Sialidases immobilized

The following experiments arc at variance with such a clearance mechanism. Incubation of rabbit erythrocytes with V. cholerae sialidase in the presence of inhibiting concentrations of Neu2en5Ac protects surface sialic acids and, correspondingly, survival of the cells in circulation.142 Treatment of erythrocytes by V. cholerae sialidase immobilized on Sepharose 4B, allowing complete separation of the enzyme from the cells after incubation, resulted in engulfment of these cells by macrophages at a rate similar to that observed with cells treated with the soluble enzyme. 1 1... 

An interesting electrochemical method for the determination of bound sialic acid has been developed, making use of a potentiometric four-channel thick-film sensor [236]. The sialidase sensor consists of a bilayer of a membrane containing Clostridium perfringens sialidase immobilized in a poly(vinyl acetate)-polyethylene copolymer, which is placed on top of an fT -selective poly(vinyl chloride)-poly(vinyl acetate) indicator membrane. The enzyme-induced release of bound sialic acid leads to a concomitant decrease in pA a of the carboxyl function of sialic acid. This decrease affords a local pH change inside the sialidase-containing sensor membrane, which is monitored by the H -selective indicator membrane. The pH optimum of the sialidase sensor was pH 4 for sialyllactose, mucin and colominic acid. 

Problems arising due to the removal of sialidase from substrates or its adherence to cells or isolated membranes can be overcome with immobilized sialidase. The use of V. cholerae and C. perfringens sialidases immobilized on Sepharose 4 B or controlled-pore glass has been demonstrated with a variety of substrates including cells (Parker et al. 1977, Corfield et al. 1978, Bazarian and WiNGARD 1979, Depierre / al. 1980). These enzymes can be used repeatedly and if suitable, also at higher temperatures. Complete desialylation, dependent on enzyme specificity, has been demonstrated using small columns of immobilized... 

Thiem,J. and Sauerbrei, B. (1991) Chemoenzymic synthesis of sialylohgosaccharides with immobilized sialidase. Angevr. Chem. Int. Ed. Engl., 30, 1521-1523. 

Complete desialylation of erythrocytes is not necessary for erythrocyte sequestration liberation of 15-20% of membrane sialic acids was found to be sufficient for a significant diminution of the life-time of erythrocytes.481,482 For an in vitro system with rat-peritoneal macrophages (see later), this threshold value was found to be only 10% when using soluble V. cholerae sialidase, but it was —30% when the erythrocytes were treated with the same enzyme immobilized on Sepharose.488... 

Thiem J, Sauerbrei B. Chemoenzymatic syntheses of sialy- 27. loligosaccharides with immobilized sialidase. Angew. Chem. Int. 

Thiem and Sauerbrei examined this concept to determine whether various sial-idases could be used synthetically [52]. The rate of condensation, or reverse hydrolysis, was found to be negligible. However, the product hydrolysis rate was competitive with the rate of transsialylation to an alcohol acceptor. In an attempt to minimize product loss, reactions were stopped after 65-75% of the starting material had been consumed. Interestingly, a mixture of a-2,3 and a-2,6 regioisomers was obtained for reactions with an immobilized Vibrio cholerae sialidase (Scheme 41). In all cases, the a-2,6 isomer predominated, probably because of a combination of faster hydrolysis of the a-2,3 products (123 —> 1 + 125) and lesser steric hinderance of the primary alcohol. Variations in the donor/acceptor ratio (1 7 optimized) had an effect on both reaction yield and regioselectivity, although most of the examples afforded only 2-3 1 a-2,6/a-2,3 product ratios in 14-20% overall yield. 

Once chemoenzymatically produced, these unnatural sialosides have demonstrated utility in a variety of biological applications. A sialoside consisting of 9-0-acetylated sialyl Tn antigen (sTn) was produced by one-pot synthesis and conjugated to human serum albumin to be used to evaluate the role of acetylation in carbohydrate-protein interactions. Para-nitrophenol-tagged sialosides can be used in a colorimetric assay to evaluate sialidase specificity. Biotin-tagged sialosides have been immobilized on a streptavidin-coated surface to study protein-carbohydrate interactions with surface plasmon resonance techniques. Size-defined... 

Recent studies have implicated the existence of a hydrophobic centre distinct from the catalytic centre in V. cholerae (Keilich et al. 1979) and C. perfringens (Corfield et al. 1980) sialidases. The detection of multiple sialidase activities in bacterial, protozoan and mammalian sialidase preparations using immobilized N-(4-nitrophenyl)-oxamic acid columns has supported these observations (Brossmer et al. 1977 b, Crampen et al 1979, Ziegler et al 1980). Those sialidases showing affinity for the immobilized ligand also showed low activity with synthetic... 

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