Vapor diffusion, crystallization

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.)...

Crystallization of CaCOj is highly dependent on nucleation condition. The precipitation of CaCOj in the absence or the presence of the G4.5 PAMAM dendrimer was carried out by a carbonate diffusion method similar to the method described by Addadi et al. [35]. A solution of the dendrimer with calcium chloride in 200 ml of distilled water was adjusted to pH 8.5 with aqueous NHj, and then placed in a closed desiccator containing crushed ammonium carbonate (Fig. 5). Carbon dioxide was introduced to the solution via vapor diffusion. The critical point of the appearance in the turbidity of the solution was observed at around 5 min. These solutions were kept at 30 °C under N2 for one day. The crys-... 

Figure 3 Protein crystallization diagrammatic representation of the hanging-drop method of vapor diffusion.

Purified MeHNL was crystallized by the sitting-drop vapor-diffusion method. The 10-20 mm bipyramidal crystals formed were cross-linked with glutaraldehyde and used as biocatalyst for the synthesis of optically active cyanohydrins. The cross-linked crystals were more stable than Celite-immobilized enzymes when incubated in organic solvents, especially in polar solvents. After six consecutive batch reactions in dibutyl ether, the remaining activity of the cross-linked crystals was more than 70 times higher than for the immobilized enzymes. Nevertheless, the specific activity of the cross-linked crystals per milligram protein was reduced compared with the activity of Celite-immobilized enzymes [53],... 

Typical methods for generation of polymorphs include sublimation, crystallization from different solvents, vapor diffusion, thermal treatment, melt crystallization, and rapid precipitation. High-throughput screening methods have been reported for polymorph screening.50... 

As described earlier, there are a number of different ways of crystallizing proteins. By far, the most common approach is the vapor-diffusion method, as mentioned earlier. Approximately, 70% of the crystal structures reported have been crystallized through variations of the vapor-diffusion method. The technique can be carried out in a number of ways the simplest two being the hanging drop method, and the sitting drop ... 

Microbatch crystallization has recently become a popular choice, for a number of reasons. First, microbatch generally uses much less protein and reagents than vapor diffusion. In addition, microbatch is much more amenable to using robotics, and there are a number of proteomics groups that have turned to microbatch for their high-throughput programs. Lastly, microbatch will often yield different crystallization conditions than vapor diffusion (Baldock et al., 1996), so the two methods can be complementary to each other. 

Baldock P, Mills V, Stewart PS. 1996. A comparison of microbatch and vapor diffusion for initial screening of crystallization conditions. J Cryst Growth 168 170-174. 

B. Zheng, J.D. Tice, L.S. Roach, and R.F. Ismagilov A Droplet-Based, Composite PDMS/Glass Capillary Microfluidic System for Evaluating Protein Crystallization Conditions by Microbatch and Vapor-Diffusion Methods with on-Chip X-Ray Diffraction. Angew. Chem. Int. Ed. 43, 2508 (2004). 

Saridakis, E. and Chayen, N. E. (2000). Improving protein crystal quality by decoupling nucleation and growth in vapor diffusion. Protein Sci. 9, 755-757. 

Lusty, C. J. (1999). A gentle vapor-diffusion technique for cross-Unking of protein crystals for cryocrystallography. /. Appl. Cryst. 32,106-112. 

Aza[60]fulleronium ion 28 has been isolated and characterized by X-ray crystallography via the oxidation of 2 with the radical cation hexabromo(phenyl)carbazole (HBPC ) in dry ODCB [27]. The counter-ion is the silver(I) complex carborane ion Ag(CBjjHgCl5)2]. The salt was crystallized as dark green crystals by diffusion of hexane vapor. The solid is reasonably air stable. has almost the same structure... 

Lounaci, M., Rigolef P., CasquiUas, G. V., Huang, H. W., and Chen, Y. 2006. Toward a comparative smdy of protein crystallization in microfluidic chambers using vapor diffusion and batch techniques. Microelect. Eng. 83 1673-76. 

Crystal structure of a protein molecule can also be determined by x-ray crystallography. Purified protein is crystallized either by batch methods or vapor diffusion. X-rays are directed at a crystal of protein. The rays are scattered depending on the electron densities in different positions of a protein. Images are translated onto electron density maps and then analyzed computationally to construct a model of the protein. It is especially important for structure-based drug designs. 

Boc-(R,/ )-/ra s-ACHC-OBzl (31 0.047 g, 0.14 mmol) was dissolved in 4M HCl/dioxane (0.5 mL) and stirred for 1 h. The solvent was then removed under a stream of N2, and the residue 34 was dried under vacuum. Boc-(f ,f )-trans-ACHC-OH (33 0.034g, 0.14mmol) and DMAP (0.023g, 0.19mmol) were added followed by DMF (lmL). EDC (0.059 g, 0.31 mmol) was added, and the reaction was stirred for 48 h under N2. Solvent was removed under a stream of N2, and the residue was further dried under vacuum. To this residue was added 1M HQ ( 3mL) and the solid that did not dissolve was isolated by suction filtration and washed with additional 1M HC1. The solid was dried under vacuum yield 0.051 g (79%). Fluffy crystals were grown by vapor diffusion of heptane into a soln of 35 in 1,2-dichloroethane mp 195 °C. 

Another type of motion of crystal/vapor interfaces occurs when a supersaturation of vacancies anneals out by diffusing to the surface where they are destroyed. In this process, the surface acts as a sink for the incoming vacancy flux and the surface moves inward toward the crystal as the vacancies are destroyed. This may be regarded as a form of crystal dissolution, and the kinetics again depend upon the type of surface that is involved. 

Interfaces may be sharp or diffuse. A sharp interface possesses a relatively narrow core structure with a width close to an atomic nearest-neighbor separation distance. Examples of sharp crystal/vapor and crystal/crystal interfaces are shown in Figs. B.l and B.2. 

One widely used technique is vapor diffusion, in which the proteinlprecipitant solution is allowed to equilibrate in a closed container with a larger aqueous reservoir whose precipitant concentration is optimal for producing crystals. An example of this technique is the hanging-drop method (Fig. 3.2). 

Less than 25 pL of the solution of purified protein is mixed with an equal amount of the reservoir solution, giving precipitant concentration about 50% of that required for protein crystallization. This solution is suspended as a droplet underneath a cover glass, which is sealed onto the top of the reservoir grease. Because the precipitant is the major solute present, vapor diffusion in this closed system results in net transfer of water from the protein solution to the reservoir, until the precipitant concentration is the same in both solutions. Because the reservoir is much larger than the protein solution, the final concentration of the precipitant in the protein solution is nearly equal to that in the reservoir. When the system comes to equilibrium, net transfer of water ceases, and the protein solution is maintained at the optimal precipitant con-... 

Crystals were grown by vapor diffusion in hanging drops from solutions containing methylpentanediol (MPD), polyethylene glycol) (PEG), ammonium sulfate (AS), or low-molecular-weight alcohols (A). 

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