Chromatofocusing profile of purified human α-fetoprotein and albumin differs from those of crude samples: Effect of protein concentration on the elution of the sample

Juan A. Leal, Kevan B. Eddy, Brooks Allen Keel

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)411-418
Number of pages8
JournalAnalytical Biochemistry
Volume192
Issue number2
DOIs
StatePublished - Feb 1 1991
Externally publishedYes

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Fetal Proteins
Serum Albumin
Albumins
Protein Isoforms
Proteins
Fetal Blood
Blood
Amniotic Fluid
Carrier Proteins

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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title = "Chromatofocusing profile of purified human α-fetoprotein and albumin differs from those of crude samples: Effect of protein concentration on the elution of the sample",
abstract = "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.",
author = "Leal, {Juan A.} and Eddy, {Kevan B.} and Keel, {Brooks Allen}",
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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 Allen

PY - 1991/2/1

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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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