Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates.

Alexa 350, Alexa 430, Alexa 488, Alexa 532, Alexa 546, Alexa 568, and Alexa 594 dyes are a new series of fluorescent dyes with emission/excitation spectra similar to those of AMCA, Lucifer Yellow, fluorescein, rhodamine 6G, tetramethylrhodamine or Cy3, lissamine rhodamine B, and Texas Red, respectively (the numbers in the Alexa names indicate the approximate excitation wavelength maximum in nm). All Alexa dyes and their conjugates are more fluorescent and more photostable than their commonly used spectral analogues listed above. In addition, Alexa dyes are insensitive to pH in the 4-10 range. We evaluated Alexa dyes compared with conventional dyes in applications using various conjugates, including those of goat anti-mouse IgG (GAM), streptavidin, wheat germ agglutinin (WGA), and concanavalin A (ConA). Conjugates of Alexa 546 are at least twofold more fluorescent than Cy3 conjugates. Proteins labeled with the Alexa 568 or Alexa 594 dyes are several-fold brighter than the same proteins labeled with lissamine rhodamine B or Texas Red dyes, respectively. Alexa dye derivatives of phalloidin stain F-actin with high specificity. Hydrazide forms of the Alexa dyes are very bright, formaldehyde-fixable polar tracers. Conjugates of the Alexa 430 (ex 430 nm/em 520 nm) and Alexa 532 (ex 530 nm/em 548 nm) fluorochromes are spectrally unique fluorescent probes, with relatively high quantum yields in their excitation and emission wavelength ranges.

A stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: applications in detecting the activity of phagocyte NADPH oxidase and other oxidases.

The enzymatic determination of hydrogen peroxide can be accomplished with high sensitivity and specificity using N-acetyl-3, 7-dihydroxyphenoxazine (Amplex Red), a highly sensitive and chemically stable fluorogenic probe for the enzymatic determination of H2O2. Enzyme-catalyzed oxidation of Amplex Red, which is a colorless and nonfluorescent derivative of dihydroresorufin, produces highly fluorescent resorufin, which has an excitation maximum at 563 nm and emission maximum at 587 nm. The reaction stoichiometry of Amplex Red and H2O2 was determined to be 1:1. This probe allows detection of 5 pmol H2O2 in a 96-well fluorescence microplate assay. When applied to the measurement of NADPH oxidase activation, the Amplex Red assay can detect H2O2 release from as few as 2000 phorbol myristate acetate-stimulated neutrophils with a sensitivity 5- to 20-fold greater than that attained in the scopoletin assay under the same experimental conditions. Furthermore, the oxidase-catalyzed assay using Amplex Red results in an increase in fluorescence on oxidation rather than a decrease in fluorescence as in the scopoletin assay. In comparison with other fluorometric and spectrophotometric assays for the detection of monoamine oxidase and glucose oxidase, this probe is also found to be more sensitive. Given its high sensitivity and specificity, Amplex Red should have a broad application for the measurement of H2O2 in a variety of oxidase-mediated reactions and very low levels of H2O2 in food, environmental waters, and consumer products.

A one-step fluorometric method for the continuous measurement of monoamine oxidase activity.

We have developed a one-step fluorometric method for the measurement of monoamine oxidase (MAO) activity in 96-well microplates with sensitivity 10-fold higher than the conventional spectrophotometric assay method. This assay is based on the detection of H2O2 in a horseradish peroxidase-coupled reaction using N-acetyl-3, 7-dihydroxyphenoxazine (Amplex Red), a highly sensitive and stable probe for H2O2. With a single sampling, this assay is useful for performing both end-point and continuous measurements of MAO activity. Using a commercially available enzyme, our assay allows the detection of MAO B activity as low as 1.2 x 10(-5) U/ml. When applied to crude tissue homogenates, we have been able to selectively detect both MAO A and MAO B from cow brain tissue with protein content as low as 200 microgram per sample. The potential applications of this assay include the measurement of MAO activity in normal and diseased tissues, blood samples, and other biological fluids and the screening of drugs for the treatment of MAO-mediated diseases.

Quenched BODIPY dye-labeled casein substrates for the assay of protease activity by direct fluorescence measurement.

We have prepared casein conjugates of two BODIPY dyes for use as fluorogenic protease substrates in homogeneous assays. Both conjugates are labeled to such an extent that the dyes are efficiently quenched in the protein, yielding virtually nonfluorescent substrate molecules. These fluorogenic substrates release highly fluorescent BODIPY dye-labeled peptides upon protease digestion, with fluorescence increases proportional to enzyme activity. These quenched substrates are suitable for the continuous assay of enzymatic activity using standard fluorometers, filter fluorometers, or fluorescence microplate readers using either fluorescein excitation and emission wavelengths to measure BODIPY FL casein hydrolysis or Texas Red wavelengths to detect proteolysis of BODIPY TR-X casein. Most current techniques for detecting protease activity, such as the fluorescein thiocarbamoyl casein (FTC-casein) protease assay, require extensive manipulation, including separation steps, and are therefore labor intensive and error-prone. In comparison, we found the BODIPY dye-labeled casein protease assays to be simple and precise and to have greater sensitivity and a broader dynamic range of detection than the FTC-casein assay. We were able to sensitively detect the activities of a wide variety of enzymes with these new substrates, including serine, acid, sulfhydryl, and metalloproteases. We also found the assay suitable for quantitating protease inhibitor concentrations and for real-time analysis of proteolysis.