Surface tension of protein solutions

Changes in the surface tension of protein solutions occur over long periods of time and this makes it difficult to know with certainty whether constant values are reached. 

A critical examination of the above four criteria reveal that none is necessarily a proof of irreversibility. The changes in surface tension of protein solutions over long periods can be attributed to slowness in adsorption due to the relatively high activation energy barriers (Sections III,B and III,C). Slowness in approaching adsorption equilib-... 

Surface tension of protein solutions changes with hydrogen ion concentration. A minimum in the surface tension is observed in the isoelectric zone. 

Retention in HIC can be described in terms of the solvophobic theory, in which the change in free energy on protein binding to the stationary phase with the salt concentration in the mobile phase is determined mainly by the contact surface area between the protein and stationary phase and the nature of the salt as measured by its propensity to increase the surface tension of aqueous solutions [331,333-338]. In simple terms the solvopbobic theory predicts that the log u ithn of the capacity factor should be linearly dependent on the surface tension of the mobile phase, which in turn, is a llne2u function of the salt concentration. At sufficiently high salt concentration the electrostatic contribution to retention can be considered constant, and in the absence of specific salt-protein interactions, log k should depend linearly on salt concentration as described by equation (4.21)... 

Surfactants are well-known protein denaturants. However, when sufficiently dilute, some surfactants (e.g. polysorbate) exert a stabilizing influence on some protein types. Proteins display a tendency to aggregate at interfaces (air—liquid or liquid—liquid), a process that often promotes their denaturation. Addition of surfactant reduces surface tension of aqueous solutions and often increases the solubility of proteins dissolved therein. This helps reduce the rate of protein... 

The aim of the present study is to demonstrate that the corresponding equations can be derived similarly to expressions for the surface tension of mixed solutions of molecules with different sizes.29 The same approach was also applied to protein solutions.30 The derived equations are compared with some experimental Il-A isotherms of monolayers of micro- and nanoparticles and proteins reported in the literature. 

The results on formation and stability of black foam films, on the first place those on bilayer foam films (NBF) (see Sections 3.4.1.2 and 3.4.4) have promoted the development of methods which enable lung maturity evaluation. The research on stability of amphiphile bilayers and probability for their observation in the grey foam films laid the grounds of the method for assessment of foetal lung maturity created by Exerowa et al. [20,24]. Cordova et al. [25] named it Exerowa Black Film Method. It involves formation of films from amniotic fluid to which 47% ethanol and 7-10 2 mol dm 3 NaCl are added [20,24]. In the presence of alcohol the surface tension of the solution is 29 mN m 1 and the adsorption of proteins from the amniotic fluid at the solution/air interface is suppressed, while that of phospholipids predominates. On introducing alcohol, the CMC increases [26], so that the phospholipids are present also as monomers in the solution. The electrolyte reduces the electrostatic disjoining pressure thus providing formation of black foam lipid films (see Sections 3.4.1.2 and 3.4.4). 

Bovine serum albumin (BSA), a globular protein, is often applied as a model protein for foam formation. The surface tension of BSA solutions as a function of time indicates that, depending on the BSA concentration, it can take 15 to 20 h to attain an equihbrium surface tension which is independent of the BSA concentration. The coagulation rates are shght and the loss of native protein in the svuface film due to adsorption and denaturation is compensated by the quick and continuous diffusive transport of native protein to the surface. Therefore, the critical micelle concentration (CMC) and Ocmc can be evaluated from these measmements. 

The spreading of proteins should be carefully attempted. Certain proteins can be spread on the water surface from the solid state, but, it is preferable to spread them from a dilute aqueous solution (0.05% or less) with the help of micropipette. The spreading of globulins poses some problems, as in their native state the molecules are water-soluble but on unfolding they are completely insoluble. This difficulty can be circumvented by adding a small amount of amyl alcohol (0.1%) to the protein solution. This reduces the surface tension of the solution considerably enabling it to spread rapidly on the water surface. 

