Proteins/enzymes stabilization

A simple way of evaluating the affect of a single nucleotide polymorphism on enzyme function is to perform transient transfection assays in COS-1 cells. Western blot analysis, with anti-UGTl A antibody, is used to determine protein expression levels of the variants, which often correlate with protein stability. Enzyme kinetic analysis is used to investigate the functional impact of amino acid substitutions. 

In acidic solution, the degradation results in the formation of furfural, furfuryl alcohol, 2-furoic acid, 3-hydroxyfurfural, furoin, 2-methyl-3,8-dihydroxychroman, ethylglyoxal, and several condensation products (36). Many metals, especially copper, cataly2e the oxidation of L-ascorbic acid. Oxalic acid and copper form a chelate complex which prevents the ascorbic acid-copper-complex formation and therefore oxalic acid inhibits effectively the oxidation of L-ascorbic acid. L-Ascorbic acid can also be stabilized with metaphosphoric acid, amino acids, 8-hydroxyquinoline, glycols, sugars, and trichloracetic acid (38). Another catalytic reaction which accounts for loss of L-ascorbic acid occurs with enzymes, eg, L-ascorbic acid oxidase, a copper protein-containing enzyme. 

Y. Mozhaev, I. Berezin, and K. Martinek, Structure stability relationship in proteins Fundamental tasks and strategies for the development of stabilized enzyme catalysts for biotechnology, CRC Crit. Rev. Biochem., 23, 235 (1988). 

Enzymatic reactions are influenced by a variety of solution conditions that must be well controlled in HTS assays. Buffer components, pH, ionic strength, solvent polarity, viscosity, and temperature can all influence the initial velocity and the interactions of enzymes with substrate and inhibitor molecules. Space does not permit a comprehensive discussion of these factors, but a more detailed presentation can be found in the text by Copeland (2000). Here we simply make the recommendation that all of these solution conditions be optimized in the course of assay development. It is worth noting that there can be differences in optimal conditions for enzyme stability and enzyme activity. For example, the initial velocity may be greatest at 37°C and pH 5.0, but one may find that the enzyme denatures during the course of the assay time under these conditions. In situations like this one must experimentally determine the best compromise between reaction rate and protein stability. Again, a more detailed discussion of this issue, and methods for diagnosing enzyme denaturation during reaction can be found in Copeland (2000). 

Phytostabilization on the root membranes. Proteins and enzymes directly associated with the root cell walls can bind and stabilize the contaminant on the exterior surfaces of the root membranes. This prevents the contaminant from entering the plant. 

In general, biomolecules such as proteins and enzymes display sophisticated recognition abilities but their commercial viability is often hampered by their fragile structure and lack of long term stability under processing conditions [69]. These problems can be partially overcome by immobilization of the biomolecules on various supports, which provide enhanced stability, repetitive and continuous use, potential modulation of catalytic properties, and prevention of microbial contaminations. Sol-gel and synthetic polymer-based routes for biomolecule encapsulation have been studied extensively and are now well established [70-72]. Current research is also concerned with improving the stability of the immobilized biomolecules, notably enzymes, to increase the scope for exploitation in various... 

Kumar, C.V. and Chaudhari A. (2003) Unusual thermal stabilities of some proteins and enzymes bound in the galleries of layered alpha-Zr(IV) phosphate/phosphonates. Microporous and Mesoporous Materials, 57,181-190. 

Mesoporous silicas have characteristics of high specific surface areas and pores with defined dimensions and uniform distribution. These features make mesoporous systems ideal candidates as host materials to guest bio-molecules. Protein stability may be enhanced due to reduced autolysis in the case of protease enzymes, and more generally reduced protein aggregation, as a result of the separation of the molecules adsorbed on the surface. 

The are several clearance and toxicological aspects that have to be considered in the drug discovery process such as metabolic stability, enzyme selectivity, CYP inhibition and type of inhibition. Among these factors, the prediction of the site of metabolism has become one of the most successful parameters for prediction. The knowledge of the site of metabolism enhances the opportunity to chemically modify the molecule to improve the metabolic stability. There are several approaches based on database mining, chemical reactivity, protein interaction or both that have been developed for the prediction of this property, with different degree of success and applicability. 

The desymmetrization of l-alkylbicyclo[3.3.0]octane-2,8-diones can be achieved in a facile coenzyme-independent enzymatic reaction in buffer. Alkyl chains in the 1 -position of up to at least five carbon atoms are tolerated. The yields of the crude keto-acids are essentially quantitative, and the enantiotopic discrimination by the enzyme is usually excellent." Work remains to be done on the optimization of this biocatalyst with respect to protein stability and reaction engineering, but it remains a unique and intriguing possibility for the generation of interesting intermediates bearing multiple chiral centres. 

Although in humans only MsrBl is a selenoprotein, the depletion of selenium from the diet of mice led to increases in both R and S stereoisomers. This was not initially explained, yet a subsequent study has shown that small molecule selenols (organic selenocysteine homologues) could act as efficient electron donors in vitro for MsrA enzymes. ° This effect has only been shown in vitro, but the possibility that small molecular selenium reductants, or more likely that some selenoproteins that contain reduced selenols (in redox-active motifs) is quite intriguing. Several small selenoproteins do not have real roles and reside in nearly all subcompartments of the cell (mitochondria, ER) where electron donors for Msr enzymes are probably critical to maintain protein stability. Low selenium nutritional status would then have a significant impact on all methionine oxidation, as Future studies to address selenium nutrition and methionine oxidation could prove to be... 

Persons with mutations that partially destroy G6PDH activity may develop an acute, episodic hemolysis. Certain mutations affect the stability of G6PDH, and, because erythrocytes cannot synthesize proteins, the enzyme is gradually lost over time and older red blood cells lyse. This process is accelerated by certain drugs and, in a subset of patients, ingestion of fava beans. In the United States, the most likely cause of a hemolytic episode in these patients is overwhelming infection, often pneumonia (viral and bacterial) or infectious hepatitis. 

The ability to biosynthetically incorporate noncoded amino acids into proteins site-specifically has facilitated studies not previously possible. These include studies of protein stability, the initiation of protein translation, electron transfer, protein-protein and protein-membrane interactions, reversal of enzyme substrate specificity, and structure-function relationships, among others. A growing number of research labs have begun to report applications of this technique. A brief look at some recent applications of the suppression mutagenesis technique follows. 

General Protease Cocktails. These should contain the following inhibitors AEBSF, franx-epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64), bestatin, leupeptin, aprotinin (and sodium EDTA if the protein or enzyme being purified does not require a divalent metal cofactor for stability or activity). 

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