Continuous‐flow bioluminescent dtermination of ATP in platelets using firefly luciferase immobilized on epoxy methacrylate

Carrea, Giacomo; Bovara, Roberto; Girotti, Stefano; Ferri, Elida Nora; Ghini, Severino; Roda, Aldo

doi:10.1002/bio.1170030103

Cited by 10 publications

(5 citation statements)

References 15 publications

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“…Carrea et al showed decreased K M(ATP) (51-22 mmol/L) and increased K M(luciferin) (2.4-28 mmol/L) for FL immobilized on nylon (7). A similar change was found on epoxy methacrylate polymer (8). K M values for collagen-bound FL (24 and 11 mmol/L for ATP and luciferin, respectively) showed no difference from the free enzyme under mechanical stirring (26).…”

Section: Kinetic Constants Of Bccp-flmentioning

confidence: 86%

“…The generally accepted K M(ATP) and K M(luciferin) for Photinus pyralis FL are in the ranges 25-250 and 2-10 mmol/L, respectively (7,8,(21)(22)(23)(24). Table 1 shows the K M and k cat values for both immobilized and free BCCP-luciferase calculated using Leonora software (25).…”

Section: Kinetic Constants Of Bccp-flmentioning

confidence: 99%

“…Weinhausen et al observed 44-84% activity with Photinus pyralis luciferase on cyanogen bromideactivated Sepharose (6). Other solid supports, such as collagen strips, epoxy methacrylate polymer and nylon tubes, were also reported (7)(8)(9). Filippova et al immobilized L mingrelica luciferase on lipidated membrane and retained 100% activity because no covalent linkage was involved (10).…”

Section: Introductionmentioning

confidence: 99%

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Properties of firefly luciferase immobilized through a biotin carboxyl carrier protein domain

Andrade

2001

Luminescence

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A fusion protein, consisting of biotin carboxyl carrier protein (BCCP) domain from Escherichia coli and firefly luciferase (FL) from Photinus pyralis, was immobilized through the biotin-avidin interaction on 6% cross-linked agarose beads. Several properties of the immobilized BCCP-FL were studied. Immobilized and free enzymes showed no significant difference in thermal stability; both retained at least 91% activity after incubation at 4 degrees C and 25 degrees C for 22 h. Incubation at 37 degrees C for 22 h caused significant activity loss. K(M) and k(cat) values were determined for both free and immobilized enzymes. K(M) values were similar between free and immobilized enzymes; however, k(cat) of immobilized BCCP-FL was one-third of the k(cat) of the free enzyme. 294 micromol/L Co-enzyme A (CoA) and 44 mmol/L dithiothreitol (DTT) enhanced the total bioluminescence output. Triton X-100, Tween 20, PEG 8,000, PVP 40,000 and PVP 360,000 did not enhance the bioluminescence reaction of immobilized BCCP-FL.

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Section: Kinetic Constants Of Bccp-flmentioning

confidence: 86%

Section: Kinetic Constants Of Bccp-flmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Properties of firefly luciferase immobilized through a biotin carboxyl carrier protein domain

Andrade

2001

Luminescence

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“…which was then positioned inside the luminometer in front of the photomultiplier window (Carrea et al, 1989).…”

Section: Methacrylate Reactor Preparationmentioning

confidence: 99%

Coupled reactions for the determination of analytes and enzymes based on the use of luminescence

Roda

Girotti

Ghini

et al. 1989

J. Biolumin. Chemilumin.

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Immobilized enzymes are widely used in the clinical laboratory in the assay of several analytes and enzymes. The use of immobilized enzymes makes these reagents recoverable and re-usable, and in most cases increases their stability and catalytic activity. In conjunction with bioluminescent enzymes (firefly and bacterial luciferases) and chemiluminescent catalyst (peroxidase) we set up high-sensitive flow methods based on the use of nylon tube coil or epoxy methacrylate column as solid support. All the NAD(P)/NAD(P)H-dependent dehydrogenases (bacterial luciferase), ATP-dependent enzymes (firefly luciferase) and oxidases producing H2O2 (peroxidase) can be immobilized and a large variety of analytes have been sensitively measured. As an alternative format we also reported a dry chemistry method in which all the enzymes, substrates and cofactors are ready to use, supported on dry cellulose disks. Methodological problems such as flow conditions, stability, pH, ionic strength and analytical performances are also reported.

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“…We previously described bioluminescent flow systems using immobilized luciferase for ATP and ADP determination in platelets after extraction with ethanoCEDTA (Carrea et al, 1986;Carrea et al, 1989). The aim of this work was to improve a method for bioluminescence determination of ATP concentrations in platelets and erythrocytes.…”

Section: Introductionmentioning

confidence: 99%

Methodological problems of direct bioluminescent ATP assay in platelets and erythrocytes

Girotti

Ferri

Cascione

et al. 1989

J. Biolumin. Chemilumin.

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Direct bioluminescent ATP determination in platelets and erythrocytes involves the study of different parameters which are discussed here. Some parameters are linked to the bioluminescent reaction and to the analyte (ATP); others have regard to the biological matrix. The composition of bioluminescent reagents and the preparation and conservation of the ATP standard, also in the presence of excipients, are among the first given. Matrix problems involve cell characteristics related to age and form, lysis resistance and the possible formation of aggregates (platelets) that may inhibit the complete release of ATP. For these reasons we used the most efficient ATP release agent with the lowest inhibitory effect on luciferase. The data obtained correlate well with a bioluminescent method requiring extraction with ethanol/EDTA, and therefore more time, for ATP determination in platelets and erythrocytes.

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Continuous‐flow bioluminescent dtermination of ATP in platelets using firefly luciferase immobilized on epoxy methacrylate

Cited by 10 publications

References 15 publications

Properties of firefly luciferase immobilized through a biotin carboxyl carrier protein domain

Properties of firefly luciferase immobilized through a biotin carboxyl carrier protein domain

Coupled reactions for the determination of analytes and enzymes based on the use of luminescence

Methodological problems of direct bioluminescent ATP assay in platelets and erythrocytes

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