[Histonet] Methanol in H2O2 explanation--Summary

Wed Jul 11 14:14:21 CDT 2012

Hi Histonetters,
This has been a fun conversation.  I called up a technical support scientist at Vector Labs with the question and was told basically "let me get back to you on that."  :)
But with some digging and the reference from Gudrun (thanks!) I've pieced together the following narrative.

1) Excess H2O2 leads to accumulation of Peroxidase compound III.

The Third Intermediate Compound of Horseradish Peroxidase and Hydrogen
Peroxide

Philip George

J. Biol. Chem. 1953 201: 427-434.

www.jbc.org/content/201/1/427.full.pdf

2) Compound III is inactive as a peroxidase and can transfer of electron to
hydrogen peroxide forming hydroxyl radical which attacks the heme porphoryin
ring causing irreversible inactivation of the enzyme.

Femji et-al
The Open Enzyme Inhibition Journal, 2009, 2, 28-35

3) Methanol itself effectively (ethanol not so much) cleaves Heme groups from
Horseradish peroxidase.

 Werner Straus J Histochem Cytochem, 1974 22:908

4) As the Vector Labs Quenching Protocols cheat sheet explains: "Methanol accelerates the destruction of the heme groups so a lower
concentration of

H2O2
can be used for a longer period of time."

 www.vectorlabs.com/Protocols/Supprotocols/quenchHRP.pdf

So Methanol actually is a "magic chemical" in this case!

Jerry Ricks
Research Scientist
University of Washington
Department of Pathology

> From: gu.lang <@t> gmx.at
> To: histonet <@t> lists.utsouthwestern.edu
> Date: Wed, 11 Jul 2012 20:22:38 +0200
> Subject: [Histonet] Re: Methanol in H2O2 explanation
> 
> Ha! I've found it. It can be found in Dr. Kiernan's book in the chapter
> enzymehistochemistry - the thing, that an electron-donator is used for
> demonstration.
>  And he states, that methanol alone also inhibits peroxidase - perhaps by
> the fixation = denaturation effect on the protein?
> 
> In the following publication one can see the equations for the
> peroxidase-reaction and this phrase: 
> "In this study, we have used a steady state kinetics approach to demonstrate
> that MPO is irreversibly inactivated by excess H2O2 in the absence of
> reducing substrate."
> The Open Enzyme Inhibition Journal, 2009, 2, 28-35 Mechanism-Based
> Inhibition of Myeloperoxidase by Hydrogen Peroxide: Enhancement of
> Inactivation Rate by Organic Donor Substrates
> 
> It's rather exhausting to prove the own statements  ;-) puh.
> 
> Gudrun
> 
> 
> -----Ursprüngliche Nachricht-----
> Von: Morken, Timothy [mailto:Timothy.Morken <@t> ucsfmedctr.org] 
> Gesendet: Mittwoch, 11. Juli 2012 18:12
> An: gu.lang <@t> gmx.at; histonet <@t> lists.utsouthwestern.edu
> Betreff: RE: [Histonet] RE: Re: Methanol in H2O2 explanation
> 
> It's funny how histology lore gets passed down, passed around and
> misunderstood over the decades. When I ask questions in labs about why they
> do a certain procedure they can rarely name a source (besides a particular
> long-retired particular tech who wrote the original procedure) or give
> reason as to why it is done that way. Even if it works well, at least people
> could take the time to understand why!
> 
> When I was researching buffer compositions long ago I found a dozen
> variations of PBS alone (and only a dozen because I just stopped looking).
> When you delve into these things it becomes clear that people made their own
> solutions for a particular purpose. Maybe they wrote a paper, taught a
> course or wrote a book. Then that formula was copied by many others and
> their particular formulation became the "state of the art" even if the "art"
> practiced by someone else had little to do with the original pursuit (the
> Sheehan book is full of variations of stains that were originally for
> specific research purposes. There is precious little guidance in any books
> about what is good for a particular use!).
> 
> The reason for using methanol / H2O2  that I was told long ago was prevent
> frozen sections from being damaged by H2O2 bubbling. In fact, we would fix
> frozens in cold acetone and THEN put the slides in cold methanol/H2O2
> (double fixation?)  In that lab we never used methanol for deparaffinized
> sections. Since I had no exposure to the clin lab I never knew about using
> methanol for blood smear fixation. From literature searches it seems
> methanol, rather than ethanol, is used primarily because it is cheaper. In
> other words, it does not appear to be a magical substance!
> 
> The questions asked here are good ones. However, hopefully those reading and
> responsible for producing procedure manuals will include the reasoning for
> each step of the method and not just assume it is necessary, or "common
> knowledge," just because it has been handed down through generations of long
> suffering techs!
> 
> 
> Tim Morken
> 
> 
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Gudrun Lang
> Sent: Wednesday, July 11, 2012 7:17 AM
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: AW: [Histonet] RE: Re: Methanol in H2O2 explanation
> 
> For the enzymatic activity of peroxidase it needs an electron-donator (or
> receptor - I can't find the literature...) in the vicinity; therefore H2O2
> and DAB are added ad once, and DAB is oxidized and transformed into the
> insoluble, amorphe substance through polymerization.
> Without the donator H2O2 in excess works as inhibitor and blocks the
> activation-side of the enzyme.
> I think H2O2 in methanol was primarly preferred, because the frozen slides
> were fixed at the same time.
> 
> Rehydration after dewaxing depends on the following reagens.
> 
> Gudrun
> 
> -----Ursprüngliche Nachricht-----
> Von: histonet-bounces <@t> lists.utsouthwestern.edu
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] Im Auftrag von Tony
> Henwood (SCHN)
> Gesendet: Mittwoch, 11. Juli 2012 06:21
> An: 'Hobbs, Carl'; histonet <@t> lists.utsouthwestern.edu
> Betreff: [Histonet] RE: Re: Methanol in H2O2 explanation
> 
> Hi Carl,
> 
> What do you mean by "Why do people rehydrate after dewaxing?" Do you really
> mean that slides do not require rehydration or do you mean that slides can
> be left to dry after de-waxing prior to staining.
> 
> Re-hydration is necessary, otherwise xylene will prevent aqueous stains from
> doing their thing efficiently.
> 
> I was lead to believe that as H2O2 was catalysed by endogenous peroxidase,
> the reactive oxygen reacted with the methanol to then degrade the enzyme,
> but I need to look closer at this chemistry.
> 
> 
> Regards
> Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
> Laboratory Manager & Senior Scientist
> Tel: 612 9845 3306
> Fax: 612 9845 3318
> the children's hospital at westmead
> Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001,
> Westmead NSW 2145, AUSTRALIA 
> 
> 
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Hobbs, Carl
> Sent: Wednesday, 11 July 2012 1:32 PM
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] Re: Methanol in H2O2 explanation
> 
> Why do some people use methanolic H2O2?
> Why do people rehydrate after dewaxing?
> Both are unnecessary, under usual conditions.
> 
> Methanol was used in the early days as a peroxidase blocker by itself.
> The combination was devised as a "belt and bracer" method.
> As you stated, you use aq H2O2 effectively.
> So do I and many others.
> 
> However, for unfixed frozen sections, I would never use aq H2O2, if I wanted
> sections remaining on my slides....
> 
> After dewaxing, rinse sections in 4x 100% alcohol, rinse in water, endog. Px
> block while you make up you AR solutions....
> 
> Carl Hobbs
> Histology and Imaging Manager
> Wolfson CARD
> School of Biomedical Sciences
> Kings College London
> Guys Campus
> SE1 1UL
> Tel: 020 78486813
> Fax: 020 78486816
> 020 78486813
> 
> 
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