto-N-fucopentaose II, did exhibit activity in this assay. On screening 22 invertebrate extracts, Baldo and coworkers found eel-serum-reactive material in 15 species.881... 

Arachis hypogaea Bandeiraea simplicifolia neuraminidase-digested A, B, O, or T antigen 202,710 /3-D-Galp-( 1—>3)-D-GalNAcp 201,617-619... 

Svasti, J., and Bowman, B. H., 1978, Human group-specific component. Changes in electrophoretic mobility resulting from vitamin D binding and from neuraminidase digestion. /. Biol. Chem. 253 4188. 

A glycoprotein that reacted with P. vulgaris lectin is secreted in all human saliva and ovarian-cyst fluid.739 Binding to P. vulgaris lectin was abolished by proteolysis (with trypsin or pronase), or mild, alkaline hydrolysis, but was unaffected by sequential digestion with neuraminidase (all detectable sialic acid was liberated) and /3-d-galactosidase (less than 1% of the galactose was released).739... 

Figure 1. Stepped collision energy LC-ESMS for gD-2 glycoprotein digested with neuraminidase/trypsin illustrating selectivity of the m/z 204 (A) and 366 (B) carbohydrate-selective markers when compared to the summed trace for m/z 400-2000 (C).

Figure 21-5 A, Poiyacrylam id e-gel electrophoresis of bone and liver alkaline phosphatases in human serum. Left, Mixture of two sera containing, respectively, entirely bone phosphatase and entirely liver phosphatase. Right, Mixture of the same two sera after each has been treated with neuraminidase for 0 minutes at 37 C.The anodai direction is downward.The more anodal zone is liver phosphatase. B, Densltometric scans of electrophoretic patterns shown in A. Broken line, Scan of mixture of untreated sera solid line, scan of mixture of sera treated briefly with neuraminidase. The anode is to the left. (From Moss DW, Edwords RK. In proved electrophoretic resolution of bone and liver alkaline phosphatases resulting from partial digestion, with neuraminidose. Clin Chim Acta 1984 143 i 77-82.) ...

Moss DW, Edwards RK. Improved electrophoretic resolution of bone and liver alkaline phosphatases resulting from partial digestion with neuraminidase. Clin Chim Acta 1984 143 177-82. 

Of interest are recent observations from Harris laboratory demonstrating that graded neuraminidase concentrations in placental alkaline phosphatase digests yielded up to eight bands (R16). Participation of neuraminidase-sensitive sialic acid in the K -dependent nitrophenyl phosphatase in isolated rat liver plasma membranes (El) has been reported. 

The acid is most conveniently obtained, in yields - of 40-60%, from milk, colostrum, submaxillary mucin, and meconium by hydrolysis of the constituent sialoproteins and oligosaccharides with 0.01 N sulfuric acid for one hour at 70°. It may also be obtained from these sources by enzymic digestion with neuraminidase (see Section VII). 

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