Hydrazide proteins using

Derivatization with biotin hydrazide can be done directly on blots after transfer of proteins from SDS-PAGE gels. In general, a method described in ref, 2 can be followed with the exception that biotin hydrazide is used instead of... 

In glycoprotein detection systems the carbohydrate portions of proteins are oxidized with sodium metaperiodate to generate aldehydes that can react with hydrazides. A biotin hydrazide is used to attach biotin onto the oxidized carbohydrates and horseradish peroxidase-conjugated streptavidin is used for chemiluminescence-based detection (Glycoprotein Detection Module, Amersham Biosciences, Uppsala, Sweden). 

Zheng J-S, Tang S, Qi Y-K, Wang Z-P, Liu L (2013) Chemical synthesis of proteins using peptide hydrazides as thioester surrogates. Nat Protocols 8 2483-2495... 

TNBS has been used to measure the free amino groups in proteins (Habeeb, 1966 Kakade and Liener, 1969), as a qualitative check for the presence of amines, sulfhydryls, or hydrazides (Inman and Dintzis, 1969), and to specifically determine the number of s-amino groups of L-lysine in carrier proteins (Sashidhar et al., 1994). 

In aqueous solutions, the easiest method for forming this type of bond is to use the water-soluble carbodiimide EDC (Chapter 3, Section 1.1). For proteins and other water-soluble macromolecules, EDC reacts with their available carboxylate groups to form an intermediate, highly reactive, o-acylisourea. This active ester species may further react with nucleophiles such as a hydrazide to yield a stable imide product (Figure 1.109). 

Most proteins contain an abundance of carboxylic acid groups from C-terminal functionalities and aspartic and glutamic acid side chains. These groups are readily modified with bis-hydrazide compounds to yield useful hydrazide-activated derivatives. Both carbohydrazide and adipic acid dihydrazide have been employed in forming these modifications using the carbodi-imide reaction (Wilchek and Bayer, 1987). 

Calculate the protein concentration in the final preparation using its absorbance at 280nm or a colorimetric method, such as the Coomassie assay. (Note The presence of hydrazine or hydrazide groups on the protein will interfere with the BCA assay for total protein concentration.)... 

Add to the glycan solution the molecule to be labeled containing an available amine, hydrazine, or hydrazide group. For small molecule derivatization, the final concentration of the nucleophile in the glycan solution should be about 0.3 M to result in maximal efficiency of labeling. For protein modification, an aqueous reaction buffer should be used, and the protein should be as concentrated as possible. 

The following protocol describes the use of biotin-hydrazide to label glycosylated proteins at their carbohydrate residues. Control of the periodate oxidation level can result in specific labeling of sialic acid groups or general sugar residues (Chapter 1, Section 4.4). 

In some cases, the ability to modify glycans at the reducing end without reduction preserves the carbohydrate s native structure sufficiently to allow interactions with proteins that would otherwise not interact if the bond were reduced. Therefore, depending on the ultimate use of the biotinylated carbohydrate, using a hydrazide mediated conjugation process can have advantages over the use of amine-biotin compounds. 

Aldehyde particles are spontaneously reactive with hydrazine or hydrazide derivatives, forming hydrazone linkages upon Schiff base formation. Reactions with amine-containing molecules, such as proteins, can be done through a reductive amination process using sodium cyanoborohydride (Figure 14.21). 

Figure 17.4 Ketone derivatives of phenylalanine and mannose can be fed to cells to incorporate the monomers into proteins and glycans. The resultant modifications can be probed using hydrazide-containing reagents.

Hydrazide groups can react with carbonyl groups to form stable hydrazone linkages. Derivatives of proteins formed from the reaction of their carboxylate side chains with adipic acid dihydrazide (Chapter 4, Section 8.1) and the water-soluble carbodiimide EDC (Chapter 3, Section 1.1) create activated proteins that can covalently bind to formyl residues. Hydrazide-modified enzymes prepared in this manner can bind specifically to aldehyde groups formed by mild periodate oxidation of carbohydrates (Chapter 1, Section 4.4). These reagents can be used in assay systems to detect or measure glycoproteins in cells, tissue sections, or blots (Gershoni et al., 1985). 

Ahn, B., Rhee, S.G., and Stadtman, E.R. (1987) Use of fluorescein hydrazide and fluorescein thiosemicar-bazide reagents for the fluorometric determination of protein carbonyl groups and for the detection of oxidized proteins on polyacrylamide gels. Anal. Biocbem. 161, 245-257. 

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