Affinity chromatography of naturally occurring folate derivatives.
In a previous report (1980, in Methods in Enzymology, Vol. 66, p. 686, Academic Press, New York) we described the preparation and use of purified milk folate-binding protein covalently linked to a Sepharose matrix. The present study was undertaken to test the capacity of this preparation to purify quantitatively folates from tissue extracts. In experiments that employed tissue extracts containing biologically synthesized [3H]folates, recovery of radioactivity from columns of the immobilized folate-binding protein (affinity columns) was consistently 90-95%. Folates eluted from the affinity column were practically pure. Anion-exchange chromatography of the purified fraction from rat liver extract yielded a number of uv-absorbing peaks which corresponded to the elution profile based on 3H counts, with respect to both the position and the area of the peaks. Treatment of the original extracts with folylpolyglutamate hydrolases resulted in the shift of these peaks to lower retention times, which corresponded to mono- or diglutamyl folate derivatives. Similar results were obtained with extracts from Lactobacillus casei. The purification of tissue folates by affinity chromatography allows determination of folate activity by direct physiochemical methods, which is particularly useful for analysis of folate composition in tissues.
Selhub
J
J
Department of Medicine, University of Chicago, Illinois 60637.
Darcy-Vrillon
B
B
Fell
D
D
D000818:Animals; D002352:Carrier Proteins / Q000494:pharmacology; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D005492:Folic Acid / Q000031:analogs & derivatives / Q000032:analysis*; D008297:Male; D011624:Pteroylpolyglutamic Acids / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains; D012685:Sepharose / Q000494:pharmacology; D011623:gamma-Glutamyl Hydrolase / Q000494:pharmacology
D002352:Carrier Proteins; D011624:Pteroylpolyglutamic Acids; D012685:Sepharose; D005492:Folic Acid; D011623:gamma-Glutamyl Hydrolase