While evaluating pH as a crystallization variable, one should also consider the buffer. When reproducing and optimizing a condition, be sure to use the same buffer in the original screen producing the hit. If HEPES buffer was used, uses HEPES, do not at first, use HEPES sodium. The same is true for Tris and TRIS hydrochloride, Citric acid and Sodium citrate, and other buffer pairs. The measured conductivity of 1.0 M HEPES pH 7.5 is 13 mS/cm, whereas 1.0 M HEPES sodium pH 7.5 is 43 mS/cm. Different buffers require different volumes of the acid or base pair of the buffer, or HCl or NaOH to be titrated to a specific pH. This in turn can result in a reagent with a unique ionic strength. This subtle change in ionic strength can influence crystallization and can make the difference in being able to successfully reproduce a hit or not.

The buffer molecule itself can be a crystallization variable. 1.0 M Tris pH 7.5 has a measured conductivity of 43 mS/cm where 1.0 M HEPES pH 7.5 13 mS/cm. While this difference in ionic strength could be masked in a high salt condition, it might be significant in a low ionic strength polymer based condition. While it is important to at first use the same buffer used to produce the hit, evaluating a different buffer is something that should be considered during optimization. Until evaluated, one does not know if varying ionic strength will worsen or improve crystal quality. The unique chemical structure of different buffers effective over the same pH can also be a crystallization variable. The buffers Citrate, Malate, Succinate, Phosphate, Cacodylate, MES, Bis-Tris, and ADA are all appropriate buffers at pH 6. The unique chemical properties and structure of each of these buffers may influence crystal quality. Screen both pH and buffer type during optimization.

The ionizable amino acid side chains in proteins are aspartic and glutamic acid (pKa values of about 4.5), histidine (pKa = 6.02), cysteine (pKa = 8.2), lysine (pKa = 10.5), tyrosine (pKa = 10.2), and arginine (pKa = 12.2). Although the pKa of an ionizable group on a protein may be strongly influenced by its chemical environment, it is worth keeping these pKa values in mind, as it is in their immediate neighborhoods that the charges on a protein, their distribution and their electrostatic consequences may be most sensitive. Buffer pH can determine the ionization state of the side chains, and that can determine sample-sample and sample-solvent interactions, as well as lattice interactions, all having the potential to manipulate crystal quality.

Keep in mind the actual, measured pH of the crystallization reagent (condition, cocktail) may be different from that of the buffer stock used in formulation. This is okay, as long as one can reproduce the result. Most screen reagents are made from 0.5 or 1.0 M titrated buffer stocks, diluted to a final concentration of 0.1 M or lower. Dilution alone can change the pH, as well as the effect of additional chemicals, including salts, polymers, non-volatile organics, and additives. Unless otherwise indicated, or a Grid Screen kit which are titrated to pH after buffer and chemicals are added, the pH indicated on a tube label or formulation is that of a 1.0 M stock prior to the addition of other chemicals. Formulation details for screen buffer is available in Crystal Growth 101Buffer Formulation.

pH & Buffer focused kits

CAT. No. NAME DESCRIPTION pH RANGE
HR2-455 GRAS Screen 5 1 mL, Deep Well block format 4.5 - 10
HR2-456 GRAS Screen 6 1 mL, Deep Well block format 4.5 - 10
HR2-457 GRAS Screen 7 1 mL, Deep Well block format 4.5 - 10
HR2-458 GRAS Screen 8 1 mL, Deep Well block format 4.5 - 10
HR2-080 PEG/pH 10 mL, tube format 3.5 - 7.4
HR2-081 PEG/pH 2 10 mL, tube format 6.4 - 9.6
HR2-461 PEG/pH HT 1 mL, Deep Well block format 3.5 - 9.6
HR2-211 Grid Screen Ammonium Sulfate 10 mL, tube format 4 - 9
HR2-219 Grid Screen Sodium Chloride 10 mL, tube format 4 - 9
HR2-247 Grid Screen Sodium Malonate 10 mL, tube format 4 - 9
HR2-217 Grid Screen PEG/LiCl 10 mL, tube format 4 - 9
HR2-213 Grid Screen PEG 6000 10 mL, tube format 4 - 9
HR2-215 Grid Screen MPD 10 mL, tube format 4 - 9
HR2-248 Grid Screen Salt HT 10 mL, tube format 4 - 9
HR2-221 Quik Screen 10 mL, tube format 5 - 8.2
HR2-070 Slice pH 0.5 mL, Deep Well block format 3.5 - 9.6
HR2-104 StockOptions Citric Acid 10 mL, tube format 2.2 - 6.5
HR2-233 StockOptions Sodium Acetate 10 mL, tube format 3.6 - 5.6
HR2-240 StockOptions Malic Acid 10 mL, tube format 3.7 - 6.0
HR2-235 StockOptions Sodium Citrate 10 mL, tube format 4.2 - 6.5
HR2-249 StockOptions Succinic Acid 10 mL, tube format 4.3 - 6.6
HR2-251 StockOptions Phosphate 10 mL, tube format 5.0 - 8.2
HR2-239 StockOptions Sodium Cacodylate 10 mL, tube format 5.1 - 7.4
HR2-243 StockOptions MES 10 mL, tube format 5.2 - 7.1
HR2-106 StockOptions Bis-Tris 10 mL, tube format 5.5 - 7.5
HR2-255 StockOptions ADA 10 mL, tube format  5.6 - 7.5
HR2-257 StockOptions PIPES 10 mL, tube format  6.1 - 7.5
HR2-095 StockOptions Imidazole 10 mL, tube format  6.2 - 7.8
HR2-103 StockOptions Bis-Tris Propane 10 mL, tube format  6.3 - 9.5
HR2-252 StockOptions MOPS 10 mL, tube format  6.5 - 7.9
HR2-102 StockOptions Hepes 10 mL, tube format  6.8 - 8.2
HR2-231 StockOptions Sodium HEPES 10 mL, tube format  6.8 - 8.2
HR2-100 StockOptions Tris 10 mL, tube format  7.0 - 9.0
HR2-237 StockOptions Tris Hydrochloride 10 mL, tube format  7.0 - 9.0
HR2-101 StockOptions Bicine 10 mL, tube format  7.4 - 9.3
HR2-253 StockOptions Tricine 10 mL, tube format  7.4 - 8.8
HR2-254 StockOptions AMPD 10 mL, tube format  7.8 - 9.7
HR2-256 StockOptions CHES 10 mL, tube format  8.6 - 10.0
HR2-250 StockOptions Glycine 10 mL, tube format  8.6 - 10.6
HR2-241 StockOptions pH 10 mL, tube format  2.2 - 11.0

