Correction of inner filter effects in fluorescence spectrometry

Mode, V.A.; Sisson, D.H.

doi:10.1021/ac60338a013

Cited by 22 publications

(10 citation statements)

References 10 publications

(3 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A concentration of 30 µM L was used to ensure formation of enzyme-L complexes with the mutants. At this concentration of L, there was no evidence of an inner filter effect (38).…”

Section: Methodsmentioning

confidence: 75%

The Role of Active Site Residue Arginine 218 in Firefly Luciferase Bioluminescence

et al. 2001

View full text Add to dashboard Cite

Firefly luciferase catalyzes the highly efficient emission of yellow-green light from substrate firefly luciferin by a sequence of reactions that require Mg-ATP and molecular oxygen. We had previously developed a working model of the luciferase active site based on the X-ray structure of the enzyme without bound substrates. In our model, the side chain guanidinium group of Arg218 appears to be located in close proximity to the substrate's hydroxyl group at the bottom of the luciferin binding pocket. A similar role for Arg337 also has been proposed. We report here the construction, purification, and characterization of mutant luciferases R218A, R218Q, R218K, R337Q, and R337K. Alteration of the Arg218 side chain produced enzymes with 15-20-fold increases in the Km values for luciferin. The contrasting near-normal Km values for luciferin determined with the Arg337 enzymes support our proposal that Arg218 (and not Arg337) is an essential luciferin binding site residue. Bioluminescence emission studies indicated that in the absence of a positively charged group at position 218, red bioluminescence was produced. Based on this result and those of additional fluorescence experiments, we speculate that Arg218 maintains the polarity and rigidity of the emitter binding site necessary for the normal yellow-green emission of P. pyralis luciferase. The findings reported here are interpreted in the context of the firefly luciferase X-ray structures and computational-based models of the active site.

show abstract

“…A concentration of 30 µM L was used to ensure formation of enzyme-L complexes with the mutants. At this concentration of L, there was no evidence of an inner filter effect (38).…”

Section: Methodsmentioning

confidence: 75%

The Role of Active Site Residue Arginine 218 in Firefly Luciferase Bioluminescence

et al. 2001

View full text Add to dashboard Cite

show abstract

“…If this is not possible, the cell holder may be modified so that the path length within the cell is reduced (12). An a posteriori suitable correction may also be satisfactorily applied (13)(14)(15)(16).…”

Section: Experiments 2: Evidence Of Inner-and Post-filter Effectsmentioning

confidence: 99%

Are Fluorescence Quantum Yields So Tricky to Measure? A Demonstration Using Familiar Stationery Products

1999

View full text Add to dashboard Cite

Fluorescence quantum yields are used to quantify the efficiency of the emission process. In spite of the importance of these data, experimental directions for their acquisition are rarely given. A general procedure for determining the relative fluorescence quantum yield of solutions is described here, drawing attention to the many pitfalls that students may encounter. Starting materials are common yellow and pink highlighter pens.

show abstract

“…In addition to assuming the validity of Equation 2, we assume that self-absorption of fluorescence by M is negligible. This assumption can virtually always be satisfied experimentally by employing a small concentration of M and by proper choice of emission wavelength; while correction procedures have been developed for reabsorption (17)(18)(19)(20), correction for simultaneous inner-filter action by M and Q would be very cumbersome. Corrections for changes in refractive index as a function of [Q] are likewise not considered here; if required, the treatment of Rohatgi and Singhal (17) could readily be applied to the equations derived herein.…”

Section: General Considerations and Assumptionsmentioning

confidence: 99%

Corrections for inner-filter effects in fluorescence quenching measurements via right-angle and front-surface illumination

Leese¹,

Wehry²

1978

Anal. Chem.

View full text Add to dashboard Cite

A procedure for the analysis of the metallic phase In multiphase systems Is described, with elemental determination by flame atomic absorption spectrometry. A potassium cuprochlorlde-tartarlc acid phase selective leach Is employed at room temperature to dissolve the metallic phase In the presence of sulfides, oxides, silicates, and carbonaceous rich material. Partial attack of the oxides, ZnO, MgO, and CdO, was observed and these oxides, when present, were therefore removed by an Initial treatment with a potassium dlchromate-tartarlc acid solution. The method can be applied to geological and Industrial materials to determine the elements Bl, Cd, Co, Fe,

show abstract

Correction of inner filter effects in fluorescence spectrometry

Cited by 22 publications

References 10 publications

The Role of Active Site Residue Arginine 218 in Firefly Luciferase Bioluminescence

The Role of Active Site Residue Arginine 218 in Firefly Luciferase Bioluminescence

Are Fluorescence Quantum Yields So Tricky to Measure? A Demonstration Using Familiar Stationery Products

Corrections for inner-filter effects in fluorescence quenching measurements via right-angle and front-surface illumination

Contact Info

Product

Resources

About