Supersaturation Factors affecting the solubility of proteins

Protein concentration Concentrations in the range 5 to 50 mg/ml are usually used. Concentrations less than 2 mg/ml usually are not effective [31]. In general, it is best to keep the protein concentration as high as possible and vary other parameters. 

Ammonium sulphate is commonly used as a precipitant because of its great solubility and ready availability. It can liberate ammonia, which reacts with heavy atom derivatives. This problem can in some circumstances be alleviated by changing the mother liquor of the crystals to phosphate. Ammonium sulphate caused further problems in the analysis of the crystal structure of oxyhaemoglobin. In the presence of this salt and under irradiation, crystals of oxyhaemoglobin were oxidised rapidly to aqua-met-haemoglobin. The problem was solved by growing the crystals under usual conditions [34] and then transferring them to 3 M phosphate [16]. 

Poly(ethylene glycol) (PEG) McPherson [35] has advocated the use of PEG for protein crystallisation. Out of 22 proteins chosen to test PEG as a precipitant, 13 yielded crystals, six for the first time. The general formula of PEG is 

Poly(ethylene glycol) produced an unexpected result with haemoglobin. Crystals grown in the presence of air were found to have the protein in the T state, in which the a-subunits were oxygenated and the jS-subunits oxygen free [37]. The result has important implications for allostery. 

Temperature The temperature coefficient of solubility varies greatly from protein to protein. At high ionic strengths many proteins are less soluble at 25°C than at 4°C. This may be exploited by forming a super-saturated solution at 4°C and then allowing it to warm up slowly. At low ionic strength solubility usually increases with temperature. 

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