 

Suggested Readings

Structural studies on the adenovirus hexon. Franklin RM, Harrison SC, Pettersson U, Philipson L, Brändén CI, Werner PE. Cold Spring Harb Symp Quant Biol. 1972;36:503-10.

Increasing the size of microcrystals by fine sampling of pH limits. A. McPherson. J. Appl. Cryst. (1995). 28, 362-365.

Acid pH crystallization of the basic protein lysin from the spermatozoa of red abalone (Haliotis rufescens). T. C. Diller, A. Shaw, E. A. Stura, V. D. Vacquier and C. D. Stout. Acta Cryst. (1994). D50, 620-626.

Protein Isoelectric Point as a Predictor for Increased Crystallization Screening Efficiency. K. A. Kantardjieff and B. Rupp. Bioinformatics 20(14): 2162-2168 (2004).

Distributions of pI vs pH provide strong prior information for the design of crystallization screening experiments. K. A. Kantardjieff, M. Jamshidian and B. Rupp. Bioinformatics 20(14): 2171-2174 (2004).

Novel buffer systems for macromolecular crystallization. J. Newman. Acta Cryst. (2004). D60, 610-612.

Optimization of buffer solutions for protein crystallization. R. A. Gosavi, T. C. Mueser and C. A. Schall. Acta Cryst. (2008). D64, 506-514.

Optimum solubility (OS) screening: an efficient method to optimize buffer conditions for homogeneity and crystallization of proteins. J. Jancarik, R. Pufan, C. Hong, S.-H. Kim and R. Kim. Acta Crystallographica Section D, Biological Crystallography, Volume 60, Part 9 (September 2004).

Crystallization Optimum Solubility Screening: using crystallization results to identify the optimal buffer for protein crystal formation. Bernard Collins, Raymond C. Stevens, and Rebecca Page. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2005 December 1; 61(Pt 12): 1035–1038.

Introduction of Fluorometry to the Screening of Protein Crystallization Buffers. Takamitsu Ikkai and Katsuhiko Shimada. Journal of Fluorescence, Volume 12, Number 2, June, 2002, Pages 167-171.

Lepre, C. A., Moore, J. M.; Microdrop screening: A rapid method to optimize solvent conditions for NMR spectroscopy of proteins; Journal of Biomolecular NMR, 12: 493-499, 1998.

Optimization of Met8p crystals through protein-storage buffer manipulation. H. L. Schubert, E. Raux, M. J. Warren and K. S. Wilson. Acta Cryst. (2001). D57, 867-869.

The Effect of Temperature and Solution pH on the Nucleation of Tetragonal Lysozyme Crystals. Russell A. Judge, Randolph S. Jacobs, Tyralynn Frazier, Edward H. Snell, and Marc L. Pusey.

A protein crystallization strategy using automated grid searches on successively finer grid screens. Patricia C. Weber. Methods: A Companion to Methods in Enzymology. Vol. 1, No. 1, August, pp. 31-37, 1990.

Buffer Solutions The Basics. R.J. Beynon and J.S. Easterby. 1996. IRL Press.