Conjugated enzyme preparations

Tests for extents of coupling are conveniently carried out by molecular-sieve chromatography of conjugated enzyme preparations on appropriate gel columns, and comparison of the elution... 

One limitation to this method should be noted. If the antibody-enzyme conjugate is prepared using antibody fragments such as Fab or F(ab )2, then nickel-chelate affinity chromatography will not work, since the requisite Fc portion of the antibody necessary for complexing with the metal is not present. 

We have examined in detail the properties of the conjugated enzymes we have synthesized. The results of carbohydrate attachment may be illustrated by considering typical properties that are changed by the modification process, with appropriate illustrations from the range of conjugates we have prepared and characterized. 

Effect of Protein Denaturants. Most of the conjugated enzymes we have prepared show greater resistance to inactivation than do the corresponding native enzymes when treated with protein... 

The formation of the GSH conjugate (or the J4 isomer of androstene-3,17-dione) is monitored at 30°C in a jacketed spectrophotometer micro-cell. The GSH-containing sodium phosphate buffer (0.1 M, 0.9 ml) of appropriate GSH concentration, is added first to the cuvette followed by the substrate in 0.05 ml ethanol (or methanol for d5-an-drostene-3,17-dione). The reaction is initiated by the addition of the enzyme preparation in the appropriate sodium phosphate buffer and... 

If liver cells (hepatocytes) are isolated and grown in culture, drugs are exposed to a similar array of enzymes. Because CYP enzymes are bound to the internal membrane fraction of hepatocytes, the liver can be homogenized and a preparation of vesicles of the hepatocyte endoplasmic reticulum called microsomes can be incubated with drug molecules. This preparation suffers because many of the soluble Phase II conjugation enzymes that are found in the cellular cytoplasm are lost. An alternative method for measuring microsomal metabolism involves isolation of the so-called S9 fraction , which includes the cytosolic soluble conjugation enzymes. 

Many HPLC analyses are carried out on biological samples such as blood, plasma, semm or urine. If urine analysis is to be carried out, the compound of interest is often excreted as the glucuronide or sulphate conjugate. These are very polar and are rarely determined directly. Samples are usually hydrolysed by acid, or an enzyme preparation to release the parent compound before analysis proceeds. 

An early indication that a widely used agricultural chemical might be metabolized to a nonpolar conjugate in plants came from an in vitro enzyme study with C-labeled surfactants of the Triton family. A crude particulate enzyme preparation from com shoots catalyzed the formation of fatty acid ester conjugates from the two 1"c-labeled polyethoxylated surfactants indicated below (Equation 32). The ester conjugates were formed primarily from palmitic and linoleic acids 0851). They were identified by mass spectrometry and by GLC analysis of hydrolysis products (J48). In vljro, rice and... 

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