Hydrazide-Activated Enzymes

These techniques have been used to target, detect, or assay glycoproteins in solution or on cell surfaces by using hydrazide-activated enzymes, avidin, or streptavidin (Chapter 23, Section 5) (Bayer and Wilchek, 1990 Bayer et al., 1987a, b, 1990) and to form conjugates with glycoproteins. 

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). 

Dissolve a hydrazide-containing enzyme or other protein at a concentration of 10 mg/ml in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2. For the preparation of a hydrazide-activated enzyme see Chapter 16, Section 2.4. For modification with a hydrazide-containing probe, such as biotin-hydrazide, use a concentration of 5 mM in the phosphate buffer. For conjugation through the amine groups of a secondary molecule, dissolve the amine-containing protein at 10 mg/ml in 0.2 M sodium carbonate, pH 9.6. 

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 activation of enzymes using adipic acid dihydrazide and EDC is identical to the procedure outlined for the modification of (strept)avidin (Chapter 23, Section 5). Alternatively, hydrazide groups may be created on enzymes using the heterobifunctional chemoselective reagents described in Chapter 17, Section 2. 

As proof of principle, Lehn and coworkers individually synthesized all acyl hydrazone combinations from the 13 DCL building blocks and measured their inhibition of acetylthiocholine hydrolysis by ACE in a standard assay. They then established a dynamic deconvolution approach whereby the pre-equilibrated DCL containing all members is prepared, frozen, and assayed. Thirteen sublibraries were then prepared containing all components minus one hydrazide or aldehyde component, and assayed. Active components in the DCL were quickly identified by an increase in ACE activity, observed in sublibraries missing either hydrazide 7 or dialdehyde i, pointing to the bis-acyl hydrazone 7-i-7 as the most likely active constituent. This was in line with the individual assay data recorded earlier resynthesis of this compound characterized it as a low nanomolar inhibitor of the enzyme. 

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. 

Esters, amides, hydrazides, and carbamates can all be metabolized by hydrolysis. The enzymes, which catalyze these hydrolytic reactions, carboxylesterases and amidases, are usually found in the cytosol, but microsomal esterases and amidases have been described and some are also found in the plasma. The various enzymes have different substrate specificities, but carboxylesterases have amidase activity and amidases have esterase activity. The two apparently different activities may therefore be part of the same overall activity. 

Major hydrolysis reactions are ester and amide hydrolysis. These are catalyzed by a group of enzymes with overlapping substrate specificity and activity. Hydrazides can also undergo hydrolysis. Some of the newer drugs such as hormones, growth factors, and cytokines now being produced are peptides, and certain toxins are also peptides or proteins, so the role of peptidases may be important. 

Alexander"2 has tried to assess the effect of indole-3-acetic acid, (2,4-dichlorophenoxy) acetic acid, and maleic hydrazide on the soluble carbohydrates and enzyme systems in sugarcane leaves. All of the chemical treatments increased sucrose, total reducing value, D-fructose, and D-glucose in leaves (compared with the controls), with a maximum at nine days after applying about two g. per plant. The indole auxin increased sucrose most, followed by the phenoxy compound and the hydrazide D-glucose increased in the reverse order. Poor translocation from the leaves may have caused the temporary increase in leaf photosynthate. Alterations in the enzyme activities as a result of the chemical treatment are difficult to interpret, partly since so little is known about their relative importance, and partly because the activity in the controls varied by as much as 100% from one sampling period to the next. 

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. 

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