Protein-water solutions

For an understanding of protein-solvent interactions it is necessary to explore the modifications of the dynamics and structure of the surrounding water induced by the presence of the biopolymer. The theoretical methods best suited for this purpose are conventional molecular dynamics with periodic boundary conditions and stochastic boundary molecular dynamics techniques, both of which treat the solvent explicitly (Chapt. IV.B and C). We focus on the results of simulations concerned with the dynamics and structure of water in the vicinity of a protein both on a global level (i.e., averages over all solvation sites) and on a local level (i.e., the solvent dynamics and structure in the neighborhood of specific protein atoms). The methods of analysis are analogous to those commonly employed in the determination of the structure and dynamics of water around small solute molecules.163 In particular, we make use of the conditional protein solute -water radial distribution function,... 

Ethoxylated nonionics from aqueous protein solutions Water (17% MeOH added if necessary to break emulsions) 1,2-dichloroethane Proteins Nonionics Nonethoxylated nonionics removed with much less efficiency. 127... 

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... 

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.)...

A biochemist isolates a new protein and determines its molar mass by osmotic pressure measurements. A 50.0-mL solution is prepared by dissolving 225 mg of the protein in water. The solution has an osmotic pressure of 4.18 mm Hg at 25°C. What is the molar mass of the new protein ... 

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. 

FIGURE 9.11 Relative mass change as a result of water absorption and loss in a solid piece of recombinant resilin prepared from a 20% protein solution in phosphate-buffered sahne (PBS), cross-linked using peroxidase (see [29] supplementary material). The fuUy swollen sample is designated 100. (Data courtesy of Shekibi, Y., Naim, K., Bastow, T.J., and HiU, A.J., The states of water in a protein based hydrogel. Internal CSIRO... 

The rejection coefficient R) was calculated according to the following equation / = In (Cr/C )/ln (VJVr). Cr or Vr represent the protein concentration in the retentate or the volume of the retentate Co is the concentration of the protein in the solution before filtration 1 is the initial volume of the feed. The pH value of each protein solution was immediately measured after dissolving the proteins in distilled water. 

Wang, JH, Theory of the Self-Diffusion of Water in Protein Solutions. A New Method for Studying the Hydration and Shape of Protein Molecules, Journal of the American Chemical Society 76, 4755, 1954. 

Laboratory Microfiltration membranes have countless laboratory uses, such as recovering biomass, measuring particulates in water, clarifying and sterilizing protein solutions, and so on. There are countless examples for both general chemistry and biology, especially for analytical proc ures. Most of these apphcations are run in dead-end flow, with the membrane replacing a more conventional medium such as filter paper. 

After heating at 40° C, liquid anhydrous milk fat (1 v) and the different protein solutions (10 v) were premixed using a polytron (PT 3000, Kinematica) and emulsified with a homogenizer (ALMO, Legrand, France) at about 40°C in order to obtain oil-in-water emulsions. After separation from the aqueous phase by centrifugation for 5 min at lOOOg, milk fat droplets stabilized by different proteins were washed twice with a phos-... 

We present a molecular theory of hydration that now makes possible a unification of these diverse views of the role of water in protein stabilization. The central element in our development is the potential distribution theorem. We discuss both its physical basis and statistical thermodynamic framework with applications to protein solution thermodynamics and protein folding in mind. To this end, we also derive an extension of the potential distribution theorem, the quasi-chemical theory, and propose its implementation to the hydration of folded and unfolded proteins. Our perspective and current optimism are justified by the understanding we have gained from successful applications of the potential distribution theorem to the hydration of simple solutes. A few examples are given to illustrate this point. 

Dissolve the Traut s reagent (Thermo Fisher) in water at a concentration of 2mg/ml (makes a 14.5 mM stock solution). The solution should be used immediately. For the modification of IgG at a concentration of lOmg/ml using a 10-fold molar excess of Traut s reagent, add 45.8 pi of the stock solution to each ml of the protein solution. 

Add 25 pi of the stock solution of either SPDP or LC-SPDP in DMSO to each ml of the protein to be modified. If sulfo-LC-SPDP is used, add 50 pi of the stock solution in water to each ml of protein solution. 

Since SMCC is a water-insoluble crosslinker, it must be dissolved first in organic solvent (DMSO or DMF) before adding it to a protein to be modified. In some cases, addition of even a small amount of organic solvent to a protein solution may be detrimental to activity. To be safe, no more than 10-20 percent solvent should be present in the aqueous reaction medium. 

Add 50 pi of the NHS-LC-biotin solution in DMF to each ml of the protein solution in two aliquots apportioned 10 minutes apart. Alternatively, add a quantity of the sulfo-NHS-biotin solution prepared in water to the protein solution to obtain a 12- to 20-fold molar excess of biotinylation reagent over the quantity of protein present. For instance, for an immunoglobulin (MW 150,000) at a concentration of 10 mg/ml, 20 pi of the sulfo-NHS-biotin solution (8 X 10-4 mmol) should be added per ml of antibody solution to obtain a 12-fold molar excess. 

Quickly wash the particles with ice-cold deionized water and then with a volume of cold coupling buffer. Resuspend the particles in the protein solution prepared in step 2. 

Reduce disulfides in the two protein samples by the addition of 2 pi of 50 mM TCEP (Thermo Fisher) to each 100 pi aliquot of protein solution. Cover and boil the samples for 10 minutes in a water bath to completely denature and reduce the proteins. Avoid the use of thiol-containing reductants, such as DTT, as these will react with the iodoacetyl group on the ICAT compound. 

Add to the solution in step 1 a quantity of EDC and sulfo-NHS (both from Thermo Fisher) to obtain a concentration of 2mM EDC and 5mM sulfo-NHS. To aid in aliquot-ing the correct amount of these reagents, they may be quickly dissolved in water at a higher concentration, and then immediately a volume pipetted into the protein solution to obtain the proper molar quantities. 

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