Protein different temperatures

Works where study the hydrodynamic properties of a biopolymers in aqueous solution at different temperatures are made by Guner (1999), and Guner Kibarer (2001) for dextran Ghen Tsaih (1998) and Kasaii (2008) for chitosan, Bohidar for gelatin (1998), and Monkos for serum proteins (1996,1997,1999, 2000, 2004 and 2005). 

There is a continuing interest to improve and extend the fimctional properties range of dairy proteins to provide both health benefits and their characteristic physical behaviors under different temperature, moisture, and pH conditions so that they may be included in foods that ordinarily do not contain them. One such research area is the extrusion texturization of whey proteins, which have resulted in dairy proteins with new characteristics imparted by a controlled texturization process, depending on the application desired (Hale et al., 2002 Manoi and Rizvi, 2008 Onwulata, 2009 Onwulata et al., 1998). Protein texturization is a two-step process that involves, first, the unfolding of the globular structure (denaturation) and, second, the alignments of the partially unfolded structures in the direction of mass flow in the extruder. The surface characteristics are imparted at the extruder die as the molten mass exits (Onwulata et al., 2003a). 

FIGURE 5.6 Solubility of texturized dairy protein products extruded at different temperatures, 25 (control), 50, 75, and 100 C Nonfat dried milk (NDM) whey protein concentrate (WPC80), containing 80% protein and whey protein isolate (WPl), containing 95% protein (Onwulata et at, 2003a). 

Mishra, N.P., Mishra, R.K. and Singhal, C.S. (1993). Changes in the activities of antioxidant enzymes during exposure of intact wheat leaves to strong visible light at different temperatures in the presence of protein synthesis inhibitors. Plant Physiology 102 903-910. 

This strategy was first realized by Lozinsky et al., who studied the redox-initiated free-radical copolymerization of thermosensitive N-vinylcaprolactam with hydrophilic N-vinylimidazole at different temperatures, as well as by Chi Wu and coworkers. Lozinsky presents an extensive review of the experimental approaches, both already described in the literature and potential new ones, to chemical synthesis of protein-like copolymers capable of forming core-shell nanostructures in a solution. 

Conformational and phase transitions can potentially be indicative of the primary structure of thermosensitive macromolecules. Indeed, depending on the relative location of H- and P-blocks, as well as on the variation of their length, the chains can either undergo conformational transition accompanied by phase separation, or they can exhibit only the conformational changes without macroscopic phase transitions, i.e. the behaviour observed in the case of protein-like HP-copolymers. Therefore, the solution behaviour of separated fractions of these NVCl/NVIAz-copolymers in an aqueous medium at different temperatures is very important. 

As the temperature drops still lower, some of the solutes present may also crystallize, thus being effectively removed from the solution. In some cases, individual buffer constituents can crystallize out of solution at different temperatures. This will dramatically alter the pH values of the remaining solution and, in this way, can lead to protein inactivation. 

In addition to the dynamic disorder caused by temperature-dependent vibration of atoms, protein crystals have static disorder due to the fact that molecules, or parts of molecules, do not occupy exactly the same position or do not have exactly the same orientation in the crystal unit cell. However, unless data are collected at different temperatures, one cannot distinguish between dynamic and static disorder. Because of protein crystal disorder, the diffraction pattern fades away at some diffraction angle 0max. The corresponding lattice distance <7mm is determined by Bragg s law as shown in equation 3.7 ... 

Wang L., Duan Y., Shortle R., Imperiali B. and Kollman P. A. Study of the stability and unfolding mechanism of BBA1 by molecular dynamics simulations at different temperatures. Proteins Science (1999) 8 1292-1304. 

Figure 2.11. The dependence of the position of the fluorescence spectrum maximum on excitation wavelength for tryptophan in a model medium (glycerol) at different temperatures (a) and singletryptophan proteins (b). 1, Whiting parvalbumin, pH 6.S in the presence of Ca2+ ions 2, ribonuclease Th pH 6.5 3, ribonuclease C2, pH 6.5 4, human serum albumin, pH 7.0, +10"4 M sodium dodecyl sulfate 5, human serum albumin, pH 3.2 6, melittin, pH 7.5, +0.15 M NaCl 7, protease inhibitor IT-AJ from Actinomyces janthinus, pH 2.9 8, human serum albumin, pH 7.0 9, -casein, pH 7.5 10, protease inhibitor IT-AJ, pH 7.0 11, basic myelin protein, pH 7.0 12, melittin in water. The dashed line is the absorption spectrum of tryptophan.

