BioUltra Reagents

Denaturation


Principles and Definition
Denaturation of Proteins and Nucleic Acids
References
Product List



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Principles and Definition

The biological activity of a biopolymer is closely linked to its native structure which is retained only if critical factors such as temperature, pH, solvent and solute are kept within narrow limits. Any alteration of the native structure causing a loss of biological activity is called denaturation. However, it should be noted that denaturation does not imply a modification of the primary structure (sequence) of the biopolymer.
Reversibility: under appropriate conditions, the renaturation of various denatured proteins is possible, e.g. the renaturation of Iysozyme [1]. Denaturation of nucleic acids was thought to be irreversible until 1961 [2].


Denaturation of Proteins

Denaturation of proteins can be achieved by various means [3]:


  • Chaotropic agents [4], such as urea and guanidine hydrochloride, are effective at high concentration, i.e. 4 M to 8 M. The study of chaotropic effects now allows a better understanding of the amino acid sequence of a protein and its 3-dimensional structure [5]
  • Detergents, e.g. sodium dodecyl sulfate [6], which is commonly used in gel-electrophoresis [7].
  • Heat, usually leads to precipitation.
  • Reagents which cleave disulfide bridges, e.g. dithiothreitol or dithioerythritol [8].
  • Acids or bases which cleave salt bridges. Moderate concentrations of certain acids, e.g. trichloroacetic acid, usually lead to complete denaturation and precipitation and can therefore be used to stop enzymatic reactions. Specific denaturation may be useful in protein purification [9].

Denaturation of Nucleic Acids

Effective denaturation of nucleic acids occurs with extreme pH, low ionic strength, and heat [10]. Denaturation of DNA is usually achieved by heat treatment or high pH, which causes the double-stranded helix to dissociate into single strands.

A reversible method of denaturing tRNA is by removal of magnesium ions with a chelator, e.g. ethylenediaminetetraaceticacid [11]. Denaturation of DNA is a prerequisite for the hybridization of originally double-stranded DNA with other nucleic acids [12]. Specific denaturing conditions can be chosen aimed at dissociating only local parts of double-stranded DNA. In denaturation mapping of DNA [13] position and size of denatured sites are measured.


References

  1. C. C. McDonald, W. D. Phillips, J. D. Glickson, J.Am.Chem.Soc.93, 235 (1971).
  2. J. Marmur, P. Doty, J. Mol. Biol. 3, 585 (1961 ).
  3. J. [3] J.C.Bennett, Methods Enzymol. 11, 211 (1967).
  4. T. Arakawa, S.N.Timasheff, Biochemistry 23, 5924 (1984).
  5. C.B.Anfinsen, Science 181, 223 (1973).
  6. J.A.Reynolds, C.Tanford, J.Biol.Chem. 245, 5161 (1970).
  7. U. K. Laemmli, Nature 227, 680 (1970).
  8. R. L. Lundblad, C. M. Noyes, Chemical Reagents for Protein Modification, vol. 1, p. 95-98, CRC Press, Boca Raton, Florida (1984).
  9. R. K. Scopes, Protein Purification, p. 60-66, Springer, New York (1982).
  10. A. M. Michelson The Chemistry of Nucleosides anl Nucleotides, Academic Press, London and New York (1963).
  11. T.lshida, J.L.Arceneaux, N.Sueoka, Methohs Enzymol.20, 98 (1971).
  12. D.Gillespie, Methods Enzymol. 12B, 641 (1968).
  13. S. Dasgupta, R. B. Inman, Methods Enzymol.65, 429 (1980).



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