Time‐resolved fluorescence studies on site‐directed mutants of human serum albumin

Helms, Michael K.; Petersen, Charles E.; Bhagavan, N.V.; Jameson, David M.

doi:10.1016/s0014-5793(97)00389-x

Cited by 76 publications

(59 citation statements)

References 32 publications

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“…The fluorescence decay of the unquenched Trp-214 was biexponential ( χ 2 = 1.007) (with τ 1 = 3.06 ns [ α 1 (fraction) = 0.64], τ 2 = 7.80 ns [ α 2 (fraction) = 0.36], and 〈 τ 〉 = 5.37 ns). These results are in agreement with those obtained for Trp-214 by Helms et al [39]. As indicated in Table 2, the average fluorescence lifetime of Trp-214 decreased slightly with increasing patulin concentration.…”

Section: Resultssupporting

confidence: 93%

Investigation of the Interaction between Patulin and Human Serum Albumin by a Spectroscopic Method, Atomic Force Microscopy, and Molecular Modeling

You

Yang

et al. 2014

BioMed Research International

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The interaction of patulin with human serum albumin (HSA) was studied in vitro under normal physiological conditions. The study was performed using fluorescence, ultraviolet-visible spectroscopy (UV-Vis), circular dichroism (CD), atomic force microscopy (AFM), and molecular modeling techniques. The quenching mechanism was investigated using the association constants, the number of binding sites, and basic thermodynamic parameters. A dynamic quenching mechanism occurred between HSA and patulin, and the binding constants (K) were 2.60 × 104, 4.59 × 104, and 7.01 × 104 M−1 at 288, 300, and 310 K, respectively. Based on fluorescence resonance energy transfer, the distance between the HSA and patulin was determined to be 2.847 nm. The ΔG 0, ΔH 0, and ΔS 0 values across various temperatures indicated that hydrophobic interaction was the predominant binding force. The UV-Vis and CD results confirmed that the secondary structure of HSA was altered in the presence of patulin. The AFM results revealed that the individual HSA molecule dimensions were larger after interaction with patulin. In addition, molecular modeling showed that the patulin-HSA complex was stabilized by hydrophobic and hydrogen bond forces. The study results suggested that a weak intermolecular interaction occurred between patulin and HSA. Overall, the results are potentially useful for elucidating the toxigenicity of patulin when it is combined with the biomolecular function effect, transmembrane transport, toxicological, testing and other experiments.

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Section: Resultssupporting

confidence: 93%

Investigation of the Interaction between Patulin and Human Serum Albumin by a Spectroscopic Method, Atomic Force Microscopy, and Molecular Modeling

You

Yang

et al. 2014

BioMed Research International

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“…The peak-height ratios were plotted versus total protein concentration and fit to eq 8 using the program KaleidaGraph version 3.52 for Windows (Synergy Software., Reading, PA) to estimate the K D value for each protein-ligand complex. The average NMR-linewidth ratio (c) for each ligand was estimated by using eq 6, where ν B was taken to be approximately 94.2 Hz using a previously measured correlation time for HSA of 41 ns 32. The value for ν F was calculated as described in the next section.…”

Section: Methodsmentioning

confidence: 99%

Estimating Protein−Ligand Binding Affinity Using High-Throughput Screening by NMR

et al. 2008

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Many of today's drug discovery programs utilize high-throughput screening methods that rely on quick evaluations of protein activity to rank potential chemical leads. By monitoring biologically relevant protein-ligand interactions, NMR can provide a means to validate these discovery leads and to optimize the drug discovery process. NMR-based screens typically use a change in chemical shift or linewidth to detect a protein-ligand interaction. However, the relatively low throughput of current NMR screens and their high demand on sample requirements generally makes it impractical to collect complete binding curves to measure the affinity for each compound in a large and diverse chemical library. As a result, NMR ligand screens are typically limited to identifying candidates that bind to a protein and do not give any estimate of the binding affinity. To address this issue, a methodology has been developed to rank binding affinities for ligands based on NMR-based screens that use 1D 1 H NMR line-broadening experiments. This method was demonstrated by using it to estimate the dissociation equilibrium constants for twelve ligands with the protein human serum albumin (HSA). The results were found to give good agreement with previous affinities that have been reported for these same ligands with HSA.

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“…The analysis of the time-resolved decay data in terms of a Gaussian lifetime distribution is a simpler model where the widths of the recovered lifetime distributions can be related to the degree of heterogeneity (and/or mobility) of the probe environment [9,38], and the mean values of the decay times to the degree of water exposure of the bound ANS. In the 1:1 complex (Fig.…”

Section: Time-resolved Fluorescencementioning

confidence: 99%

Monitoring Local Unfolding of Bovine Serum Albumin During Denaturation Using Steady-State and Time-Resolved Fluorescence Spectroscopy

2009

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In a previous report (J. Fluoresc. 16, 153, 2006) we studied the chaotropiclly induced denaturation of Bovine Serum Albumin (BSA) using the fluorescence decay kinetics at different stages in the denaturation of BSA by guanidinium hydrochloride (GuHCl). In this work, we gain a more detailed insight into the BSA denaturation process by investigating the thermodynamics of the process. Structural changes were monitored spectrophotometrically via the intrinsic protein fluorescence from tryptophan residues, and the extrinsic fluorescence from 1,8-anilinonaphthalene sulphonate (ANS). ANS tends to locate in a variety of binding sites in BSA which are located in different domains, and these can be selectively populated using different, 1:1 and 1:10 molar ratios of BSA to ANS. The data from steady-state and time-resolved fluorescence spectroscopy were analyzed using thermodynamic two-state and three-state models and the lifetime data clearly indicated the formation of an intermediate state during denaturation. A global analysis using non-linear regression gave a . The lifetime analysis of the BSA-ANS complexes also shows clearly that there are differences in stability of the BSA domains, with domain III unfolding first at low GuHCl concentrations (<1.5M).

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Time‐resolved fluorescence studies on site‐directed mutants of human serum albumin

Cited by 76 publications

References 32 publications

Investigation of the Interaction between Patulin and Human Serum Albumin by a Spectroscopic Method, Atomic Force Microscopy, and Molecular Modeling

Investigation of the Interaction between Patulin and Human Serum Albumin by a Spectroscopic Method, Atomic Force Microscopy, and Molecular Modeling

Estimating Protein−Ligand Binding Affinity Using High-Throughput Screening by NMR

Monitoring Local Unfolding of Bovine Serum Albumin During Denaturation Using Steady-State and Time-Resolved Fluorescence Spectroscopy

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