In article <3um32f$e8j at sun4.bham.ac.uk> altabios at bham.ac.uk (John E. Fox) writes:
>We are doing some amino acid analysis on a protein which
>has been purified in a solution containing glycerol. On acid
>hydrolysis in constant boiling HCl, the glycerol appears to
>destroy many of the amino acids. Has anyone any idea what is
>happening?
>When the protein has been dialysed to remove the glycerol,
>the amino acid analysis still seems to give lower values for the
>concentration, compared to other protein assays or OD280 measurement.
>The theory so far, is that some glycerol is being trapped in the
>protein and is still degrading the amino acids on hydrolysis.
>>Has anyone out there experienced these sort of problems?
>>John Fox
hi John. Acid hydrolysis of proteins and peptides often destroys some
amino acids--tryptophan will never survive an acid hydrolysis, and
serine and threonine can be partially destroyed as well. AA's like
leucine and alanine tend not to be destroyed as readily and would be
better bets for a quantitative analysis of protein concentration.
Thorough degassing of your HCl solution prior to hydrolysis can help--
freeze the solution, pump on it, let it thaw (you can bleed nitrogen
or argon back into the analysis tube), and repeat two more times
before hydrolysis. Pierce sells tubes that can be hooked up easily
to vacuum tubing for this purpose.
I can't think of any reason why glycerol would be promoting destruction
of the amino acids. If you want to check that, you could take two tubes
of amino acids, add glycerol to only one tube, and boil away for 24hrs or
48hrs or however long you're doing the real hydrolysis for, and see if
there's any difference in the AAA.
I hope this is of some use to you (i.e. I hope I haven't just repeated
stuff you know already!)
Robert Cerpa
cerpa at cgl.ucsf.edu
Graduate Group in Biophysics, UC San Francisco
