Aqueous protein solutions

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. 

Like the carbodiimide method, the mixed anhydride method results in an amide complex (Table 5, Figure 17). The acid-containing hapten is dissolved in a dry, inert, dipolar, aprotic solvent such as p-dioxane, and isobutyl chloroformate is added with an amine catalyst. The activated mixed anhydride is chemically stable and can be isolated and characterized. The aqueous protein solution is added to the activated acid and the pH is maintained at around 8.5. A low temperature (around 10 °C) is necessary during the reaction to minimize side reactions. 

The coprecipitation technique was based on the dropwise addition of a synthetic polymer solution, in a solvent mixture, into an aqueous protein solution under magnetic stirring. The progressive interaction between the water insoluble polymer and the protein gave rise to the microsphere formation. The glycolipid was then added as an aqueous dispersion to the nanoparticle suspension. No sedimentation was observed after several weeks of storage at room temperature. 

Fig. 13. Typical experimental results (adapted from ref. (50)) showing the dispesion curve of water protons in an aqueous protein solution. Vc = (Ocf2K is the cut-off frequency (see Eq. (66)).

Curtis, R. A. Blanch, H. W. Prausnitz, J. M. Calculation of Phase Diagrams for Aqueous Protein Solutions. ]. Phys. Chem. B 2001, 105, 2445-2452. 

Crystallization is a phase transition phenomenon. Crystals grow from an aqueous protein solution when the solution is brought into supersaturation (Ataka, 1993). Supersaturation is achieved by varying the concentrations of precipitant, protein and additives, pH, temperature, and other parameters (McPherson, 1999 Ducruix and Giege, 1992 Ducruix and Giege, 1999). 

If a compound dissociates in a solvent and one part of the pair has another absorption than the other (e.g., p-nitrophenol/p-nitrophenolate), the absorption coefficient changes with dilution. This should be taken into consideration when different dilutions of a compound are compared. The concentration of an aqueous protein solution can be estimated by reading the UV absorption. The aromatic amino acids (phenylalanine, tryptophan, tyrosine)... 

F ure 4.20 Variation of intrinsic viscosity of aqueous protein solutions with axial ratio and extent of solvation. Reprinted, by permission, from P. Hiemenz, Polymer Chemistry, p. 598. Copyright 1984 by Marcel Dekker, Inc. 

The essence of this model for the second virial coefficient is that an excluded volume is defined by surface contact between solute molecules. As such, the model is more appropriate for molecules with a rigid structure than for those with more diffuse structures. For example, protein molecules are held in compact forms by disulfide bridges and intramolecular hydrogen bonds by contrast, a randomly coiled molecule has a constantly changing outline and imbibes solvent into the domain of the coil to give it a very soft surface. The present model, therefore, is much more appropriate for the globular protein than for the latter. Example 3.3 applies the excluded-volume interpretation of B to an aqueous protein solution. 

FIG. 4.13 Intrinsic viscosity of a protein solution (a) variation of the intrinsic viscosity of aqueous protein solutions with axial ratio a/b and extent of hydration mlb/m2 (redrawn from L. Oncley, Ann. NY Acad. Sci., 41, 121 (1941)) (b) superposition of the [r ] = 8.0 contour from Fig. 4.13a and the f/f0 = 1.45 contour from Figure 2.9. The crossover unambiguously characterizes particles with respect to hydration and axial ratio. 

Metaphosphoric Acid. Reactions jor Determining Metaphosphoric Acid and Its Salts. 1. Pour about 1 ml of an aqueous protein solution into a test tube and add to it approximately the same amount of a sodium metaphosphate solution acidified with acetic acid. What do you observe See whether the protein solution is affected in the same way by sodium metaphosphate and acetic acid solutions taken separately. 

Preparation oj Metaphosphoric Acid. 1. Dissolve 0.1 g of phosphoric anhydride in water and test the solution with an aqueous protein solution. What do you observe ... 

Alternatively, dissolve DPH (at this same concentration) in tetrahydrofuran and add directly to the aqueous protein solution. Similar hydrophobicity results can be obtained this way (author s unpub. observ.). 

R. A. Curtis, H. W. Blanch, and J. M. Prausnitz, Calculation of phase diagrams for aqueous protein solutions,... 

Protein Adsorption. The development of medical implant polymers has stimulated interest in the use of ATR techniques for monitoring the kinetics of adsorption of proteins involved in thrombogenesis onto polymer surfaces. Such studies employ optical accessories in which an aqueous protein solution (93) or even ex - vivo whole blood (94-%) can be flowed over the surface of the internal reflection element (IRE), which may be coated with a thin layer of the experimental polymer. Modem FT-IR spectrometers are rapid - scanning devices, and hence spectra of the protein layer adsorbed onto the IRE can be computed from a series of inteiferograms recorded continuously in time, yielding ah effective time resolution of as little as 0.8 s early in the kinetic runs. Such capability is important because of the rapid changes in the composition of the adsorbed protein layers which can occur in the first several minutes (97). 

