Protein crystallization dialysis

Zeppezauer, M., Eklund, H., and Zeppezauer, E. S. Micro diffusion cells for the growth of single protein crystals by means of equilibrium dialysis. Arch. Biochem. Biophys. 126, 564-573 (1968). 

Fig. 2. Crystals of EFTu and the absorption spectrum of the purified protein. Left panel crystals of EFTu GDP formed in 0.35 saturated ammonium sulfate. Right panel absorption spectrum of EFTu. Top curve, spectrum of isolated EFTu middle curve, spectrum of protein after dialysis in the absence of Mg + lower curve, difference spectrum. (From Miller and Weissbach. )...

As noted earlier, one of the most powerful techniques for producing a supersaturated protein solution is adjustment of the pH to values where the protein is substantially less soluble. This may be done in the presence of a variety of precipitants so that a spectrum of possibilities can be created whereby crystals might form. The gradual alteration of pH is particularly useful because it may be accomplished by a variety of gentle approaches that do not otherwise perturb the system or introduce unwanted effects. Chief among these are dialysis and vapor diffusion. 

Electrophoresis of a crystalline preparation revealed one major enzymatically active component and a minor inactive one (Negelein and Wulff, 1937). The smaller, inactive constituent varied from 5% to 20% of the total protein. Re-examination of the electrophoretic properties of yeast ADH crystals confirmed the presence of one slow, presumably active, and one fast-moving, presumably inactive component. Their relative amounts did not change systematically with recrystallization as shown by area analysis. The percentage of inactive component was a function of age of the solution and duration of preliminary dialysis. Preparations dialyzed for 20 hours at pH 5 contained as much as 14 % to 25 % of the inactive component, while dialysis for 4 hours showed as little as 6 %. The second component was assumed to be an inactive transformation product of the active enzyme (Hayes and Velick, 1954). 

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