Fractionation of serum transcobalamins on charged cellulose filters.
A simple and rapid fractionation procedure of the three transcobalamins, TCI, TCII, and TCII, of human serum was achieved by filtration through a stack of charged cellulose filters composed of one cellulose-nitrate and three DEAE-cellulose (DE-81) disks. A reaction mixture containing microliter amounts of serum was incubated with excess of 57Co B12 of high specific activity, diluted with 0.1 M sodium borate buffer (pH 8.5), and passed through the filter stack by applying vacuum. Under these conditions TCII is selectively and quantitatively adsorbed to the cellulose-nitrate filter while both TCI and TCIII adsorb to the DE-81 filters. In the second step TCIII is selectively desorbed from the latter filters by a 0.05 M monopotassium phosphate solution of pH 4.6. Using sera of different distribution of transcobalamins the data obtained were comparable to those determined by the more laborious methods employing DE-52 column chromatography combined with procedures to remove TCII.
Fractionation of serum transcobalamins on charged cellulose filters. A simple and rapid fractionation procedure of the three transcobalamins, TCI, TCII, and TCII, of human serum was achieved by filtration through a stack of charged cellulose filters composed of one cellulose-nitrate and three DEAE-cellulose (DE-81) disks. A reaction mixture containing microliter amounts of serum was incubated with excess of 57Co B12 of high specific activity, diluted with 0.1 M sodium borate buffer (pH 8.5), and passed through the filter stack by applying vacuum. Under these conditions TCII is selectively and quantitatively adsorbed to the cellulose-nitrate filter while both TCI and TCIII adsorb to the DE-81 filters. In the second step TCIII is selectively desorbed from the latter filters by a 0.05 M monopotassium phosphate solution of pH 4.6. Using sera of different distribution of transcobalamins the data obtained were comparable to those determined by the more laborious methods employing DE-52 column chromatography combined with procedures to remove TCII.