Rapid paper chromatographic separation of [14C] angiotensen II from some metabolites: application to organ...

A rapid, inexpensive method for the separation of 5-1-isoleucyl[14C] angiotensin II (A-II) from its various metabolites has been devised. A-II was extracted from tissues with absolute methanol (recovery 96%) and paper chromatographed in a butanol-acetic acid-water (18:2:5) medium for two ascents at 60 degrees C. The resulting RF for A-II of 0.45 was then compared with the RF values of three A-II metabolites produced by enzymatic degradation of the 14C-A-II and [14C]isoleucine. Trypsin degradation produced the [14C]hexapeptide metabolite, chymotryptic degradation produced the [14C]tetrapeptide metabolite and carboxypeptidase A degradation produced the [14C]heptapeptide. Increases in temperature produced a continuous increase in RF values for all the substances examined but the resolution decreased above 60 degrees C. Similarly, increases in the temperature caused the appearance of secondary peaks with some but not all peptides. The tryptic digest (hexapeptide) and the chymotryptic digest (tetrapeptide) are apparently acid- and heat-stable under the experimental conditions. All of the peptides examined failed to produce secondary peaks when heated at neutral pH. The method was used to study the tissue distribution of 14C-A-II after intravenous injection.