Fluorescence Polarization: Analysis of Carbohydrate–Protein Interaction

Kakehi, Kazuaki; Oda, Yasuo; Kinoshita, Mitsuhiro

doi:10.1006/abio.2001.5309

Cited by 48 publications

(31 citation statements)

References 37 publications

(19 reference statements)

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“…The power and sensitivity of fluorescence spectroscopy in investigating conformation of proteins and other biological compound is well known. 18,19,[45][46][47][48][49][50] In our analyses, intrinsic fluorescence spectroscopy did not reveal any significant difference in the protein after oxidation. The position of the emission maximum did not undergo any shift, and the intensity remained constant.…”

Section: Discussionmentioning

confidence: 78%

Oxidized Recombinant Human Growth Hormone That Maintains Conformational Integrity

Mulinacci

Bell

Capelle

et al. 2011

Journal of Pharmaceutical Sciences

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Section: Discussionmentioning

confidence: 78%

Oxidized Recombinant Human Growth Hormone That Maintains Conformational Integrity

Mulinacci

Bell

Capelle

et al. 2011

Journal of Pharmaceutical Sciences

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“…Glycosaminoglycan Binding-CXCL11 binding to fluorescein-heparin (Molecular Probes) was determined using fluorescence polarization analysis on the Polarstar Optima 96-well fluorimeter (BMG) essentially as described previously for other binding proteins (56). Incubations contained 0.1 M heparinfluorescein isothiocyanate and chemokines at concentrations of 0 to 2 M, performed in 100 mM Tris, 150 mM NaCl, pH 7.4, for 1 h at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Matrix Metalloproteinase Processing of CXCL11/I-TAC Results in Loss of Chemoattractant Activity and Altered Glycosaminoglycan Binding

Cox

Dean

Roberts

et al. 2008

Journal of Biological Chemistry

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The CXCR3 chemokine receptor regulates the migration of Th1 lymphocytes and responds to three ligands: CXCL9/MIG, CXCL10/IP-10, and CXCL11/I-TAC. We screened for potential regulation of T cell responses by matrix metalloproteinase (MMP) processing of these important chemokines. The most potent of the CXCR3 ligands, CXCL11, was identified by matrixassisted laser desorption ionization time-of-flight mass spectrometry as a substrate of the PMN-specific MMP-8, macrophage-specific MMP-12, and the general leukocyte MMP-9. The 73-amino acid residue CXCL11 is processed at both the amino and carboxyl termini to generate CXCL11-(5-73), -(5-63), and -(5-58) forms. NH 2 -terminal truncation results in loss of agonistic properties, as shown in calcium mobilization and chemotaxis experiments using CXCR3 transfectants and human T lymphocytes. Moreover, CXCL11-(5-73) is a CXCR3 antagonist and interestingly shows enhanced affinity to heparin. However, upon COOH-terminal truncation to position 58 there is loss of antagonist activity and heparin binding. Together this highlights an unexpected site for receptor interaction and that the carboxyl terminus is critical for glycosaminoglycan binding, an essential function for the formation of chemokine gradients in vivo. Hence, MMP activity might regulate CXCL11 tissue gradients in two ways. First, the potential of CXCL11-(5-73) to compete active CXCL11 from glycosaminoglycans might lead to the formation of an antagonistic haptotactic chemokine gradient. Second, upon further truncation, MMPs disperse the CXCL11 gradients in a novel way by proteolytic loss of a COOH-terminal GAG binding site. Hence, these results reveal potential new roles in down-regulating Th1 lymphocyte chemoattraction through MMP processing of CXCL11.Chemokines are a superfamily of low molecular weight chemotactic cytokines that function in directing the migration of leukocytes and other cell types in a multitude of processes including development, lymphocyte homing, inflammation, and wound repair (1). Chemokines form haptotactic gradients in vivo through associations with proteoglycan glycosaminoglycans (2). Upon interaction with 7-transmembrane G proteincoupled receptors, chemokines induce a chemotactic response. The expression and secretion of inducible chemokines is stimulated during infection or injury to promote rapid and efficient inflammatory and immune responses. Conversely, dampening of inflammation, a critical event in allowing tissue repair to continue unimpeded and in preventing excessive tissue damage and autoimmunity, is known to involve coordinated down-regulation of chemokine expression (3), receptor internalization (4), scavenger receptors (5), and proteolytic mechanisms of inactivation and conversion to antagonists (6).Specific and limited proteolysis, termed processing (7), of chemokines results in altered bioactivity with functional consequences such as increased or decreased receptor binding (8), conversion of an agonist to an antagonist (6, 9), shedding of membrane-anchored chemokines (10, 11),...

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“…However, when the fluorescentlabeled small molecules are bound to the antigen/antibody, the emitted light of the conjugates is obviously less depolarized due to the larger mass. Difference between these states is dependent on the number of bound molecules and the binding constant or affinity constant (Kakehi et al 2001). Yadavalli and Pishko (2004) demonstrated that fluorescence polarization can be used to reliably and accurately study binding events and detect analytes in microfluidic devices.…”

Section: Fluorescence Detectionmentioning

confidence: 99%

“…13a Illustration of a fluorescence polarization apparatus. b Principle of the assay of binding reaction using fluorescence polarization (Source:Kakehi et al 2001) …”

mentioning

confidence: 99%

Microfluidic whole-blood immunoassays

Jiang

Weng

2010

Microfluid Nanofluid

100

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Immunoassay is one of the most widely used biomedical diagnostic methods due to its sensitivity and specificity. Microfluidic lab-on-a-chip technology has the advantages of portability, integration, and automation. The combination of these two technologies leads to a pathway for point-of-care diagnostics using the unprocessed samples such as the whole blood. This article reviews the recent advancement and the major development in the microfluidic-based whole-blood immunoassays. After a survey of the recent studies on microfluidic whole-blood immunoassays, an in-depth review about the detection methods that can be miniaturized and integrated in the immunoassay chips is provided. Point-of-care diagnostics applications require developing a fully integrated, disposable, low-cost, and handheld microfluidic device for the whole-blood immunoassay. In this regard, some comments and suggestions for future research are given.

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Fluorescence Polarization: Analysis of Carbohydrate–Protein Interaction

Cited by 48 publications

References 37 publications

Oxidized Recombinant Human Growth Hormone That Maintains Conformational Integrity

Oxidized Recombinant Human Growth Hormone That Maintains Conformational Integrity

Matrix Metalloproteinase Processing of CXCL11/I-TAC Results in Loss of Chemoattractant Activity and Altered Glycosaminoglycan Binding

Microfluidic whole-blood immunoassays

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