Enzymes salts using

Levels of a number of metabolites as well as a number of enzymes in body fluids are indicative of disease conditions. Many of the enzymatic reactions mentioned above have been used in solution clinical assays as well as in test strips.446,497-508 512-515 Assays for hydrogen peroxide and the enzyme peroxidase using NADH and a tetrazolium salt have been de-scribed.509,5io Assays of exogenous substances (e.g., drugs or their metabolites) also utilize this chemistry. The determination of alcohol using alcohol dehydrogenase is an example.511 As mentioned above, the assay of enzyme levels can also be achieved using tetrazolium salts.516-520... 

Synthesis of sucrose 6 -phosphate by an enzymic method using uridine 5 -(a-D-glucopyranosyl diphosphate) plus D-glucose 6-phosphate has been reported.126-131 The first, unambiguous, chemical synthesis of sucrose 6 -phosphate was achieved by Buchanan and coworkers.18 The reaction of 2,3,4,6,l, 3, 4 -hepta-0-acetylsucrose, prepared by five steps of synthesis, with cyanoethyl phosphate in pyridine gave a crude product from which sucrose 6 -phosphate was isolated as the barium salt. 

The authors then used a modification of their Lipase-AK route to produce the natural enantiomer, as described in detail in the chapter by Kenji Mori in this volume. Instead of using the enzyme to execute a stereoselective monohydrolysis of meso diacetate 140, the enzyme was used to esterify selectively one of the hydroxy groups of meso diol 128, resulting in the antipodal hydroxyester. After oxidation of the free hydroxyl to the acid, and recrystallization of its salt with (JR)-l-naphthylethylamine, the purified acid was then carried through the remaining steps to furnish the chiral pheromone compound (see the chapter by Kenji Mori in this volume). 

A particularly interesting, and extremely simple, method of activating enzymes for use in nonaqueous media is lyophilization in the presence of nonbuffer salts. In the process, a great deal about excipient-enzyme-water interactions has been learned along with an appreciation of how enzymes adjust to novel microenvironments while retaining their intrinsic catalytic properties. 

Eppler et al. [103] viewed these results as having a potential relationship to salt-activated enzyme preparations, particularly in relation to the mobility of enzyme-bound water. Specifically, the authors examined both water mobility [as measured by T2-derived correlation times, (tc)D20] and NaF-activated enzyme activity and observed a linear relationship. This suggests that the salt-activated enzymes contain a more mobile water population than salt-free enzymes, which facilitates a more aqueous-like local environment and dramatically increases enzyme activity through increased flexibility. Therefore, enzyme activation appears to correlate with the properties of enzyme-associated water. Once again, the physicochemical properties of water dictate enzyme structure, function, and dynamics. Hence, salt activation has proven to be a useful technique in activating enzymes for use in organic solvents and has provided a quantitative tool to better understand the role of water in enzymatic catalysis in dehydrated media. 

In the tanning process hides are first washed or soaked, hair and keratinous debris are removed, bated (enzymes are used to break down non-collagenous components, which are washed out) and the hide is acid-pickled to prepare for the addition of the chromium salt. Contemporary processes are exclusively based on one-bath procedures and utilize chromium(III). The older two-bath process is now obsolete, mainly because it involved the in situ reduction of chromate, a major environmental and toxicological hazard (cf. chromate toxicity p. 947) to chromium(III) on the hide. A useful review of the history of chromium tannage processes is available.1205... 

DNA is precipitated from aqueous solutions by ethanol or isopropanol in the presence of salt. The amount of alcohol and salt depends on the type of salt that one wishes to use (Table 6.2). The type of salt used depends largely on downstream applications for which the DNA is to be used. For example, precipitation in the presence of ammonium acetate removes small molecules such as nucleotides, and the DNA can be used for many enzymatic reactions. On the other hand, the phosphorylating enzyme, T4 kinase, is inhibited by ammonium ions, and unless DNA is reprecipitated in the presence of salts other than ammonium acetate, the phosphorylation reaction may be inhibited. For most routine purposes, alcohol precipitation of DNA with sodium acetate is preferred over sodium chloride because of the higher solubility of the acetate salt in ethanol. Selection of isopropanol or ethanol is more of a convenience than a rule. Although isopropanol precipitation requires an equal volume of isopropanol for the precipitation of DNA, that with ethanol requires 2 volumes and hence can increase the total volume... 

