>
>We are trying to develop an assay to detect a low level of pepsin
activity
>in the presence of a large amount of protein. The approach of
measuring
>production of TCA soluble peptides from hemoglobin is not sensitive
enough.
>Does anyone know of a suitable colorimetric substrate, such as the
BAEE
>assay for trypsin? Or have any other types of assays to suggest we
look at?
>
>Many thanks,
>
>Beth Fowler
>Elizabeth Fowler
>Director, Biochemistry and Process Development
>AutoImmune Inc.
>128 Spring Street
>Lexington, MA 02173
>Phone: 781-860-0710 x 230
>FAX: 781-860-0705
<underline>>Email: efowler@ma.ultranet.com
</underline>
Dear Beth: At what level of sensitivity are you trying to assay. One
possibility is to use fluorescently-labeled (FITC) myoglobin or
hemoglobin. Pepsin cleaves at the C-terminal of Phe, Leu, Tyr and Trp
preferentially. Double blocked amino acid substrates for all of these
compounds should be available with the MCA leaving group (fluorescent)
(Try Bachem). I do not remember if Pepsin displays good activity on
simple substrates (this should be readily available info.) One problem
with the double-blocked hydrophobic AA substrates is solubility. I
know the FITC-Mb and Hb are quite soluble, these occasionally give rise
to high blanks, but generally work quite well. I recall that that I
was able to get about 0.1 mM concentrations of Bz-Phe-NHNan in
solution. All these beasties dissolve quite well in DMF or DMSO. I
will try to locate more info and send you an update if they are are
useful, gautam
Gautam Sarath
N-226, Beadle Center
Protein Core Facility - Center for Biotechnology &
Department of Biochemistry
University of Nebraska-Lincoln
Lincoln, NE 68588-0664
Phone: 402-472-2928
FAX: 402-472-7842
http://www.biotech.unl.edu/Proteins/index.html