High-molecular-weight folates of rat liver.

PATRICIA A. BARFORD, JOHN A. BLAIR and FREDERICK J. STAFF Department of Chemistry, University of Aston in Birmingham, Gosta Greem, Birmingham B4 7ET, U.K. It has been frequently claimed that polyglutamate forms of folates are found in animals (Noronha & Silverman, 1962; Shin et al., 1972; Brown et al., 1974) on the basis of column chromatography of these folates and chromatography of 'authentic' standards and/or microbiological assay before and after conjugase treatment. The present communication describes attempts to identify folate polyglutamates in normal rat livers. Two types of columns are normally used to separate and identify folate polyglutamates : a column of Sephadex G-15, on which folate polyglutamates leave the column close to the void volume and in a smaller volume than authentic folate monoglutamate markers, or anion-exchange columns, on which folate polyglutamates are reported to be eluted from the column at higher ionic strength than folate monoglutamates. In our experiments, normal Wistar rats (150g body wt.) received oral doses of [2-'4C]foIic acid (2,uCi; 75pg/kg). Animals were killed 48h after administration of the dose, and the livers removed immediately and treated to prevent breakdown of folate polyglutamates by conjugase by placing finely divided liver pieces into boiling aqueous buffers (pH7.0,g.O and 11.0) containing suitable antioxidants (Shin et al., 1972; Brown et al., 1974; J. M. Scott, personal communication). Liver extracts were subjected to gel-filtration chromatography on columns of Sephadex G-15. On all chromatograms a large peak of radioactivity which left the column in an effluent volume slightly greater than the void volume was observed. This peak travelled in front of 4a-hydroxy-5methyltetrahydrofolic acid (Blair et al., 1975), which is the first folate monoglutamate to leave a Sephadex G-15 column. The same liver extracts were then subjected to chromatography on a DEAEcellulose column (Whatman DE52). Liver extracts were diluted to give the same conductivity as the starting buffer h fo re being loaded on to the column. Radioactive material was eluted from the colurnp by using a 0-1 .OM gradient of NaCl in phosphate buffer, pH7.0. Three peaks of radioactivity eluted from the DEAE-cellulose columns. The two major peaks co-chromatographed with authentic 5-methyltetrahydrofolic acid and folic acid, a third minor peak remained unidentified. Elution of DEAEcellulose columns with NaCl gradients up to 3M failed to produce any peaks at high ionic strength, as did chromatography at pH6.0. Rechromatography of the peak from the Sephadex G-15 column on DEAE-cellulose gave similar results. As it was possible that the high-molecular-weight folate was breaking down on the DEAE-cellulose column, the peak from a DEAE-cellulose column was rechromatographed on a column of Sephadex G-15. Chromatograms showed that the radioactivity was resolved into three peaks, one of which was eluted in the same position as 4a-hydroxy-5-methyltetrahydrofolic acid and another just after the void volume. Several groups of workers have used DEAE-Sephadex chromatography to demonstrate polyglutamates. We therefore chromatographed liver extracts on DEAE-Sephadex at pH7.0 by using a 0.3-1.5~ gradient of NaCI. The liver extract separated into two fractions, neither of which corresponded to authentic monoglutamates, but both of which were eluted from the column in the same region as folate monoglutamates. The lysosoma1 enzyme conjugase hydrolyses the peptide links in polyglutamates to produce monoglutamates. This hydrolysis is said to be very rapid. Placing livers in a boiling pH7.0 buffer should effectively prevent breakdown of folate polyglutamates. However, Scott (J. M. Scott, personal communication) suggested that conjugase may not be deactivated under these conditions at pH7.0, that its action is sufficiently fast for breakdown of folate polyglutamates to occur and therefore that pH9.0 or 11 .O might be more appropriate for the extraction procedure. Extraction of liver folates with boiling buffer at pH9.0 or 11.0 failed to reveal the presence of polyglutamates. In both cases chromatograms showed peaks of radioactivity that were eluted on DEAE-cellulose at the same ionic strength as folate monoglutamates.