Re: hydrolysis protocols

Angela c. Murphy (acmurphy@helix.nih.gov)
Wed, 4 Nov 1998 15:16:33 -0500

I too have gotten brown gunk (tar, caramel) from hydrolyzing sugar-
containing peptides or proteins, but have found that the interference
with the amino acid analysis really depends on how much carbohydrate is
there. I routinely do analyses for another lab that does NMR studies of
DNA-binding proteins, and I am often given a sample of a protein which
has the DNA bound. These samples do not give any brown or even yellow
color upon hydrolysis and the analyses are very reproducible. On the
other hand, people in my own lab have given me protein samples that were
concentrated by dialysis against sucrose - very messy. The sucrose
apparently gets through the membrane somehow, perhaps by dissolving in
the water it is supposed to remove. Has anyone else encountered this
problem and found a way to get rid of sucrose before hydrolysis?
Angela C. Murphy

On Wed, 4 Nov 1998, Richard Laursen wrote:

> Kerryn,
>
> All of the reagents you cite are acids, and of them, HCl (6N?) at 105 oC is
> by far the strongest. Therefore, it will hydrolyze both proteins and DNA.
> In the case of DNA, both the phosphodiester bonds and sugar/purine and
> probably sugar/pyrimidine links will cleave. The resulting ribose
> monophosphate esters may be stable--I'm not sure. HF will not have much
> effect on the protein (it's used to deprotect peptides, after all), and
> formic acid at 60 oC will probably cause partial hydrolysis of peptide
> bonds, particularly at Asp.
>
> If you use HCl you will end up with a mixture of sugars and amino acids,
> which are likely react with each other to form, depending on how much of
> each is present, brown "gunk"--the "browning" reaction. If you are doing
> amino acid analysis, you will probably get erratic results because of this.
> This is why one never wants to hydrolyze proteins in the presence of
> carbohydrates. I believe you would get similar results in either the gas
> phase or in solution.
>
> Richard Laursen
> -----------------------
>
>
>
> >Dear ABRF's,
> >
> >I am working with a mixture of dna and protein and need to hydrolyse both
> >consituents.
> >In the past I have used HF(room temp) or formic acid(60 degrees C) for DNA
> >hydrolysis and HCL(105 degrees C, 24 hrs) for protein hydrolysis. Will
> >HF/formic acid hydrolyse protein and will HCL hydrolyse DNA or what would
> >be an appropriate hydrolysis method?
> >Also, how does gas phase hydrolysis of these compounds compare to leaving
> >the compounds sit in the acid?
> >My review of the literature has not enabled me to answer these questions.
> >
> >Any suggestions will be gratefully received.
> >
> >
> >Thanking you,
> >Kerryn Mason
> >Senior Research Fellow
> >Ray Williams Biomedical Mass Spectrometry Facility
> >UNSW
> >Sydney
> >Australia
>
> Richard A. Laursen
> Department of Chemistry
> Boston University
> 590 Commonwealth Ave.
> Boston, MA 02215
> Tel (617) 353-2491; FAX (617) 353-6466
> email: <laursen@bu.edu>
>
>

*******************************************
Angela C. Murphy, Chemist
Lab. of Cell Biology, NHLBI, NIH
3 Center Drive, MSC 0301, Rm. B1-22
9000 Rockville Pike
Bethesda, MD 20892-0301 USA
tel.: (301) 496-2324
fax: (301) 402-1519
email: acmurphy@helix.nih.gov
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