Glycoproteins modification with

Figures Selective protein modification using a keto amino acid, p-acetyl-L-phenylalanine. (a) Labeling of fluorescein hydrazide to the Z domain protein. Only the mutant protein containing p-acetyl-L-phenylalanine was labeled and became fluorescent, (b) A general method for preparing glycoprotein mimetics with defined glycan structure.

For sialylated ai-acid glycoprotein, although the intensity modifications with temperature can be explained by the presence of different types of quenching, the global profile is more complex than in the asialylated protein as the result of the difference in the local structures, around mainly the surface Trp residue. 

The Na/K ATPase has been extensively purified and characterized, and consists of a catalytic a subunit of around 95 kDa and a glycoprotein 0 subunit of approximately 45 kDa (Skou, 1992). The functional transporter exists as a dimer with each monomer consisting of an a and /3 subunit. Hiatt aal. (1984) have su ested that the non-catalytic jS subunit may be involved in the cottect insertion of the a subunit into the lipid bilayer and, therefore, it is conceivable that a modification of the 0 subunit structure may be reflected by changes in the catalytic activity of the a subunit. Therefore, in studies involving the manipulation of tissue glutathione levels, alterations of intracellular redox state may have an effect on substrate binding at an extracellular site on this ion-translocating protein. 

This same type of modification strategy also can be used to create highly reactive groups from functionalities of rather low reactivity. For instance, carbohydrate chains on glycoproteins can be modified with sodium periodate to transform their rather unreactive hydroxyl groups into highly reactive aldehydes. Similarly, cystine or disulfide residues in proteins can be selectively reduced to form active sulfhydryls, or 5 -phosphate groups of DNA can be transformed to yield modifiable amines. 

Carbohydrates and other biological molecules that contain polysaccharides, such as glycoproteins, can be specifically modified at their sugar residues to produce reactive formyl functionalities. With proteins, this method often allows modification to occur only at specific locals, usually away from critical active centers or binding sites. 

Since sialic acid is a frequent terminal sugar constituent of the polysaccharide trees on glycoproteins, this method selectively forms reactive aldehydes on the most accessible parts for subsequent modifications. The carbohydrate polymer of a protein provides a long spacer arm that can be used to conjugate another large macromolecule, such as a second protein, with little steric problems. 

BODIPY 530/550 C3 hydrazide is insoluble in aqueous solution, but may be dissolved in DMF or methanol as a concentrated stock solution prior to addition of a small aliquot to a reaction. Coupling to aldehyde-containing molecules occurs rapidly with the formation of a hydrazone linkage. The reaction may be done in buffered environments having a pH range of 5-10. However, modification of glycoproteins with this fluorophore may not yield satisfactory... 

---