Denatured proteins, viscosity

Factors to be considered in maldng the selection of chromatography processing steps are cost, sample volume, protein concentration and sample viscosity, degree of purity of protein product, presence of nucleic acids, pyrogens, and proteolytic enzymes. Ease with which different types of adsorbents can be washed free from adsorbed contaminants and denatured proteins must also be considered. 

Filter aids are widely used in die fermentation industry to improve the efficiency of filtration. It is a pre-coated filter medium to prevent blockage or blinding of the filter by solids, which would otherwise wedge diemselves into the pores of the cloth. Filter aid can be added to the fermentation broth to increase the porosity of the cake as it formed. This is only recommended when fermentation product is extracellular. Filter aid adds to the cost of filtration. The minimum quantity needed to achieve the desired result must be established experimentally. Fermentation broths can be pretreated to improve filtration characteristics. Heating to denature proteins enhances the filterability of mycelial broths such as in penicillin production. Alternatively, electrolytes may be added to promote coagulation of colloids into larger, denser particles, which are easier to filter. The filtration process is affected by the viscosity and composition of the broth, and the cell cake.5... 

The measurements of chain stiffness of denatured proteins are made in the presence of a strong denaturant, such as 8 M urea or 6 M GdmCl, in which peptide H-bonds are weak and peptide helices unfold (Scholtz et al., 1995 Smith and Scholtz, 1996), and the possible presence of (/-helices or /3-hairpins is not an issue in these denaturants. The careful and thorough measurements of intrinsic viscosities made by Tanford and co-workers (1968), discussed above, yield a substantially lower estimate for chain stiffness than the work of Flory and co-workers. A comparison is made by Tanford (1968) between the proportionality coefficient... 

Effect of Sample Preparation It is well known that the viscosities of these concentrated slurries (8 percent) are shear rate dependent (14). What is less known is that the viscosity of denatured proteins are highly dependent upon dispersion conditions (Table II). For 12 percent slurries of Supro 620, viscosities decrease drastically from 10 to 33 cp as total shear is increased (1 ). If the slurries are allowed to stand, the viscosities increase slowly and revert to a much higher value (Table III). Thus, the highly sheared slurries are not at equilibrium. But, since the approach to equilibrium is slow, it is possible to use shear to produce a functionally desirable viscosity. 

II Figure 1). Adsorption continually occurs around the bubbles to replace protein in areas of the interface where coagulation or stretching of the film is occurring. The actual bubble size in the foam depends upon the rate of protein adsorption as well as upon the ease of film rupture. The protein films on adjacent bubbles come in contact and trap the liquid, preventing it from flowing freely. This restriction is governed by the viscosity of the colloidal solution. The polypeptides of denatured proteins situate to positions where their hydrophobic side chains are directed outward toward each other. Because liquid... 

The proteins in the liquid film should 1) be soluble in the aqueous phase, 2) readily concentrate at the liquid-air interface, and 3) denature to form cohesive layers possessing sufficient viscosity and mechanical strength to prevent rupture and coalescence of the droplet. That is, the polypeptides of the denatured proteins in the liquid film should exhibit a balance between their ability to associate and form a film and their ability to dissociate, resulting in foam instability. 

Protein denaturation. Protein denaturation is normally observed as an increase in viscosity and a decrease in wettability. It is temperature-sensitive, generally occurring between 40 and 80°C. A common drying process scheme is to dry thermally and under wet-bulb drying conditions without overheating and then vacuum, heat-pump, or freeze-dry to the target moisture. 

Generally food processing causes irreversible denaturation followed by reactions of the thermally denatured proteins with other components that may lead to loss in food quality. However, in foods denaturation may have beneficial or detrimental effects. The main effects comprise changes in pi, hydration, solubility, viscosity of solutions, biological activity, and reactivity of a.a. residues. 

Why does a denatured protein typically have a higher viscosity factor than a folded protein ... 

A folded protein is more spherical than a denatured protein and a sphere is expected to have a lower viscosity factor than non-spherical shapes. This is discussed in more detail in Chapter 27 and shown clearly in Fig. 27.11. 

In summary, due to its high UV cutoff and viscosity, DMSO has found very limited use in HPLC. One area where it could be more heavily utilized is in the separation of proteins. DMSO is one of the few solvents that does not denature proteins, even at high concentrations. Unfortunately, the very high boiling point of DMSO also precludes work where sample is to be collected from the eluant after elution. 

An important problem in the study of protein folding is the control of the initial state (i.e., obtaining as starting material a completely unfolded protein). Therefore a very careful characterization of the denatured protein is the first step of the experimental work the second step is finding good conditions for reversibility. Different methods can be used to characterize the unfolded proteins hydrodynamic methods such as viscosity and sedimentation coefficient determination spectroscopic methods (absorption and fluorescence), fluorescence depolarization, CD, ORD and optical rotation, and NMR. Chemical methods provide a more direct tool for measuring the accessibility... 

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