Proteins solutions

A review of various theoretical (molecular-statistical, scaling and thermodynamic) models which describe the adsorption of proteins at liquid/fluid interface was presented in [86]. Here the thermodynamic models derived to describe the protein adsorption are discussed briefly. The adsorption isotherm (2.27), and also the equation of state (2.26) accounting for the 

Coulomb contribution can be used as a basis to describe adsorption layers of proteins. It should be kept in mind that the subscript i refers to various states of the protein molecule at the surface. Problems arising from a non-ideality of the surface layer, the inter-ion interactions in the adsorption layer and the dependence of Kj on the adsorption state of large molecules at the surface have been addressed in [26, 85, 86,131]. 

In analogy to surfactant molecules able to reorient, we assume here that protein molecules can exist in a number of states with different molar areas and that the non-ideality of enthalpy for protein adsorption layers does not depend on the state of molecules at the surface, that is, the activity coefficients are given by Eqs. (2.72). Assuming an entropy non-ideality (the convention oio = coi) and enthalpic non-ideality contribution of the Frumkin type, and taking into account the contribution of the DEL, Eq. (2.59), one can transform the equation of state for the surface layer (2.26) into 

Here a is a constant which determines the variation in surface activity of the protein molecule in the i state with respect to the state 1 characterised by a minimum partial molar area C0[ = co j , bj = b,i . The value i can be either integer or fractional and the increment is defined by Ai = Aco/coi. For a = 0 one obtains b = bj = const, while for a 0 the bj increase with increasing coj. 

The simplifications discussed in [85, 86] allow us to transform the equation of state for protein surface layers (2.122) and the adsorption isotherm (2.123) into the simpler form 

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Koenig S H and Brown R D 1990 Field-cycling relaxometry of protein solutions and tissue— implications for MRI Prog. Nucl. Magn. Reson. Spectrosc. 22 487-567... 

Lian T, Locke B, Kholodenko Y and Hochstrasser R M 1994 Energy flow from solute to solvent probed by femtosecond IR spectroscopy malachite green and heme protein solutions J. Rhys. 

Carbon black Pastes Cosmetics Polymer solutions Drilling muds Protein solutions Fog Soils... 

Ball V and Ramsden J J 1998 Buffer dependence of refractive Index Increments of protein solutions Biopolymers 46 489-92... 

In addition to an array of experimental methods, we also consider a more diverse assortment of polymeric systems than has been true in other chapters. Besides synthetic polymer solutions, we also consider aqueous protein solutions. The former polymers are well represented by the random coil model the latter are approximated by rigid ellipsoids or spheres. For random coils changes in the goodness of the solvent affects coil dimensions. For aqueous proteins the solvent-solute interaction results in various degrees of hydration, which also changes the size of the molecules. Hence the methods we discuss are all potential sources of information about these interactions between polymers and their solvent environments. 

Figure 9.4 (a) For aqueous protein solutions, variation of [77] with the axial... 

The factor 1 - p/p2 cannot be too close to zero, nor can the refractive index of the polymer and the solvent be too similar. These additional considerations limit the choice of solvents for a synthetic polymer, while their values are optimal for aqueous protein solutions. 

Plasteins ate formed from soy protein hydrolysates with a variety of microbial proteases (149). Preferred conditions for hydrolysis and synthesis ate obtained with an enzyme-to-substrate ratio of 1 100, and a temperature of 37°C for 24—72 h. A substrate concentration of 30 wt %, 80% hydrolyzed, gives an 80% net yield of plastein from the synthesis reaction. However, these results ate based on a 1% protein solution used in the hydrolysis step this would be too low for an economical process (see Microbial transformations). 

Fig. 32. Maximum flux obtained with various protein solutions as a function of protein concentration according to equation 3. Feed flow rates, cm /min = A, 3000 B, 2000 C, 1000 and D, 500. The flux decreases exponentially as the protein concentration increases. The extrapolated protein concentration at no flux is the gel point for this type of solution (approx 28%). These results were obtained in a flow-through cell and demonstrate the...

Electroultrafiltration (EUF) combines forced-flow electrophoresis (see Electroseparations,electrophoresis) with ultrafiltration to control or eliminate the gel-polarization layer (45—47). Suspended colloidal particles have electrophoretic mobilities measured by a zeta potential (see Colloids Elotation). Most naturally occurring suspensoids (eg, clay, PVC latex, and biological systems), emulsions, and protein solutes are negatively charged. Placing an electric field across an ultrafiltration membrane faciUtates transport of retained species away from the membrane surface. Thus, the retention of partially rejected solutes can be dramatically improved (see Electrodialysis). 

Electroultrafiltration has been demonstrated on clay suspensions, electrophoretic paints, protein solutions, oil—water emulsions, and a variety of other materials. Flux improvement is proportional to the appHed electric field E up to some field strength E where particle movement away from the membrane is equal to the Hquid flow toward the membrane. There is no gel-polarization layer and (in theory) flux equals the theoretical permeate flux. It... 

The gel-like, bead nature of wet Sephadex enables small molecules such as inorganic salts to diffuse freely into it while, at the same time, protein molecules are unable to do so. Hence, passage through a Sephadex column can be used for complete removal of salts from protein solutions. Polysaccharides can be freed from monosaccharides and other small molecules because of their differential retardation. Similarly, amino acids can be separated from proteins and large peptides. 

