Synthesis of dansyl ribonucleotides and their use in steady-state fluorescence anisotropy studies of nucleotide binding by initiation factor-2 (eif-2) and histone hi

Timothy C. Mueser, Lawrence J Parkhurst

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

1. 1. Fluorescent analogs of GDP and ATP were prepared with DANSYL-β-alanine (DβA) coupled to the (2′)3′ hydroxyl of the ribose. 2. 2. Observation of changes in both total fluorescence and anisotropy accompanying the binding of DβA-GDP to eIF-2 allowed determination of Kd (33 nM). 3. 3. When DβA-ATP bound to HI histone, the fluorescence quantum yield increased and the emission was blue shifted. Analysis yielded a Kd of 3.4 μM and 20 binding sites per histone. At high levels of ATP, fluorescence anisotropy values and light scattering intensities pointed to significant aggregation of H1 that is strongly dependent on ATP concentration.

Original languageEnglish (US)
Pages (from-to)1689-1696
Number of pages8
JournalInternational Journal of Biochemistry
Volume25
Issue number11
DOIs
StatePublished - Nov 1993

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Prokaryotic Initiation Factor-2
Ribonucleotides
Fluorescence Polarization
Histones
Anisotropy
Nucleotides
Adenosine Triphosphate
Fluorescence
Alanine
Ribose
Quantum yield
Hydroxyl Radical
Light scattering
Agglomeration
Binding Sites
Observation
Light
1-myristoyl-2-(12-((5-dimethylamino-1-naphthalenesulfonyl)amino)dodecanoyl)-sn-glycero-3-phosphocholine

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Synthesis of dansyl ribonucleotides and their use in steady-state fluorescence anisotropy studies of nucleotide binding by initiation factor-2 (eif-2) and histone hi",
abstract = "1. 1. Fluorescent analogs of GDP and ATP were prepared with DANSYL-β-alanine (DβA) coupled to the (2′)3′ hydroxyl of the ribose. 2. 2. Observation of changes in both total fluorescence and anisotropy accompanying the binding of DβA-GDP to eIF-2 allowed determination of Kd (33 nM). 3. 3. When DβA-ATP bound to HI histone, the fluorescence quantum yield increased and the emission was blue shifted. Analysis yielded a Kd of 3.4 μM and 20 binding sites per histone. At high levels of ATP, fluorescence anisotropy values and light scattering intensities pointed to significant aggregation of H1 that is strongly dependent on ATP concentration.",
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T1 - Synthesis of dansyl ribonucleotides and their use in steady-state fluorescence anisotropy studies of nucleotide binding by initiation factor-2 (eif-2) and histone hi

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AU - Parkhurst, Lawrence J

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N2 - 1. 1. Fluorescent analogs of GDP and ATP were prepared with DANSYL-β-alanine (DβA) coupled to the (2′)3′ hydroxyl of the ribose. 2. 2. Observation of changes in both total fluorescence and anisotropy accompanying the binding of DβA-GDP to eIF-2 allowed determination of Kd (33 nM). 3. 3. When DβA-ATP bound to HI histone, the fluorescence quantum yield increased and the emission was blue shifted. Analysis yielded a Kd of 3.4 μM and 20 binding sites per histone. At high levels of ATP, fluorescence anisotropy values and light scattering intensities pointed to significant aggregation of H1 that is strongly dependent on ATP concentration.

AB - 1. 1. Fluorescent analogs of GDP and ATP were prepared with DANSYL-β-alanine (DβA) coupled to the (2′)3′ hydroxyl of the ribose. 2. 2. Observation of changes in both total fluorescence and anisotropy accompanying the binding of DβA-GDP to eIF-2 allowed determination of Kd (33 nM). 3. 3. When DβA-ATP bound to HI histone, the fluorescence quantum yield increased and the emission was blue shifted. Analysis yielded a Kd of 3.4 μM and 20 binding sites per histone. At high levels of ATP, fluorescence anisotropy values and light scattering intensities pointed to significant aggregation of H1 that is strongly dependent on ATP concentration.

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