Dye precipitation

For the reaction:-

Protein in solution -------- Protein precipitate

most of the methods discussed above may be called “pushing”  methods, in that the properties  of the solution  are changed, making these unsuitable for protein dissolution,  i.e. the protein  is  pushed  out of solution.  If  the protein is present at a very low concentration,  in a large volume of solution, “pushing” methods can be quite uneconomical as the amount of precipitant required is proportional to the total  solution volume, and often inversely proportional to the protein  concentration.  The alternative is a “pulling” method, in which properties of the protein are changed so that it comes out of solution.  The push/pull terminology  is due to Dr Rex Lovrien, of the University of Minnesota.

An example of a pulling method is dye precipitation or,  as it has been called, “matrix  co-precipitation”. Proteins are kept in solution by the disposition of charged, hydrophilic, groups on their  surfaces.  At  low pH these are mainly positive and at high pH mainly negative.  Dyes,  on the other hand, are typically salts of strong  acids or bases with attached aromatic groups having extended conjugation, which gives rise to their colour. If a dye having a negatively charged sulfonic acid group is added to a positively charged protein,  ionic bonds will form between the  dye and the  protein.  As a result,  bulky,  hydrophobic  groups  will become attached to the protein at its previously positive sites and the protein will be precipitated out of solution.  An  advantage  of this  method  is that  the amount of dye required is proportional  to the moles of protein present, not to the  volume of solution,  so  it  is particularly  suitable for  harvesting proteins from dilute solutions.

After precipitation,  it is necessary to  separate  the  protein  from  the complexed dyestuff. This can be accomplished either by ion-exchange or by TPP.  In  TPP,  the  high  salt  concentration  breaks the  ionic  bonds and the released dye is extracted into the t-butanol layer.  Since dye precipitation is a  pulling  method  and TPP  is a “pushing”  method,  the sequential application  of these  two  could  be  described  as  a  “pull -push” method.

References

-Dennison, C. (2002). A guide to protein isolation . School of Molecular mid Cellular Biosciences, University of Natal . Kluwer Academic Publishers new york, Boston, Dordrecht, London, Moscow .