Characterization of the binding and chiral separation of d- and l-tryptophan on a high-performance immobilized human serum albumin column

Ju Yang, David S. Hage

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108 Citations (Scopus)

Abstract

High-performance affinity chromatography was used to study the separation and binding of d- and l-tryptophan on an immobilized human serum albumin (HSA) column. Frontal analysis and zonal elution studies indicated that both d- and l-tryptophan were binding to single but distinct sites on HSA. l-Tryptophan bound to the indole site of HSA. d-Tryptophan had indirect interactions with the warfarin site of HSA but no interactions with the indole site. The association constants for the binding of d- and l-tryptophan at pH 7.4 and 25°C were 0.4·104 and 2.7·104 M-1, respectively. The value of ΔG for these sites ranged from -5.2 to -5.7 kcal/mol (1 cal = 4.184 J) and had a significant entropy component. The effects of varying the pH, phosphate concentration, temperature and polarity of the mobile phase on the binding of d- and l-tryptophan to HSA were examined. The role of sample size in determining peak shape and retention was also considered. From these data, general guidelines were developed for the separation of d- and l-tryptophan on immobilized HSA. Under optimized conditions the enantiomers were separated in less than 2 min with baseline resolution.

Original languageEnglish (US)
Pages (from-to)241-250
Number of pages10
JournalJournal of Chromatography A
Volume645
Issue number2
DOIs
StatePublished - Aug 20 1993

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Serum Albumin
Tryptophan
Affinity chromatography
Enantiomers
Entropy
Warfarin
Affinity Chromatography
Sample Size
Phosphates
Association reactions
Guidelines
Temperature

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry

Cite this

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abstract = "High-performance affinity chromatography was used to study the separation and binding of d- and l-tryptophan on an immobilized human serum albumin (HSA) column. Frontal analysis and zonal elution studies indicated that both d- and l-tryptophan were binding to single but distinct sites on HSA. l-Tryptophan bound to the indole site of HSA. d-Tryptophan had indirect interactions with the warfarin site of HSA but no interactions with the indole site. The association constants for the binding of d- and l-tryptophan at pH 7.4 and 25°C were 0.4·104 and 2.7·104 M-1, respectively. The value of ΔG for these sites ranged from -5.2 to -5.7 kcal/mol (1 cal = 4.184 J) and had a significant entropy component. The effects of varying the pH, phosphate concentration, temperature and polarity of the mobile phase on the binding of d- and l-tryptophan to HSA were examined. The role of sample size in determining peak shape and retention was also considered. From these data, general guidelines were developed for the separation of d- and l-tryptophan on immobilized HSA. Under optimized conditions the enantiomers were separated in less than 2 min with baseline resolution.",
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N2 - High-performance affinity chromatography was used to study the separation and binding of d- and l-tryptophan on an immobilized human serum albumin (HSA) column. Frontal analysis and zonal elution studies indicated that both d- and l-tryptophan were binding to single but distinct sites on HSA. l-Tryptophan bound to the indole site of HSA. d-Tryptophan had indirect interactions with the warfarin site of HSA but no interactions with the indole site. The association constants for the binding of d- and l-tryptophan at pH 7.4 and 25°C were 0.4·104 and 2.7·104 M-1, respectively. The value of ΔG for these sites ranged from -5.2 to -5.7 kcal/mol (1 cal = 4.184 J) and had a significant entropy component. The effects of varying the pH, phosphate concentration, temperature and polarity of the mobile phase on the binding of d- and l-tryptophan to HSA were examined. The role of sample size in determining peak shape and retention was also considered. From these data, general guidelines were developed for the separation of d- and l-tryptophan on immobilized HSA. Under optimized conditions the enantiomers were separated in less than 2 min with baseline resolution.

AB - High-performance affinity chromatography was used to study the separation and binding of d- and l-tryptophan on an immobilized human serum albumin (HSA) column. Frontal analysis and zonal elution studies indicated that both d- and l-tryptophan were binding to single but distinct sites on HSA. l-Tryptophan bound to the indole site of HSA. d-Tryptophan had indirect interactions with the warfarin site of HSA but no interactions with the indole site. The association constants for the binding of d- and l-tryptophan at pH 7.4 and 25°C were 0.4·104 and 2.7·104 M-1, respectively. The value of ΔG for these sites ranged from -5.2 to -5.7 kcal/mol (1 cal = 4.184 J) and had a significant entropy component. The effects of varying the pH, phosphate concentration, temperature and polarity of the mobile phase on the binding of d- and l-tryptophan to HSA were examined. The role of sample size in determining peak shape and retention was also considered. From these data, general guidelines were developed for the separation of d- and l-tryptophan on immobilized HSA. Under optimized conditions the enantiomers were separated in less than 2 min with baseline resolution.

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