Hydrazides, immobilization

Another route to the formation of a hydrazide on a surface is to use an aldehyde-containing particle (such as HEMA/acrolein copolymers) and subsequently modify the aldehydes to form hydrazone linkages with bis-hydrazide compounds, which then can be stabilized by reduction with sodium cyanoborohydride (Chapter 2, Section 5). The resulting derivative contains terminal hydrazides for immobilization of carbonyl ligands (see Figure 14.18). 

Brillhart, K.L., and Ngo, T.T. (1991) Use of microwell plates carrying hydrazide groups to enhance antibody immobilization in enzyme immunoassays./. Immunol. Meth. 144, 19-25. 

Hoffman, W.L., and O Shannessy, D.J. (1988) Site-specific immobilization of antibodies by their oligosac-chanide moieties to new hydrazide derivatized solid supports. J. Immunol. Meth. 112, 113-120. 

Horak, D., Karpisek, M., Turkova, J., and Benes, M. (1999) Hydrazide-functionalized poly(2-hydroxye-thyl methacrylate) microspheres for immobilization of horseradish Peroxidase. Biotechnol. prog. 15, 208-215. 

D.J.O. Shannessy and W.L. Hoffman, Site-directed immobilization of glycoproteins on hydrazide-containing solid supports. Biotechnol. Appl. Biochem. 9, 488—496 (1987). 

Hydrazide method, for covalent ligand immobilization, 6 396t Hydrazine, 70 727 73 562-607. See also Anhydrous hydrazine analytical methods for, 73 588-589 aqueous grades of, 73 585 chemical properties of, 73 566-576 as a corrosion inhibitor, 73 595 economic aspects of, 73 584-585 electrode potentials of, 73 566t explosive limits of, 73 566 handling and storage of, 73 586-588 as a hazardous material, 73 585... 

Figure 3.10 Immobilization of aldehyde oligonucleotides in hydrazide gel. (From Khrapko, K.R. et al.,/. DNA Sequencing Mapping, 1,375-388,1991 [www.tandf.co.uk/ journals]. With permission.)...

Carbohydrates are easily oxidized by periodate to aldehydes which react with primary amines or hydrazines. If the formed hydrazide carries a specific ligand, e.g., biotin or digoxigenin, these ligands immobilized via the blotted macromolecule are very sensitively detected by the respective enzyme conjugates. 

Covalent immobilization of lipase on nylon fibers has been done, using the enzymes carbohydrate groups as chemical link. Oxidation of the lipases carbohydrates with periodate provides aldehyde groups for the binding to hydrazide activated nylon (Lopez, Braun Klein, 1996). 

While the batch process is the dominant one in current use, researchers and companies have attempted to create continuous bioreactor systems. Lopez et al. immobilized Candida rugosa in polymethacrylamide hydrazide beads and polyurethane foam 3 with the intent to achieve the continuous production of lipase enzymes. Despite flow problems with the polyurethane foam, it showed high lipolytic activity. Biomass buildup was problematic. Feijoo et al. immobilized Phanerochaete chry-sosporium on polyurethane foam in packed bed bioreactors under near-plug flow conditions. Continuous lignin peroxidase production was accomplished, the rate of which was studied as a function of recycle ratio. 

Arenkov et al. prepared poly(acrylamide) gel pads for use in protein microarrays [199], The gels were prepared by photopolymerization of acrylamide and crosslinkers. Capture probes were immobilized, either by use of glutaraldehyde or by converting some of the acrylamide groups into hydrazides and subsequent coupling of aldehyde-modified antibodies to the pending hydrazide groups. Then, immunoassays were performed on the pads, either assays with directly labeled analytes or sandwich assays. Furthermore, the gel pads were used for enzyme activity studies. 

DNA-conjugated polymers were prepared from polyallylamine or polyacrylic acid (PAA) modified with ssDNA as a probe, and thioctic acid (TA) or 3-(pyridyldithio)propionyl hydrazide (PDPH) for self-assembled immobilization. The oligonucleotides sequences of the probes and of the fully matched and unmatched DNAs were S -TCC-TCT-TCA-TCC-TGC-TGC-TAT-GCC-TCA-TCT-T-R, 5 -AGA-TGA-GGC-ATA-GCA-GCA-GGA-TGA-AGA-GGA-3, ... 

Lee M, Shin I. Eacile preparation of carbohydrate microarrays by site-specific, covalent immobilization of unmodified carbohydrates on hydrazide-coated glass slides. Org. Lett. 2005 7 4269-4272. 

Lee MR, Shin I. Fabrication of chemical microarrays by efficient immobilization of hydrazide-linked snbstances on epoxide-coated glass snrfaces. Angew. Chem. Int. Ed. Engl. 2005 44 2881-2884. 

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]. 

Periodate Oxidation mainly of polysaccharide supports has become a popular activation technique for protein immobilization [149]. Sodium periodate (Nal04) can react with vicinal cw-hydroxyl groups on cellulose, dextran, or any other diols to produce aldehyde groups. These aldehyde groups can easily be transformed into secondary amines by reductive oxidation or to hydrazides by reaction with dihydrazine. Further attachment of ligands or spacer molecules can be performed via primary amino groups. 

---