TY - JOUR
T1 - Chromatofocusing profile of purified human α-fetoprotein and albumin differs from those of crude samples
T2 - Effect of protein concentration on the elution of the sample
AU - Leal, Juan A.
AU - Eddy, Kevan B.
AU - Keel, Brooks A.
PY - 1991/2/1
Y1 - 1991/2/1
N2 - Chromatofocusing was utilized to characterize charge microheterogeneity of purified human α-fetoprotein (AFP) and human serum albumin (HSA). Crude cord blood samples yielded three isoforms: AFP-IA, IB, and II, with pIs 4,57 (52%), 4,27 (43%), and <4,00 (5%), respectively. In contrast, 10 μg of purified AFP or 250,000 cpm of 125I-AFP eluted entirely as isoform AFP-II. 125I-AFP focused in the presence of crude cord blood, amniotic fluid, adult male serum, or 25 mg purified HSA resulted in elution profiles similar to those of crude cord blood. Pure AFP focused along with 0.1, 1.0, 5.0, or 10 mg HSA showed a gradual shift from AFP-II to AFP-I. With ≥5 mg HSA, isoform I was further resolve into AFP-IA and IB. Similarly, 250,000 cpm of 125I-HSA, which also eluted entirely as isoform II, showed a gradual shift to isoform I when increasing concentrations of unlabeled HSA were added. The resolution of isoform HSA-I in HSA-IA, IB, and IC was again improved with ≥5 mg unlabeled HSA. When carrier proteins of varying pI values were chromatofocused along with purified AFP, it was observed that only those proteins withpIs in the range of AFP caused significant alteration in the relative distribution of AFP. We conclude that sample protein concentration and composition must be carefully considered when chromatofocusing is being used for purified samples and when the elution profiles of samples from different origins and varying protein concentrations are being compared.
AB - Chromatofocusing was utilized to characterize charge microheterogeneity of purified human α-fetoprotein (AFP) and human serum albumin (HSA). Crude cord blood samples yielded three isoforms: AFP-IA, IB, and II, with pIs 4,57 (52%), 4,27 (43%), and <4,00 (5%), respectively. In contrast, 10 μg of purified AFP or 250,000 cpm of 125I-AFP eluted entirely as isoform AFP-II. 125I-AFP focused in the presence of crude cord blood, amniotic fluid, adult male serum, or 25 mg purified HSA resulted in elution profiles similar to those of crude cord blood. Pure AFP focused along with 0.1, 1.0, 5.0, or 10 mg HSA showed a gradual shift from AFP-II to AFP-I. With ≥5 mg HSA, isoform I was further resolve into AFP-IA and IB. Similarly, 250,000 cpm of 125I-HSA, which also eluted entirely as isoform II, showed a gradual shift to isoform I when increasing concentrations of unlabeled HSA were added. The resolution of isoform HSA-I in HSA-IA, IB, and IC was again improved with ≥5 mg unlabeled HSA. When carrier proteins of varying pI values were chromatofocused along with purified AFP, it was observed that only those proteins withpIs in the range of AFP caused significant alteration in the relative distribution of AFP. We conclude that sample protein concentration and composition must be carefully considered when chromatofocusing is being used for purified samples and when the elution profiles of samples from different origins and varying protein concentrations are being compared.
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U2 - 10.1016/0003-2697(91)90557-A
DO - 10.1016/0003-2697(91)90557-A
M3 - Article
C2 - 1709797
AN - SCOPUS:0026020835
SN - 0003-2697
VL - 192
SP - 411
EP - 418
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -