Deglycosylation chemical

The once popular deglycosylation with hydrofluoric acid requires longer response times in comparison to trifiuoromethanesulfonic acid and has smaller yields. In addition, special equip- 

The fourth method comes from Rademaker et al. (1998). These people remove 0-glycosidi-cally bound sugar chains via P-elimination with ammonium hydroxide. This sounds complicated, but is simple. You incubate the protein or glycopeptide in 25% ammonium hydroxide for 18 h at 45° C. The protein remains intact except for the fact that the sugar chain is replaced 

The Rademaker method has been refined by Hanisch et al. (2001). Franz-Georg Hanisch P-eliminates with ethylamine or methylamine. This has the advantage that the sugar chain bearing threonines and serines is labeled with the alkylamines, because an alkylamine is incorporated in place of the cleaved oligosaccharide. The alkylaminylated amino acid differs in MW strongly from the source amino acid (serine or threonine) and thus you can determine the location of the protein glycosylation with the mass spectrometer. 

you can choose among five methods trifluoromethanesulfonic acid, hydrofluoric acid, oxidation and P-elimination, and two P-elimination methods (overview Table 9.4). I advise against other methods. Hydrazinolysis (see Section 9.5.2.1) may split the sugar from glycoproteins, but it also cleaves peptide bindings. The same is true for treatment with NaOH/boron hydride (Rehm et al. 1986). 

Trifluoromethanesulfonic acid N-glycosidic and 0-glycosidic sugar chains Removes up to 90% of the sugar chains. Inconvenient. Peptide bindings are cleaved at temperatures over 25° C. Membrane proteins aggregate. 

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When using ricin A chains, it has been found that chemical deglycosylation of the subunit prevents its nonspecific binding to receptors for mannose on certain cells of the reticuloendothelial system (Vitetta and Thorpe, 1985 Ghetie et al., 1988, 1991). 

Ghetie, V., Ghetie, M.-A., Uhr, J. W., and Vitetta, E. S. (1988) Large scale preparation of immunotoxins constructed with the Fab fragment of IgGl murine monoclonal antibodies and chemically deglycosylated ricin A chain. /. Immunol. Methods 112, 267—277. 

T. A. Gerken, R. Gupta, and N. Jentoft, A novel approach for chemically deglycosylating O-linked glycoproteins. The deglycosylation of submaxillary and respiratory mucins. Biochemistry, 31 (1992) 639-648. 

J. C. Hong and Y. S. Kim, Alkali-catalyzed beta-elimination of periodate-oxidized glycans a novel method of chemical deglycosylation of mucin gene products in paraffin embedded sections, Glycoconj. J., 17 (2000) 691-703. 

Enzyme Isolation and Antiserum Preparation. Periplasmic CA was prepared from air-grown Chlamydomonas reinhardtii (137 mt" ") by p-methylaminobenzenesulfonamide affinity chromatography as described by Yang et al, (6). Antiserum against the isolated enzyme was prepared in rabbits as we described previously (7). Chemically deglycosylated CA was prepared by trifluoromethanesulfonic acid hydrolysis (8,9). 

Fig. 9.5 Overlay presentation for the conformational change of the Glc3 sugar ring. Panel A is for glycosylation step, and panel B is for deglycosylation step. Red color is for ES or EI2, green color is for transition states, while purple color is for Ell and EP [69]. Reprinted with permission from (Liu J, Wang X, Xu D (2010) QM/MM study on the catalytic mechanism of cellulose hydrolysis catalyzed by cellulase Cel5A from addothermus cellulolyticus, J Phys Cham B 114 1462-1470). Copyright (2010) American Chemical Society...

The toxins used for the chemical construction of ITs are bRT, RTA in deglycosylated form (dgRTA), two ribosome-inactivatiing proteins (RIPs) (PAP and saporin), and PE. The preparation of some of these toxins is described in Subheading 3.3.1. It should be noted that presently almost all plant and bacterial toxins used for the preparation of ITs can also be expressed in recombinant form in E. coli. 

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