To: Dr. Robert B. Corey From: Walter Schroeder Date: Dec. 6, 1951
Subject: Present status of the study of amino acid sequence in gelatin
About 3 months ago we began to use the ion exchanger, Dowex-50, and the methods of
Moore and Stein in an attempt to isolate peptides from partial hydrolysates of gelatin.
In our initial experiments, we compared the results which were obtained when a partial
hydrolysate and a complete hydrolysate of gelatin were chromatographed under identical
conditions. Despite the arbitrary set of conditions which was used, the results were
very gratifying: the chromatogram of the partial hydrolysate showed eleven zones which
either were absent from the chromatogram of the complete hydrolysate and thus presumably
were peptides or had characteristics indicating at least the presence of peptides.
The partial hydrolysate which was used in these experiments had been obtained by refluxing
gelatin in 6 N hydrochloric acid for half an hour. These conditions are very different from those
of Grassmann and Riederle who isolated lysyl-prolyl-glycine from a hydrolysate prepared
by heating at 37º in 3.6 N hydrochloric acid for 28 days. Recently, we have been duplicating their hydrolytic
conditions and have been interested to find that the mixture of peptides so produced
is very similar to that of our initial experiments.
Up to the present time, the scale of our experiments has been so small that further
work on the peptides which have been isolated can only e preliminary in scope. However,
one peptide zone has been subjected to further study and has been found to contain
threonylglycine in amount roughly equivalent to 10% of the theronine in gelatin.
The results of this preliminary investigation are very encouraging and permit us to
arrive at a tentative program which will make the best use of our present staff.
(1) Of most immediate interest is an attempt to improve the separation of the observed
peptide zones by variation of the conditions on the ion exchange columns. Each chromatogram
requires one to two weeks for completion and Mr. Honnen and I can run what amounts
to about one and a half chromatograms at a time. This study of the effect of variation
in conditions will require 6 to 8 weeks to complete and even then will be far from
thorough.
(2) While the above work is in progress, Dr. Green and Miss Michelson will make a
preliminary study of the small amounts of the various peptide zones which have been
isolated in order that we may have an insight into the complexity of each and be able
to decide which are most amenable to further productive study. It will probably not
be possible to complete these preliminary studies during the 6 to 8 weeks which will
be used to try to improve the separation of the peptide zones.
(3) It is also desirable for us as soon as possible to begin experiments with paper
chromatography. Paper chromatography may prove to be valuable in two ways: as a means
of separating peptide mixtures which may be well present in the various peptide zones
and also as a method for identifying the constituent amino acids in any peptide which
may be isolate. The letter can be done by the DNP technique but confirmatory evidence
from paper chromatography would be valuable. The extent to which we may be able to
venture into paper chromatographic work will depend to a large extent upon the available
space. At the present time, there is little space available in either of the laboratories
and what is available may not be suitable for paper chromatographic work.
(4) After we have completed the study which is projected in (1), larger chromatographic
columns will be prepared and at least 10 to 20 times the amounts of peptides which
are now available will be isolated. The preparation of ion exchanger for a large column
will be a major task.
(5)The larger quantities which will be isolated in (4) will then be subjected to exhaustive
study. The complexity of the mixture in any pepetide zone will determine the amount
of time which will be required to complete a study of it but it may be anticipated
that for each zone the full time of one person may be needed for as little as two
or three weeks or as much as six months.
On the basis of the results which we have already obtained, I feel that we should
be able to make a contribution to the structure of gelatin and of collagen but the
rate of progress will certainly depend upon the size of our group and space and facilities
which we can use. I have been wondering whether it might be possible to obtain financial
support to maintain or increase the present group from industrial concerns which are
interested in gelatin and collagen. I am thinking of such manufacturers as Wilson
and Company, who produce the gelatin for OPG, Knox Gelatin, leather companies, and
the like. If no support could be obtained from such sources, perhaps application should
be made for a grant from the Research Corporation.
