Digestion with neuraminidase

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).

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. 

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). 

Glycoproteins containing sialic acid may be detected by staining with a cationic carbocyanine dye before and after digestion with neuraminidase a change in the colour of the stain from blue to red-purple indicates the presence of sialic acid. The n.m.r. spectra of iV-acetylneuraminic acid and its methyl ester in DMSO and in water have indicated that the acid exists predominantly in the /S-form in solution. The finding supports the assumption that 7V-acetyl-neuraminic acid, produced by the enzymic cleavage of its a-ketosides, leaves the catalytic site of Vibrio cholerae neuraminidase as the j3-anomer. 

As reviewed by Drzeniek (1973) and discussed elsewhere in this volume, the removal of sialic acids by digestion with neuraminidases is controlled by a number of factors. 

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.) ...

Fig. 9-221. Separation of oligosaccharides of a polyclonai antibody after enzymatic digest without (a) and with neuraminidase (b) as compared to a standard (c). - Chromatographic conditions see Fig. 9-220 peaks 1 to 4 correspond to the structures 1 to 4 in Table 9-17, N-acetyineuraminic acid (5) and N-glycolyIneuraminic acid (6) (taken from [369]).

Digestion of the gonadotrophins from intact and ovariectomized monkeys with neuraminidase appeared to abolish any differences in the molecular size. The ability of hCG to bind to rat Leydig cells and to stimulate the production of testosterone and cAMP has been examined. Sequential removal of the sugars led to a progressive loss of activity, which was attributed to the inability of the modified hCGs to stimulate adenyl cyclase, rather than to a failure to bind... 

Amylases with unusually fast electrophoretic mobilities have been found in the sera of certain patients. Since digestion of the amylases with neuraminidase markedly modified the electrophoretic mobility, it was concluded that neuraminic acid residues are incorporated into the enzyme, possibly by attachment to the glycoprotein. 

Digestion with a neuraminidase is the most commonly used histochemical technique for the identification of sialic acids and when positive is the most specific. In this procedure tissue sections are incubated with the neuraminidase and the intensity of their staining with Alcian blue pH 2.5 is compared to that of a serial section incubated under identical conditions, with the buffer but without neuraminidase and also to that of a serial section subjected to no pretreatment. Reduction in staining, in the enzyme treated section only, is evidence for the presence of sialic acids but a negative result (no reduction in staining) cannot be... 

Orosomucoid. — A glycopeptide obtained by pronase digestion of orosomucoid has been obtained and the carboxy-group of its terminal A-acetylneuraminic acid esterified with a carbodi-imide and then reduced with sodium boro-hydride. The reduced glycopeptide was shown to be resistant to hydrolysis by neuraminidase and other glycosidases. 

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