Protein solutions critical point

Applying MD to systems of biochemical interest, such as proteins or DNA in solution, one has to deal with several thousands of atoms. Models for systems with long spatial correlations, such as liquid crystals, micelles, or any system near a phase transition or critical point, also must involve a large number of atoms. Some of these systems, including synthetic polymers, obey certain scaling laws that allow the estimation of the behaviour of a large system by extrapolation. Unfortunately, proteins are very precise structures that evade such simplifications. So let us take 10,000 atoms as a reasonable size for a realistic complex system. 

Figure 7.10 Effect of the thermodynamic incompatibility of otsi/p-casein + high-methoxy pectin (pH = 7.0, / = 0.01 M) on phase diagram of the mixed solutions and elastic modulus of corresponding casein-stabilized emulsions (40 vol% oil, 2 wt% protein), (a) (O) Binodal line for p-casein + pectin solution with critical point ( ) ( ) binodal line for asi-casein + pectin solution with critical point ( ). (b) Complex shear modulus G (1 Hz) is plotted against the pectin concentration (O) p-casein ( ) o i -casein. Dotted lines indicate the range of pectin concentration for phase separation in the mixed solutions. The pectin was added to the protein solution before emulsion preparation. Data are taken front Semenova et al. (1999a).

The binodal branches do not coincide with the phase diagram axes. This means that the biopolymers are limitedly cosoluble. For instance, on mixing a protein solution A and a polysaccharide solution B a mixture of composition C can be obtained. This mixed solution spontaneously breaks down into two liquid phases, phase D and phase E. Phase D is rich in protein and E is rich in polysaccharide. These two liquid phases form a water-in-water (WIW) emulsion. Hie phase volume ratio is estimated by the inverse lever rule. The phase D/phase E volume ratio equals the ratio of the tieline segments EC/CD. Point F represents the phase separation threshold, that is, the minimal critical concentration of biopolymers required for phase separation to occur. 

Constmction of phase diagrams usually starts with the preparation of series of mixed solutions sufficiently differing in bulk biopolymer concentration. Some of them can be single-phase solutions, others biphasic systems. A true equilibrium between the phases is experimentally obtained by mixing or shaking the water-in-water emulsions under different time-temperature conditions. Separation of the phases by centrifuge provides information about both the number and the volume ratio of the system phases. The closer a system composition is to the critical point, the smaller the difference in density is between the phases and the more difficult their separation is by centrifugation. The amount of each biopolymer in each phase can be quantified by various techniques. Estimation of protein concentration by UV absorbance at 280 nm is widely used because of its simplicity and sensitivity. 

Although ellipsometry is well established as an experimental technique for the investigation of adsorbed layers, the number of studies at fluid/liquid interfaces is relatively small. Ellipsometry was used for investigation of the layer thickness between two immiscible liquids near the critical point (254, 255). This technique was also quite often used for in situ studies of the adsorption kinetics at an air/protein solution surface or polymer monolayers at an air/water interface (251, 256). It was also shown that ellipsometric re-... 

Fig. 1.26 Sketch of a typical phase diagram of a globular protein solution. The critical point is marked by the asterisk...

Aside Recently, it has been found that protein salt water solutions do have liquid-liquid phase separations with true critical points, albeit sometimes only in metastable regimes at temperatures and protein concentrations at which the protein solution is supersaturated(69-71). Near critical points, Eq. 10.16 may well become valid, but only with a f that is substantially larger than molecular dimensions.)... 

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