Ammonium sulfate precipitation

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The ammonium sulfate precipitation is a biochemical method for the precipitation of proteins from a solution using ammonium sulfate . Because of its low cost, it is the most widely used method for enriching proteins on a larger scale.

principle

The precipitation of proteins with ammonium sulfate was described in the Hofmeister series as early as 1888 . Due to its low cost, it is suitable as the first purification step in the course of protein purification , in order to separate proteins from other biomolecules . Protein solutions should have a concentration of over 1 mg / ml before precipitation.

Ammonium sulphate is a cosmotrop that precipitates proteins in high concentrations through dehydration and the hydrophobic effect , which is why this precipitation represents the salting out of proteins from a solution. Ammonium sulfate has a solubility of 761 g per liter of water at 20 ° C and a solubility of 706 g per liter of water at 0 ° C. Since the ammonium sulphate increases the volume, the total solubility of the ammonium sulphate at 20 ° C is 533 g / L. Due to the high solubility of ammonium sulphate, all proteins in a solution can be precipitated with increasing concentration. The proteins are not denatured . In addition, even the density of a saturated ammonium sulfate solution is lower than that of proteins, which means that they always settle as a precipitate and can be centrifuged off. The concentration of ammonium sulfate at which a protein precipitates is usually given relative to the solubility of ammonium sulfate, i.e. i.e. 50% of the solubility corresponds to a concentration of 267 g / L at 20 ° C. The concentration of ammonium sulfate is usually determined with a refractometer . A solubility curve can be generated by creating a graph with the logarithm of the experimentally determined solubility plotted against the percentage of ammonium sulfate saturation.

The ammonium sulfate is usually added step by step in solid, finely ground form and the respective precipitated proteins are isolated (fractional precipitation), but the addition can also take place in one step. The mass of ammonium sulfate to be added must be recalculated at each step, as the volume of the solution changes. Alternatively, the mass to be added can also be read from a nomogram . After the dissolution process, the precipitated protein fraction formed in each case is concentrated and desalted by centrifugation and dissolving the precipitate in a buffer or by centrifugation, dialysis and lyophilization .

Alternative precipitations are PEG precipitation , TCA precipitation (denaturing), ethanol precipitation (denaturing) and heat precipitation (denaturing). Alternative non-denaturing methods are mostly chromatography- based.

literature

Web links

Individual evidence

  1. ↑ Franz Hofmeister: On the doctrine of the effect of salts: second communication . In: Archives for pathological anatomy and pathology (1888), Volume 24, pp. 247-260.
  2. ^ Richard R. Burgess: Richard R. Burgess and Murray P. Deutscher (eds.): Methods in Enzymology  (= Guide to Protein Purification, 2nd Edition), Volume 463. Academic Press, January 1, 2009, pp. 331-342 , doi : 10.1016 / s0076-6879 (09) 63020-2 .
  3. ↑ a b c d e f g Alfred Pingoud : Working methods of biochemistry. Walter de Gruyter, 1997, ISBN 978-3-11-016513-5 , p. 53.
  4. ↑ Krisna C. Duong-Ly, Sandra B. Gabelli: Jon Lorsch (Ed.): Methods in Enzymology  (= Laboratory Methods in Enzymology: Protein Part C), Volume 541. Academic Press, January 1, 2014, p. 85– 94, doi : 10.1016 / b978-0-12-420119-4.00007-0 .
  5. ^ A b Robert K. Scopes: Protein Purification. Springer Science & Business Media, 2013, ISBN 978-1-4757-2333-5 , p. 81.
  6. ^ Dale L. Perry: Handbook of Inorganic Compounds, Second Edition. CRC Press, 2016, ISBN 978-1-4398-1462-8 , p. 33.
  7. ↑ Martin Holtzhauer: Biochemical laboratory methods. Springer-Verlag, 2013, ISBN 978-3-642-59173-0 , p. 101.
  8. ^ John R. Whitaker, Robert Hughes: A Rapid Method for Determining the Concentration of Ammonium Sulphate Solutions. In: Nature. 183, 1959, p. 603, doi: 10.1038 / 183603a0 .
  9. ^ Richard R. Burgess: Richard R. Burgess and Murray P. Deutscher (eds.): Methods in Enzymology  (= Guide to Protein Purification, 2nd Edition), Volume 463. Academic Press, January 1, 2009, pp. 331-342 , doi : 10.1016 / s0076-6879 (09) 63020-2 .
  10. ^ Gerhard Richter: Practical Biochemistry. Georg Thieme Verlag, 2003, ISBN 978-3-13-132381-1 , p. 200.
  11. ↑ M. Dixon: A nomogram for ammonium sulphate solutions. In: The Biochemical journal. Volume 54, Number 3, June 1953, pp. 457-458, PMID 13058924 , PMC 1269013 (free full text).
  12. ↑ F. Di Jesus: ammonium sulfate concentration conversion nomograph for 0 degrees. In: The Journal of biological chemistry. Volume 243, Number 8, April 1968, pp. 2022-2023, PMID 5646491 .