These kinetics studies required development of reproducible criteria of subtraction of foe H-O-H bending band of water, which completely overlaps foe Amide I (1650 cm 1) and Amide II (1550 cm"1) bands (98). In addition, correction of foe kinetic spectra of adsorbed protein layers for foe presence of "bulk" unadsorbed protein was described (99). Examination of kinetic spectra from an experiment involving a mixture of fibrinogen and albumin showed that a stable protein layer was formed on foe IRE surface, based on foe intensity of the Amide II band. Subsequent replacement of adsorbed albumin by fibrinogen followed, as monitored by foe intensity ratio of bands near 1300 cm"1 (albumin) and 1250 cm"1 (fibrinogen) (93). In addition to foe total amount of protein present at an interface, foe possible perturbation of foe secondary structure of foe protein upon adsorption is of interest. Deconvolution of foe broad Amide I,II, and m bands can provide information about foe relative amounts of a helices and f) sheet contents of aqueous protein solutions. Perturbation of foe secondary structures of several well characterized proteins were correlated with foe changes in foe deconvoluted spectra. Combining information from foe Amide I and m (1250 cm"1) bands is necessary for evaluation of protein secondary structure in solution (100). 

The expected trends are born out for the low molecular weight enzymes ribonuclease-a, cytochrome-c, and lysozyme, as shown in Figure 2. These results are presented as the percentage of the protein transferred from a 1 mg/ml aqueous protein solution to an equal volume of isooctane containing 50 mM of the anionic surfactant Aerosol 0T, or AOT (di-2-ethylhexyl sodium sulfosuccinate). As anticipated, only at pH s lower than the pi was there any appreciable solubilisation of a given protein, while above the pi the solubilisation appears to have been totally suppressed. Note, however, that as the pH was lowered even further, there was a drop in the degree of solubilisation of the proteins. This was accompanied by the formation of a precipitate at the interface between the two phases, attributed to a denaturation of the protein. 

Values of pcr and p, r for foam films and foams obtained from aqueous protein solutions... 

For NaDoS foams at Ap0 < 20kPa an avalanche-like destruction is not realised. Critical pressure in the foam is determined by the type of foam films and the type of the surfactant solution used to obtain the foam. For example, for foams from aqueous protein solution Apcr is by two orders of magnitude less than Apcr of foams from the non-ionic surfactant NP20. 

N., and Benedek, G.B. (1995). Phase separation in multicomponent aqueous-protein solutions. J. Phys. Chem., 99, 454-461. 

Five millimeter diameter disks of a porous polyethylene membrane, used as a window material in disposable FT-IR cards manufactured by 3M Corp. (Fisher Cat. 14385-861), were prepared by pre-wetting with 2 pi of methanol. A 1 pi aliquot of the aqueous protein solution was then added, and the membrane allowed to air dry at room temperature, before addition of 2 pi of matrix solution and final air drying. For those samples that were washed, after the protein solution had dried the membrane spot was vortexed in an aqueous 50% methanol solution for 30 seconds and air dried before addition of the matrix solution. 

If the haptenic molecule is not soluble in water, it can be dissolved in water-miscible solvents, such as dime thy Iformamide, and added to the aqueous protein solution. ... 

Fig. 1 Using reconstitution to achieve high protein concentrations. The vial on the left has been filled with lOmL of a 15mg/mL aqueous protein solution. After lyophilization, this vial contains 150 mg of the protein plus any nonvolatile excipients. Reconstitution with 1 mL of water would yield a final formulation that contains 150mg/mL protein plus nonvolatile excipients at concentrations 10-fold higher than originally filled into the vial. If the formulation scientist plans to use this method for achieving a high-concentration formulation, it is necessary to consider the final concentration of excipients.

In this paper, the Kirkwood-Buff theory of solutions is used to examine the effect of PEG on aqueous protein solutions, the focus being on the local composition of the mixed solvent in the vicinity of the protein molecule and on the protein solubility. The theoretical considerations led to equations that coimect the experimental preferential binding parameter with the excess (or deficit) numbers of water and cosolvent molecules around a protein molecule. Calculations were carried out for various proteins in various PEG solutions. The results showed that in all cases the proteins were preferentially hydrated. Evidence was also brought that the hydration is a result of steric exclusion. 

The addition of a salt to an aqueous protein solution leads to a more complex behavior of the aqueous protein solubility. Old solubility measurements [10,11] suggested that (i) a small amount of salt increases the aqueous protein solubility, and (ii) a large amount of salt decreases the aqueous protein solubility. At sufficiently large salt concentrations the solubility of a protein can be expressed by the empirical Cohn equation [1] ... 

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