Ion exchange resins are also useful for demineralising biochemical preparations such as proteins. Removal of metal ions from protein solutions using polystyrene-based resins, however, may lead to protein denaturation. This difficulty may be avoided by using a weakly acidic cation exchanger such as Bio-Rex 70. 

Figure 18.4 The hanging-drop method of protein crystallization, (a) About 10 pi of a 10 mg/ml protein solution in a buffer with added precipitant—such as ammonium sulfate, at a concentration below that at which it causes the protein to precipitate—is put on a thin glass plate that is sealed upside down on the top of a small container. In the container there is about 1 ml of concentrated precipitant solution. Equilibrium between the drop and the container is slowly reached through vapor diffusion, the precipitant concentration in the drop is increased by loss of water to the reservoir, and once the saturation point is reached the protein slowly comes out of solution. If other conditions such as pH and temperature are right, protein crystals will occur in the drop, (b) Crystals of recombinant enzyme RuBisCo from Anacystis nidulans formed by the hanging-drop method. (Courtesy of Janet Newman, Uppsala, who produced these crystals.)...

Van den Berg, G.B. Hanemajer, J.H. and Smolders, C.A., "Ultrafiltration of Protein Solutions the Role of Protein Association in Rejection and Osmotic Pressure," Journal of Membrane Science, 31 (1987) 307-320. 

Eigure 3.5 presents the dependence of A.S ° on temperature for chymotryp-sinogen denaturation at pH 3. A positive A.S ° indicates that the protein solution has become more disordered as the protein unfolds. Comparison of the value of 1.62 kj/mol K with the values of A.S ° in Table 3.1 shows that the present value (for chymotrypsinogen at 54.5°C) is quite large. The physical significance of the thermodynamic parameters for the unfolding of chymotrypsinogen becomes clear in the next section. 

If a solution of protein is separated from a bathing solution by a semipermeable membrane, small molecules and ions can pass through the semipermeable membrane to equilibrate between the protein solution and the bathing solution, called the dialysis bath or dialysate (Figure 5A.2). This method is useful for removing small molecules from macromolecular solutions or for altering the composition of the protein-containing solution. 

Fig. 6.1.6 Effect of the purple protein on the luminescence of Latia luciferin (0.16 jxg) plus Latia luciferase (A280,icm 1.2, 10 pi) in 5 ml of 5mM sodium phosphate buffer, pH 6.8. The amounts of the purple protein solution ( 280,1 cm 0.6) used 20 pi (curve 1), 5 pi (curve 2), 1 pi (curve 3), 0.5 pi (curve 4), 0.2 pi (curve 5), and none (curve 6). From Shimomura and Johnson, 1968c, with permission from the American Chemical Society.

It should be noted that the flocculation effect produced by microdisperse forms of CP is applicable not only to protein solutions but also to solutions containing various organic ions. 

The qualitative thermodynamic explanation of the shielding effect produced by the bound neutral water-soluble polymers was summarized by Andrade et al. [2] who studied the interaction of blood with polyethylene oxide (PEO) attached to the surfaces of solids. According to their concept, one possible component of the passivity may be the low interfacial free energy (ysl) of water-soluble polymers and their gels. As estimated by Matsunaga and Ikada [3], it is 3.7 and 3.1 mJ/m2 for cellulose and polyvinylalcohol whereas 52.6 and 41.9 mJ/m2 for polyethylene and Nylon 11, respectively. Ikada et al. [4] also found that adsorption of serum albumin increases dramatically with the increase of interfacial free energy of the polymer contacting the protein solution. 

Fig. 10. HPLC of proteins (commercial samples) on the /V-butyl polyacrylamide coated silica gel column. Sample 20 pi of 5-15 mg/ml protein solution in buffer A. Buffer A 10% methanol, 0.2 mol/1 ammonium acetate, pH 4.5. Buffer B methanol. Gradient 50-min linear, 0-100% B. Flow rate 0.8 ml/min. Peaks (/) — lysozym, (2,3) — insulin, (4,5) — myoglobin [57]...

Precipitation is another method of concentration that is used extensively for biopolymers such as proteins, polypeptides, etc. By increasing the salt concentration of a protein solution, some proteins can be precipitated. The sample is then centrifuged and the supematent liquid removed. The protein can be reconstituted in an appropriate solution, usually the one that will subsequently be used as the mobile phase. 

This precipitation process can be carried out rather cleverly on the surface of a reverse phase. If the protein solution is brought into contact with a reversed phase, and the protein has dispersive groups that allow dispersive interactions with the bonded phase, a layer of protein will be adsorbed onto the surface. This is similar to the adsorption of a long chain alcohol on the surface of a reverse phase according to the Langmuir Adsorption Isotherm which has been discussed in an earlier chapter. Now the surface will be covered by a relatively small amount of protein. If, however, the salt concentration is now increased, then the protein already on the surface acts as deposition or seeding sites for the rest of the protein. Removal of the reverse phase will separate the protein from the bulk matrix and the original protein can be recovered from the reverse phase by a separate procedure. 

Investigation of the polypeptide folding properties of iron-sulfur via NMR spectroscopy began in 1997 (122). All studies performed to date have focused on the effect of the addition of guanidinium chloride (GdmCl hereafter) to protein solutions. Under these conditions, re-... 

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