Kato and Yutani [28] correlated the surface activity of six mutants of tryptophan synthase a-subimits with their stability, as measured by their free energy of denaturation in water (AG ater)- 0 1 measure of surface activity was given by the air-liquid siuface tension of a mutant solution. These results are in line with the previous examples and demonstiate the importance of conformational stability in predicting the difference in the adsorptive behavior among proteins. Incidentally, it serves as another example suggesting that the surface tension of protein, ypy, is related to its intrinsic conformational stability. 

Kinsella (13, 14) summarized present thinking on foam formation of protein solutions. When an aqueous suspension of protein ingredient (for example, flour, concentrate, or isolate) is agitated by whipping or aeration processes, it will encapsulate air into droplets or bubbles that are surrounded by a liquid film. The film consists of denatured protein that lowers the interfacial tension between air and water, facilitating deformation of the liquid and expansion against its surface tension. 

Now, while it is possible, by lowering the surface tension of the liquid medium to 50 dyn/cm by means of solutes of low molecular weight, to dissociate antigen-antibody precipitates (of the pure VDW type) (6), whole mammalian serum (with a surface tension < 50 dyn/cm) definitely has no such dissociating power. Upon some reflection this is not as anomalous as it might seem. Table I shows that it is mainly because of the presence of proteins (of molecular weight > 20,000) that blood (or plasma) has a surface tension y < 50 (and not y 70), and it cannot be expected that molecules with dimensions of the order of 100 A will effect a separation by purely physical means between other proteins of the same approximate size that are only about 2 A apart at their site of interaction (13). 

We now may examine the relevance of surface tension measurements to interactions of cells in contact with serum or plasma. The surface tensions reported in Table I for the unfiltered liquids are much lower than those of the ultrafiltrates, at least in part, because of the adsorption of proteins having molecular weight > 20,000 at the liquid-air interface. And the measurements made on the ultrafiltrates are likely to be a good approximation to the zero-time surface tensions of the whole serum and plasma. The solutes that remain in the solution after ultrafiltration evidently are of a relatively low level of surface activity, and do not affect the surface tension to any greater extent than would be expected from their volume fractions. 

Cryo- and Lyoprotectants and Bulking Agents Various mechanisms are proposed to explain why excipients serve as cryo- or lyoprotectants. The most widely accepted mechanism to explain the action of cryoprotection is the preferential exclusion mechanism [177]. Excipients that will stabilize proteins against the effects of freezing do so by not associating with the surface of the protein. Such excipients actually increase the surface tension of water and induce preferential hydration of the protein. Examples of solutes that serve as cryoprotectants by this mechanism include amino acids, polyols, sugars, and polyethylene glycol. 

Specific interactions between starch and proteins were observed as early as the beginning of the twentieth century. Berczeller996 noted that the surface tension of aqueous soap solutions did not decrease with the addition of protein (egg albumin) alone, but it did decrease when starch and protein were added. This effect was observed to increase with time. Sorption of albumin on starch is inhibited by bi- and trivalent ions and at the isoelectric point. Below the isoelectric point, bonding between starch and albumin is ionic in character, whereas nonionic interactions are expected above the isoelectric point.997 The Terayama hypothesis998 predicts the formation of protein complexes with starch, provided that starch exhibits the properties of a polyelectrolyte. Apart from chemically modified anionic starches (such as starch sulfate, starch phosphate, and various cross-linked starch derivatives bearing ionized functions), potato starch is the only variety that behaves as a polyelectrolyte. Its random phosphate ester moieties permit proteins to form complexes with it. Takeuchi et a/.999-1002 demonstrated such a possibility with various proteins and a 4% gel of potato starch. 

The latter, however, do not have the capacity, once adsorbed, to stabilise the foam, it is well established that pure liquids do not foam. Transient foams are obtained with solutes such as short-chain aliphatic alcohols or acids which lower the surface tension moderately really persistent foams arise only with solutes that lower the surface tension strongly in dilute solution - the highly surface-active materials such as detergents and proteins. The physical chemistry of the surface layers of the solutions is what determines the stability of the system. 

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