Yes, pH is extremely important in enzyme histochemistry. 

(disclaimer: caps are used here for emphasis only, NOT for offending anyone...)

My question was: Why are myolin-ATPase stains run at THOSE particular pHs? Why
were those pHs chosen? How do they relate to the staining mechanism (if known)?

More to my original point, do ATPase procedures take into account the
thermodynamic fact that the effective pH of most buffers declines as temperature
increases? 

For example the glycine buffer used in some ATPase stains will drop approx 0.4 -
0.5 pH units as its temp rises from 20C to 37C. According to a table I found,
only the citrate buffer used near pKa 6.40 does not change pH with temperature.
Try it: measure a buffer at room temp then put it into a 37C oven and measure it
after 10-15min - but be sure to bring your meter to the oven so you are actually
measuring the buffer at 37C. Then let it cool to room temp and measure it again.
Even easier, just look at the temperature adjustment table on the label of your
calibration buffers (Fisher prints it on the label).

My original question was essentially: Does a 9.4 stain - which is going to be
incubated at 37C - need to be pH'ed to 9.4 at room temp? OR does it actually
need to be AT pH 9.4 WHILE incubating. In the later case, the buffer actually
has to be pH'ed to something ABOVE 9.4 at room temp because the effective pH at
37C will often be significantly lower. Again, is "9.4" just another mysterious
part of the procedure, or is it somehow characteristic of a particular isoform
of the human myolin-ATPase enzyme? (Enzyme isoforms are present in different
ratios in different fiber types. Somehow this produces the light-dark patterns
in ATPase stains, but I don't know how.)

My point is emphasized by an excellent buffer design website. Given your needs
it will calculate a recipie for your buffer. You are REQUIRED to indicate the
temperature at which you will make the buffer AND THE TEMPERATURE AT WHICH YOU
WILL USE IT. The recipes state the buffer must be adjusted to a pH ABOVE the
desired pH if it is to be used at a temperature above the mixing temp. 

Check out this buffer design site:
http://www.bi.umist.ac.uk/users/mjfrbn/buffers/makebuf.asp


I hope I haven't repeated myself too much, but I realized I probably wasn't
clear in my first posting - and maybe not in this one...

Thanks again to all who respond,
Sincerely,
-brice


> -----Original Message-----
> From:	Komuves, Laszlo [SMTP:Laszlo.Komuves@corr.com]
> Sent:	Thursday, October 18, 2001 2:21 PM
> To:	Due, Brice; histonet@pathology.swmed.edu
> Subject:	RE: muscle ATPAse
> 
> 
> Skeletal muscles are made up of different fiber populations (Type1, Type 2A,
> 2B, 2C). The classification/detection/diagnosis of these fibers is largely
> based on ATPase enzyme histochemistry.
> 
> Read the  following most basic textbooks to learn more and admire the color
> microphotographs: 
> Sternberg SS Histology for pathologists, 2nd Ed, 1997, pp. 197-220. 
> Woods AE & Ellis RC, Laboratory Histopathology, 1994, pp. 7.3.8-7.3.11. 
> Carson FL, Histotechnology, 1997, pp.260-263. 
> 
> The message is: pH is not chosen arbitrarily, it has a huge importance, it
> does matter. 
> 
> Correspondence: 
> 
> László G. Kömüves, Ph.D. 
> Senior Scientist, Histology, 
> COR Therapeutics, Inc. 
> 256 East Grand Avenue, 
> South San Francisco CA 94080 
> 
> Phone: (650) 244-6855, Fax: (650) 244-9270, E-mail: lkomuves@corr.com 
>