Protein addition, enzyme stabilization

Some metabolites of curcumin (particularly tetrahydrocurcumin) may also participate in producing the observed effects of curcumin in different models because these metabolites display greater stabilities than the parent curcumin molecule at physiological pH. Recent data show similar modes of action of curcumin metabolites regarding antioxidant enzyme induction and inhibition of multidrug-resistant proteins. " Additional data indicate that curcumin may even act against other types of diseases such as atherosclerosis " " and Alzheimer s disease. " - " ... 

It should be pointed out that the addition of substances, which could improve the biocompatibility of sol-gel processing and the functional characteristics of the silica matrix, is practiced rather widely. Polyethylene glycol) is one of such additives [110— 113]. Enzyme stabilization was favored by formation of polyelectrolyte complexes with polymers. For example, an increase in the lactate oxidase and glycolate oxidase activity and lifetime took place when they were combined with poly(N-vinylimida-zole) and poly(ethyleneimine), respectively, prior to their immobilization [87,114]. To improve the functional efficiency of entrapped horseradish peroxidase, a graft copolymer of polyvinylimidazole and polyvinylpyridine was added [115,116]. As shown in Refs. [117,118], the denaturation of calcium-binding proteins, cod III parvalbumin and oncomodulin, in the course of sol-gel processing could be decreased by complexation with calcium cations. 

The majority of the published investigations are concentrated onto the reaction conditions of enzymes in reverse micelles at low substrate concentrations, because high substrate concentrations in microemulsions influence their phase behaviour. Additionally, high substrate and enzyme concentrations often lower the enzyme stability to uneconomical values. At high enzyme concentrations the activity can be lowered due to the formation of protein aggregates. 

The active site is in a cleft between a large domain with a nonpolar core and a smaller (3-sheet domain that contains many hydrogen-bonded polar side chains (Figs. 12-3,12-4). Human lysozyme has a similar structure and properties.57-59 The T4 lysozyme has an additional C-terminal domain whose function may be to bind the crosslinking peptide of the E. coli peptidoglycan. Goose lysozyme is similar in part to both hen lysozyme and T4 lysozyme. All three enzymes, as well as that of our own tears, may have evolved from a common ancestral protein.60 On the other hand, Streptomyces erythaeus has developed its own lysozyme with a completely different structure.61 An extensive series of T4 lysozyme mutants have been studied in efforts to understand protein folding and stability.61-63... 

Propenaldehyde. Aldehyde group. Typically available as a 92% active, liquid. It is flammable, volatile, lachrymatory, and a strong irritant. Acrolein is a protein and enzyme poison. It is stabilized against polymerization by the addition of hydroquinone (oxidation results in polymerization, rendering acrolein inactive as a microbiocide). Although only small dose levels are required (1.5 to 3.0 ppm) to achieve threshold toxicity, rapid microbial resistance can occur. This product is seldom used today as the effort is often not worth the gain. 

Third generation biosensors very few cases reported. Active site usually deeply buried within the protein, thus enzyme engineering is required for protein modification to allow electrical wiring between enzyme and electrode. Advantages interferences could be completely eliminated. Disadvantages large amount of coenzyme required, low stability of the enzyme and relatively high applied potential for amperometric detection. Still require the addition of the coenzyme. 

In addition to its role at the active sites of hundreds of enzymes, there are cases where zinc serves a purely structural role. The most notable of these are the zinc fingers and some related twists and clusters. These occur in DNA binding proteins where they stabilize the correct binding sites. Figure 15-7 shows... 

Methods presently employed for obtaining correctly refolded proteins from inclusion body preparations are often all-or-none propositions. They typically consist of denaturant solubilization, in urea or guanidine, followed by dilution or dialysis (2). Recovery of native activity or structure may be aided by using additives (enzyme inhibitors, co-factors, oxidation-reduction couples, etc.), which act to stabilize the native-state protein conformation. However, because such efforts are time-consuming and tedious, systematic examinations of solution conditions for protein folding/unfolding are rarely performed. 

A number of other enzymopathic substances (e.g., pyruvate kinase. Chapter 13 and pyrimidine-5 -nucleotidase. Chapter 27), abnormal hemoglobins (Chapter 28), and abnormalities of the erythrocyte cytoskeleton (Chapter 10) may cause hemolytic anemia. Because many enzymes in the red cell are identical to those in other tissues, defects in these enzymes may have pleiotropic effects. Thus, in addition to hemolytic anemia, triose phosphate isomerase deficiency causes severe neuromuscular disease, and phospho-fructokinase deficiency causes a muscle glycogen storage disease (Chapter 13). Mutations that result in decreased enzyme stability are usually most strongly expressed in erythrocytes because of their inability to synthesize proteins. 

The conjugates can be stored at — 70 C after the addition of stabilizing proteins. However, activity disappears rather fast when frozen in dilute solutions. The addition of NaNj is not recommended since it is a potent inhibitor (above 10" M) of the enzyme. The addition of an equal volume of glycerol and storage at — 20°C in convenient aliquots also proved satisfactory in our laboratory. The sample then remains liquid and the required small quantities can be retrieved without the harmful, repeated freezing-thawing or the wasteful dispensing in very small aliquots. 

In addition to its acceptance of unnatural substrates, several other characteristics make NeuAc aldolase a useful synthesis catalyst. The cloning of the enzyme has reduced its cost and offers the potential to produce large quantities of new proteins with improved stability or with altered stereoselectivity. This approach could be used to extend the chain of a variety of aldoses by two carbon units. Although the optimal pH for activity of NeuAc aldolase is near pH 7.5 at 37 °C, the enzyme is active at pH 7-9.82.83 TTie protein is stable in the presence of oxygen and does not require added cofactors. - One drawback is that an excess of pyruvate (the less expensive reagent) must be used in synthetic reactions to shift the equilibrium towards the formation of product approximately 7 equiv. of pyruvate are needed to attain 90% conversion of ManNAc to NeuAc at equilibrium. It may be possible to avoid the need for an excess of pyruvate by coupling the synthesis of NeuAc to a more thermodynamically favored process. 

Several properties of these enzymes are shared irrespective of the identity of the flavin (FMN or FAD). Many react with sulfite to form N5 flavin adducts, which is readily detectable spectroscopically. In addition, they stabilize the benzoquinoid resonance structure of 8-mercaptoflavin when it is substituted for the namral prosthetic group, producing a characteristic intense absorbance band at 600 nm. They also stabilize semi-quinone. Since the pA), of the stabilized semiquinone is often low, the anionic rather than the neutral (blue) semiquinone is detected. However, there are a few cases where the pA), is experimentally accessible and both anionic and neutral semiquinones are observed. These general features are the consequence of a positive charge (contributed by either a protein residue or the positive end of a helix dipole) at the Nl—C2=0 locus of the flavin. 

The studies of elementary films formed in inverse emulsions and stabilized by different synthetic and natural surfactants revealed that the membrane electric conductivity experiences a sharp increase upon the addition of some biologically active surfactants. For instance, membrane conductivity may increase by five orders of magnitude when trace amounts of valinomycin antibiotic are introduced into the outer aqueous medium of lipid membrane. At the same time the membrane becomes permeable to potassium and hydrogen ions but impermeable to sodium ions. A sharp decrease in electric resistance of synthetic membranes is observed when proteins and enzymes with suitable substrates are introduced into them. By studying the properties of such membranes one may model important biological processes, e.g. the transfer of neural impulses. 

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