Dorn anus el al.(74> proposed that the ratio of phosphorescence intensity to lifetime, P/t), of tryptophan phosphorescence as a function of temperature be used to distinguish heterogeneity in emission from multitryptophan proteins. Since different tryptophans within one protein show different temperature-... 

Clearly, many of these effects can be addressed through preliminary studies. Incubation of the enzyme at one temperature while assaying at a different temperature will address issues concerned with protein stabihty as well as effects of temperature on any couphng enzymes in the assay protocol. Nevertheless, temperature studies on each enzyme in a multienzyme system should be addressed individually at an early point in the investigation. Issues related to the effects on affinities can usually be minimized by assaying under saturating conditions. Care... 

Nevertheless, we know40 that cytochrome c in its two oxidation states has a different solubility, a different rate of movement on columns, and a different pH and temperature stability. It is also known that the protein binds anions (even chloride) or cations, or even other proteins differently on change of redox state. These binding differences would be sufficient to operate a relay. 

In subunit R2 of ribonucleotide reductase there is a tyrosyl radical (Y ) in close proximity to a di-iron cluster.100 In the protein from E. coli the EPR signal from Y can be observed up to room temperature. However, in the protein from yeast the Y signal broadens above 15 K and is not observable above about 60 K. Saturation recovery measurements at 140 GHz showed that at 60 K the spin-lattice relaxation rates for the Y signal in the yeast protein were about 2 orders of magnitude faster than for the E. coli protein. The temperature dependence of the relaxation enhancement was consistent with the activation energy for the first excited state of the di-iron cluster, so the relaxation enhancement was attributed to interaction with the di-iron cluster. Relaxation enhancements measured at 140 GHz showed little orientation dependence so the enhancement was assigned to isotropic exchange, which is different from the orientation-dependent dipolar interaction observed for the E. coli protein.100... 

The PEG could stabilize proteins by two different temperature-dependent mechanisms. At lower temperatures, it is preferentially excluded from the protein surface but has been shown to interact with the unfolded form of the protein at higher temperatures, given its amphipathic nature (57). Thus, at lower temperatures, it may protect proteins via the mechanism of preferential exclusion, but at higher temperatures possibly by reducing the number of productive collisions between unfolded molecules. PEG is also a cryoprotectant and has been employed in Recombinate, a lyophilized formulation of recombinant Antihemophilic Factor, which utilizes PEG 3350 at a concentration of 1.5mg/mL. The low-molecular weight liquid PEGs (PEG 300-600) can be contaminated with peroxides and cause protein oxidation. If used, the peroxide content in the raw material must be minimized and controlled throughout its shelf life. The same holds true for polysorbates (discussed below). 

Figure IS. Protein solubilities in various buffer systems of soy extrudates produced at different temperatures ( ) pH 7.2, O.OM phosphate buffer pH 10.0, 0.0IM carbonate buffer (/ ) 1% 2-mE added phosphate buffer ( ) 1% SDS added phosphate buffer (0)1% 2-mE and 1 % 2-mE added phosphate buffer.

Measurements of the steady state phosphoprotein level at different temperatures revealed that phosphoprotein formation is accompanied by a large and constant enthalpy change of 48 kJ/mol. In contrast, the likewise quite high activation energy of phosphoprotein formation exhibits a pronounced break between 20°C and 30°C. A break in the Arrhenius plot of the calcium-dependent ATPase has been observed in the same temperature range and has been interpreted as transitions between two activity states of the enzyme. Apparently, the phosphorylation of the calcium free protein by inorganic phosphate exhibits a similar kind of activity transition as observed for the calcium-dependent interaction of the transport protein with ATP131. A similar transition phenomenon complicates the time course of phosphoprotein formation... 

In the fractionation of the milk proteins, usually the first step in the process is to separate the so-called whole casein from the whey in a skim milk. A number of procedures are available (McKenzie 1971C), but the most commonly used method is based upon classical acid precipitation at the pH of minimum solubility. Several different temperatures have been employed 2, 20, and 30°C. Except for precipitation at 2°C, where minimum solubility occurs at pH 4.3, the skim milk is adjusted to pH 4.5-4.6 with hydrochloric acid (1 M). A more recent investigation of the relationship of temperature and pH to the completeness of casein precipitation indicated that optimum yield was obtained at pH 4.3 and 35°C (Helesicova and Podrazky 1980). 

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