Enzyme hydrazide derivatives

Ghosh, S.S., Kao, P.M., and Kwoh, D.Y. (1989) Synthesis of 5 -oligonucleotide hydrazide derivatives and their use in preparation of enzyme-nucleic acid hybridization probes. Anal. Biochem. 178, 43-51. 

Neuraminidase has been immobilized to give a water-insoluble derivative with retention of enzymic activity by covalent reaction with agarose cyclic imidocarbonate, a hydrazide derivative of agarose cyclic imidocarbo-nate, and periodate-oxidized controlled-pore glass (CPG)-glycophase. ... 

Figure 23.10 Glycoproteins may be oxidized with sodium periodate to generate aldehyde residues. These may be specifically labeled using a hydrazide-streptavidin derivative through hydrazone bond formation. Subsequent detection may be done using biotinylated enzymes.

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). 

The insolubilization of NAD and AMP and the uses of these supports have already been described, as has the use of hydrazide-agarose for immobilization. Other insolubilized nucleotide affinity columns have also been described. For example, Olsen [101] isolated galactosyltransferase from whey using a UDP-Sepharose affinity column. GTP coupled to a hydrazide Sepharose derivative was used to isolate D-erythro-dihydroneopterin triphosphate synthetase, the first enzyme for folate biosynthesis in Lactobacillus plantarum [102]. ATP-agarose has been used in the purification of heavy meromysin, elution being effected with ATP [103]. 

When A -nucleophiles such as ammonia, amines or hydrazine are subjected to acyl-transfer reactions, the corresponding Af-acyl derivatives - amides or hydrazides - are formed through interception of the acyl-enzyme intermediate by the iV-nucleophile (Schemes 2.1 and 3.21) [262]. Due to the pronounced difference in nucleophiU-city of the amine (or hydrazine) as compared to the leaving alcohol (R -OH) (Scheme 3.21), aminolysis reactions can be regarded as quasi-irreversible. Any type of serine hydrolase which forms an acyl-enzyme intermediate (esterases, lipases, and most proteases) is able to catalyze these reactirms. Among them, proteases such as subtilisin and peniciUin acylase and lipases from Candida antarctica and Pseudomonas sp. have been used most often. 

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