Protein stabilization oxidation

The significance of protein oxidation became paramount with the advent of recombinant protein biologies used as human therapeutics. Careful characterization of protein stability is essential to maintaining the efficacy of protein pharmaceuticals. If even a single side chain amino acid residue becomes oxidized, then a protein therapeutic may not have the same activity in vivo as the unmodified protein. 

Hora et al. [3.19] described the complexity of protein stabilization by the example of recombinant, human Interleukin-2 (rhIL-2). Formulations with amino acids and mannitol/ sucrose are sensitive to mechanical stress e. g. by pumping. Hydroxypropyl-beta-cyclodextrin (HPcD) provides stability, but increases the sensitivity to oxygen. Polysor-bate 80 forms a mechanically stable product, but results in oxidation. In both cases contamination in the HPcD or traces of H202 in the Polysorbate may have been the starter for the oxidation. Brewster [3.20] reports, that HPcD stabilizes interleukin without forming aggregations and this results in 100 % biopotency. 

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... 

Electrophoresis methods are among the most common and potent used in the evaluation of protein purity and homogeneity. They are valuable indicators of protein stability because they detect small molecular or chemical changes in the product caused by denaturation, aggregation, oxidation, deamidation, etc. One of the advantages is that they require only microgram amounts of a sample. 

The excessive replacement of Met by SeMet lowers the protein stability in vitro, but not necessarily in vivo. At the low levels of SeMet normally present in culture media, the substitution of Met by the more oxygen-sensitive SeMet is unlikely to affect the properties of enzymes in an adverse manner. Moreover, since methionine selenoxide is readily converted back to SeMet on reaction with biogenic thiols such as GSH, a mechanism of repair of this type of oxidative damage is available. The reversibility of SeMet oxidation in the... 

Knowing that peptides and amines confer thermal stability on enzymes from certain thermophilic organisms (47-49) led some workers to examine protein stabilization by antibodies. It was found that the presence of specific polyclonal antibodies stabilize several enzymes (50, 51). In addition, not only did antibodies increjise the thermostability of a-amylase, glucoamylase, and subtilisin, but some stability toward acid denaturation, oxidizing agent, and organic solvent exposure was increased in specific cases (52, 53). 

Pocock, K.F., Waters, E.J. (1998). The effect of mechanical harvesting and transport of grapes, and juice oxidation, on the protein stability of wines. Aust. J. Grape Wine Res., 4, 136-139... 

The term chemical chaperone has been proposed to describe small molecules such as glycerol, dimethylsulfoxide, and trimethylamine N-oxide that act as protein-stabilizing agents. This terminology is unfortunate and confuses students, because proteins are also chemicals. This term should be replaced by the term kosmotrope that physical chemists use to describe small molecules that stabilise proteins. 

Flavoenzymes are widespread in nature and are involved in many different chemical reactions. Flavoenzymes contain a flavin mononucleotide (FMN) or more often a flavin adenine dinucleotide (FAD) as redox-active prosthetic group. Both cofactors are synthesized from riboflavin (vitamin B2) by microorganisms and plants. Most flavoenzymes bind the flavin cofactor in a noncovalent mode (1). In about 10% of aU flavoenzymes, the isoalloxazine ring of the flavin is covalently linked to the polypeptide chain (2, 3). Covalent binding increases the redox potential of the flavin and its oxidation power, but it may also be beneficial for protein stability, especially in flavin-deficient environments. 

DAAO is one of the most extensively studied flavoprotein oxidases. The homodimeric enzyme catalyzes the strictly stere-ospecihc oxidative deamination of neutral and hydrophobic D-amino acids to give a-keto acids and ammonia (Fig. 3a). In the reductive half-reaction the D-amino acid substrate is converted to the imino acid product via hydride transfer (21). During the oxidative half-reaction, the imino acid is released and hydrolyzed. Mammalian and yeast DAAO share the same catalytic mechanism, but they differ in kinetic mechanism, catalytic efficiency, substrate specificity, and protein stability. The dimeric structures of the mammalian enzymes show a head-to-head mode of monomer-monomer interaction, which is different from the head-to-tail mode of dimerization observed in Rhodotorula gracilis DAAO (20). Benzoate is a potent competitive inhibitor of mammalian DAAO. Binding of this ligand strengthens the apoenzyme-flavin interaction and increases the conformational stability of the porcine enzyme. 

Are the irreversible denaturation pathways truly irreversible Thomas and coworkers have demonstrated that the activity of (inactive) oxidized aFGF can be recovered by the addition of the reducing agent dithiothreitol (10). Furthermore, as previously mentioned, various formulation additives have minimized thermally induced aggregation of aFGF. In addition to the physiological implications, these irreversible denaturation pathways also complicate thermodynamic analyses of protein stability, particularly those which rely on van t Hoff analysis of denaturation profiles. 

For liquid formulations, the choice of using either a salt or a carbohydrate to adjust the osmolality of the solution is made by the impact on protein stability. Sodium chloride is one of the most commonly used salts in the formulation of both traditional pharmaceutics as well as biological pharmaceutics. It is extremely safe, well tolerated, and inexpensive. However, the presence of sodium chloride in a formulation of rhuMAb HER2 was found to increase oxidation when the formulation was stored in stainless steel containers, presumably because the sodium chloride promoted corrosion of the stainless steel. Interactions of salts with the proteins must be investigated on an individual basis because the type and concentration of salt may lead to protein aggregation. 

On the other hand, long-chain oligo(ethylene oxides) in hydrophobic SAMS reject biopolymers, e.g. proteins. Stabilization of colloids by solvophilic polymers can be explained by a concept called steric repulsion which means disfavourable compression and resulting loss of chain mobilities in colliding polymers. 

Considering chemical stability, even alterations at single amino acids or the peptide bond can be detrimental [6], Chemical reactions having an impact on protein stability include hydrolysis of the peptide bond, deamidation, oxidation, p-elimina-tion, isomerization, and disulfide bond breakage and formation. The extent to which they occur is mainly influenced by the temperature and pH value of the solution [24], Bearing in mind that proteins react sensitively to the above-mentioned environmental conditions, preparation procedures for protein pharmaceuticals have to be chosen very carefully to preserve protein integrity and functionality. 

Chevalier et al. (1999) reported that high-pressure thawing of blue whiting was quicker and resulted in lower drip volume in comparison to conventional thawing. Chevalier et al. (2001), based on the analysis of physicochemical properties of fillets (such as color, lipid oxidation, and protein stability), later concluded that 140 MPa was the optimum pressure level for pressure-shift freezing of turbot fillets. 

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