That ionic immobilization is strongly dependent on the pH value and salt concentrations during immobilization and of the reaction mixture in which the enzyme is used cannot be overemphasized. If these parameters are not taken into... 

The presence of crown ethers in aqueous medium before lyophilization can greatly enhance the activity of proteases (e.g., chymotrypsin, subtilisin, trypsin) for peptide synthesis in organic solvents. - The crown ether must also be rinsed away from the lyophilized enzyme before use. The activity enhancement is solvent dependent. It is speculated that crown ethers accelerate enzymatic rates either by preventing a salt bridge from forming in the enzyme s secondary structure or inducing microscopic changes in the structure of the solid phase. ... 

A new area of research is the control of pH for nonaqueous enzyme reactions using organic pH buffers. - These buffer materials, which are soluble in nonaqueous media, strongly control the effect of pH on the reaction, and are able to override the pH memory of the enzyme before lyophilization or immobilization from an aqueous solution. - Typically, buffer salts employed are oppositely charged (e.g., / -COOH and / -COONa+, where R is hydrophobic in nature). It is the ratio of these two forms that controls the enzymatic rate. The buffers are believed to function by displacing hydrogen atoms of carboxylic acid groups on the surface of the enzyme, . - ... 

Upon an extensive study, we found a new chemiluminescence substrate for the enzyme iimnunoassay using a new 1,2-dioxetane derivative, 5-t-butyl-4,4-dimethyl-l-(3 -phosphoryloxy)phenyl-2,6,7-trioxabicyclo[3.2.0]heptane disodium salt as shown in Scheme l. °... 

Preparation of Enzyme. Separation and purification of the enzyme from the liver of the common squid were as follows Squid livers were mashed and dispersed in five volumes of distilled water, then acidified to pH 4.0. Much oil was eliminated. This was followed by ultrafiltration, salting out with ammonium sulfate (50% saturation), dialyzing and freeze-drying in vacuo, to yield a crude enzyme. A purified enzyme was obtained from this crude enzyme by using column chromatography. Active fractions were separated by cation exchange resin. Mono S (Pharmacia) and further purified by gel-flltration column of Superdex 75 (Pharmacia). The active fractions were collected as the purified enzyme. 

Physical adsorption relies on non-specific physical interaction between the enzyme protein and the surface of the matrix. In this method only weak bonds are involved, e.g. hydrogen bonds, multiple salt linkages, van der Waals forces, which on one hand is less desruptive towards enzyme, while on the other hand leads to desorption from the surfaces once there are changes in temperature, pH, ionic strength etc. Physical forces are non-specific and further adsorption of other proteins or other substances can occur as the immobilized enzyme is used which may alter the properties of the immobilized enzyme. 

There is a need to keep track of the protein purification studies if it is an enzyme then use its enzyme activity or if it is a colored protein, then monitor its specific wavelength of absorbance. But it must maintain biological conditions of pH, salt concentration, and temperature to keep the protein from denaturing and losing its biological activity [26]. 

Uses Thickener, consistency agent, stabilizer, water retention aid in water-based prods., latex paints Features Enhanced enzyme resist. delayed solubility Properties Whitish powd. 98% < 425 m particle size sol. in water vise. 3000 0600 mPa-s-(1%, 20 C), 50,000 mPa-s-(2%, 20 C) pH neutral (1%) nonionic 4% max. water, 4.5% max. salt Use Level 0.2-0.5% on total paint wt. 

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