Protein denaturation, factors affecting

Native fluorescence of a protein is due largely to the presence of the aromatic amino acids tryptophan and tyrosine. Tryptophan has an excitation maximum at 280 nm and emits at 340 to 350 nm. The amino acid composition of the target protein is one factor that determines if the direct measurement of a protein s native fluorescence is feasible. Another consideration is the protein s conformation, which directly affects its fluorescence spectrum. As the protein changes conformation, the emission maximum shifts to another wavelength. Thus, native fluorescence may be used to monitor protein unfolding or interactions. The conformation-dependent nature of native fluorescence results in measurements specific for the protein in a buffer system or pH. Consequently, protein denatur-ation may be used to generate more reproducible fluorescence measurements. 

The combination of lEF with gradient SDS-PAGE gives very high resolution. The technique was first described in 1975 (02) and has been applied to denatured (C12, 03) and nondenatured protein mixtures (SI). A definitive account of the factors affecting reproducibility, quantitation of components, resolution, and sensitivity is detailed in the paper by O Farrell (02). Subsequently, Anderson and Anderson (A8, A9) have adapted O Farrell s original method to allow the analysis of large... 

Many of the globular proteins will be met in action as enzymes and hormones in later chapters. It is worth emphasizing here, though, the particular sensitivity of the tertiary structure of these proteins to factors which affect their weak bonding, especially changes in pH and temperature, which rapidly denature most globular proteins. 

A number of factors affect the rate and extent of protein degradation by proteolytic enzymes. These include the specificity of the enzyme, the extent of denaturation of the protein used as substrate as well as substrate and enzyme concentrations, pH, temperature and ionic strength of the reaction medium, presence of inhibitory substances, etc. It is beyond the scope of this review to discuss all these factors in detail. However, we do want to call attention to some of the more important factors and to refer the reader to articles such as those of Blow (15), Keil (16), and Killheffer and Bender (17). 

Besides the effect of enzyme, substrates and modulator concentrations, the catalytic potential of enzymes is affected by several environmental factors among which, pH and temperature are outstanding. These variables not only affect enzyme activity but enzyme stability as well. Enzyme stability is regarded as the capacity of the enzyme to retain its activity. Protein denaturation is an event or sequence of events leading to structural changes (not compromising its primary structure) and in most cases... 

Many factors affect the solubility such as processing conditions, storage conditions, composition, pH, density, and particle size. It was found that increasing product temperatures is accompanied by increasing protein denaturation, which decreases solubility. Thus, more protein is denatured and its solubility gets decreased [49]. Removal of water by evaporation results in the formation of an amorphous state product. 

Another important factor affecting storage stability of dehydrated foods is temperature and period of storage. Generally, the storage stability bears an inverse relationship to storage temperature, which affects not only the rate of deteriorative reaction (enzyme hydrolysis, lipid oxidation, NEB, protein denaturation), but also the kind of spoilage mechanism. 

Although the free iron is catalytically active, the relative importance of protein-bound iron is being debated. Lipid oxidation in cooked meat is not only attributed to changes in iron distribution by protein denaturation and the release of catalytically active non-heme iron, but also to the disruption of cell membranes in meat that brings the polyunsaturated hpids in close contact to the catalysts. Unfortunately, in many studies using the TBA test to determine the effects of different forms of iron on lipid oxidation in meats, the results must be interpreted with caution, because the TBA reaction is significantly affected by iron and other metals, and is subject to serious interference by other factors. In addition to products of lipid oxidation, TB ARS are also formed from proteins and nucleic acids and other non-lipid components in meat tissues that confound the results of the TBA test. 

The shelf life of proteins in solution is short as their stability is affected by various factors once they are isolated from the living cell. The major factors affecting protein stability in solution are aeration, pH, ionic strength, temperamre, proteolysis or denaturation by exposure of protein to unsterile surfaces, etc. During electrochemical studies the protein in solution is exposed to many such physical and chemical denaturants. These factors cause rapid denaturation of proteins in solution and their reusability is further limited. It has been shown by many researchers that immobilisation of proteins in suitable matrices over electrodes reduces such effects and contributes towards the stability of proteins and their reusability [29]. 

Other factors that can impact these constants relate to reaction solution conditions. We have already discussed how temperature can affect the value of kCM and kcJKM according to the Arrhenius equation (vide supra). Because enzymes are composed of proteins, and proteins undergo thermal denaturation, there are limits on the range of temperature over which enzymes are stable and therefore conform to Arrhenius-like behavior. The practical aspects of the dependence of reaction velocity on temperature are discussed briefly in Chapter 4, and in greater detail in Copeland (2000). 

Emulsification is a stabilizing effect of proteins a lowering of the interfacial tension between immiscible components that allow the formation of a protective layer around oil droplets. The inherent properties of proteins or their molecular conformation, denaturation, aggregation, pH solubility, and susceptibility to divalent cations affect their performance in model and commercial emulsion systems. Emulsion capacity profiles of proteins closely resemble protein solubility curves and thus the factors that influence solubility properties (protein composition and structure, methods and conditions of extraction, processing, and storage) or treatments used to modify protein character also influence emulsifying properties. 

Despite some conflicting evidence (Kinsella and Fox, 1986), it appears that denaturation has little influence on the amount of water bound by whey proteins. However, other factors which may accompany denaturation (e.g. Maillard browning, association or aggregation of proteins) may alter protein sorption behaviour. Drying technique affects the water sorption characteristics of WPC. Freeze-dried and spray-dried WPC preparations bind more water at the monolayer level than do roller-, air- or vacuum-dried samples, apparently due to larger surface areas in the former. As discussed above, temperature also influences water sorption by whey protein preparations. The sorption isotherm for /Mactoglobulin is typical of many globular proteins. 

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