Re: AAA hydrolysis

John Stewart (John.Stewart@UCHSC.edu)
Mon, 17 Aug 1998 19:04:04 -0600 (MDT)

Nadine:
The best procedure is to add a reducing agent to the hydrolysis
mixture. As recommended in Stewart & Young (1984), page 109, use 6N HCl
containing 1 mg/mL each of phenol and 2-mercaptoethanol. Bubble with N2
slowly for 2min and seal. This allows analysis of Trp if you work up
rapidly and minimize exposure of the acid residue to O2. Cys2 is reduced
to CySH, which elutes near Pro; analyze it in performic oxidized samples.
We use pyrex culture tubes with Teflon-lined screw caps. No evacuation is
needed. Just dont bang the hot tubes; they might explode due to internal
pressure.

John M. Stewart, Department of Biochemistry
Univ. of Colorado Medical School, Denver, CO 80262
Phone: 303-315-7534; FAX: 303-315-8215
Email: John.Stewart@UCHSC.edu


On Mon, 17 Aug 1998, Ritter,Nadine wrote:

> Thanks for the responses on the PTC-AAA question. I suspected that they
> might not be available pre-labelled because they are not stable for long.
> This is similar to PTH-AAs, which have to undergo conversion right before
> HPLC analysis. Fortunately, the sequencer has the reaction cartridge and
> conversion flasks as separate entities, making troubleshooting a bit easier.
>
> Now for a hydrolysis question: I recall reading somewhere that one way to
> test your hydrolysis conditions for complete oxygen scavenging is to add
> sodium sulfite (or bisulfite, I can't recall) to the ?sample tubes? or
> ?acid/phenol pool in the bottom of the hydrolysis vial?. This was for the
> typical 6N HCl/phenol hydrolysis conditions whereby samples are in pyrolized
> tubes which are placed in the hydrolysis vial with the HCl and phenol at the
> bottom. The vial is purged with nitrogen several times, then a vaccum is
> drawn and the vial sealed, then placed in a heating block for the required
> time.
>
> Does anyone know of this procedure? Of course I can't locate that procedure
> in my references, now that I need it. If not this procedure, does anyone
> have a procedure to demonstrate that no oxidation is occuring during vacuum
> hydrolysis, apart from hearing the slight "pssst" as you open the
> vacuum-sealed vials after heating?
>
> Thanks again!
>
> Nadine Ritter
> Abbott Diagnostics Division
> Nadine.Ritter@ add.ssw.abbott.com
>
> Gerbil Update: The older son came home and forgave his mom for the gerbil
> incident, and the loss of his other pets. However, he did request
> verfication from his dad that they really died of natural (or accidental)
> causes, rather than being used in scientific experimentation, as his mom does
> have a history of this with chickens, rats, mice, pigeons, hamsters, sharks,
> stingrays, and ratfish...let's just say that his concerns are justified.
>
>
>
>