I have not seen a reply to your enquiry So I offer one. This is not based
on my experience but on literature references and comments from books. Th=
e
use of Pronase and aminopeptidase M is advocated.
Quotation from 'Protein Protocols on CD-ROM', Humana Press
Pronase is known to contain at least ten proteolytic components: five
serine-type proteases, two Zn2+ endopeptidases, two Zn2+ -leucine
aminopeptidases, and one Zn2+ carboxypeptidase. Pronase therefore has ve=
ry
broad specificity, and is used in cases where extensive or complete
degradation of protein is required.
To prepare a protein hydrolysate, dissolve 0.2 mmol of protein in 0.2 mL =
of
0.05M ammonium bicarbonate buffer, pH 8.0 (or 0.2M sodium phosphate pH 7.=
0
if ammonia interferes with the amino acid analysis). Add Pronase to 1%
(w/w), and incubate at 37=B0C for 24 h. To achieve complete hydrolysis, i=
t is
usually necessary to make a further addition of aminopeptidase M (see
Section 18.5) at 4% (w/w), and incubate at 37=B0C for a further 18 h. The
sample is then lyophilized and subjected to amino acid analysis. When usi=
ng
two enzymes in this way, there is often an increase in the background ami=
no
acids owing to hydrolysis of each enzyme. It is therefore important to
carry out a digestion blank to correct for these background amino acids
Hediger, H., Stevens, R. L., Bradenberger, H., and Schmid, K. (1973)
Determination of asparagine, glutamine and pyrrolidonecarboxylic acld in
total enzymic hydrolysates of peptides and glycopeptides by gas-liquid
chromatography. Biochem. J. 133, 551-561.
.Nomoto, M., Narahashi, Y. and Murakami, M. (1960) A proteolytic enzyme o=
f
Streptomyces griseus. Hydrolysis of protein by Streptomyces griseus
protease. J. Biochem. 48(4), 593-602.
Hope this helps
Len
>Dear ABRFs
>
>I would need to perform aminoacid analyses on plasma protein samples,
>searching a special residue wich is sensible to both acid and alkaline
>hidrolysis. I know that there are several methods for total hidrolysis
>by using enzyme combinations. It would be very kind of you to address me
>
>to where I can obtain these procedures.
>
>Thanking in advancement your solidarity
>
>Manel Portero
>Medical School
>University of Lleida
>SPAIN
*********************************************************************
Dr Len C. Packman
Assistant Director of Research
Protein and Nucleic Acid Chemistry Facility
Department of Biochemistry
University of Cambridge
80 Tennis Court Road
Old Addenbrookes Site
Cambridge, CB2 1GA, UK
Tel: +44 (1223) 333639
FAX: +44 (1223) 766002
e-mail: lcp2@mole.bio.cam.ac.uk
Visit my WWW page at http://www.bio.cam.ac.uk/proj/adr/PNAC/pnac.html