A long-wavelength biolabeling reagent based on the oxonol fluorophore.

A red fluorescent dye of the oxonol class, bis-[1-(carboxymethyl)barbituric acid-(5)]-pentamethinoxonol, has been synthesized and, in the form of the succinimidyl active ester, has been applied to antibody labeling for application to flow cytometry and to imaging of tissue sections. The new dye, named CMOX (for carboxymethyloxonol), shows maximum excitation at 583 nm and emission at 611 nm, with a quantum yield of 0.2 in aqueous buffer and methanol. Antibodies labeled with the new dye show favorable brightness, photostability, and low levels of nonspecific binding.

A stable diazo photoaffinity label with high absorptivity and effective photoactivation beyond 300 nm.

The sulfosuccinimidyl active ester of 3-(3-carbethoxy-4-diazo-5-oxo-2-pyrrolin-1-yl)propanoic acid (DIAZOPY-SE) has been synthesized for use as a photoaffinity labeling reagent. This compound was obtained from commercial chemicals by a four-step synthesis requiring no complex procedures or special apparatus. The active ester efficiently derivatizes protein amino groups with the chromophore 3-carbethoxy-4-diazo-5-oxo-2-pyrroline (DIAZOPY, epsilon 8800 M-1 cm-1 at lambda max 330 nm), which on irradiation yielded products expected from formation of a reactive carbene intermediate. Brief irradiation of DIAZOPY in 2-propanol using wavelengths greater than 300 nm for photolysis yielded mainly an isopropyl ether resulting from insertion of the carbene into the O-H bond of the alcohol. Formed concurrently and to a somewhat lesser extent was an isopropyl ester, resulting from a ring-contracting Wolff rearrangement of the carbene and subsequent reaction with isopropanol. Analogous products were produced by photolysis in 2-propanol of DIAZOPY-PA (for DIAZOPY propanoic acid), the carboxylic acid precursor of DIAZOPY-SE. Facile protein derivatization by DIAZOPY-SE was demonstrated using actin and sheep IgG. Actin labeled with DIAZOPY-SE and irradiated while in the F-actin (reversibly polymerized) form was crosslinked to yield a covalently-linked dimer, illustrating the potential of the reagent in photoaffinity applications. Advantages of DIAZOPY-SE as a photoaffinity labeling reagent include ease of synthesis, chemical and photostability, efficient photolysis at wavelengths greater than 300 nm, and a capacity for crosslinking by carbene insertion processes.

Cyanine dye labeling reagents--carboxymethylindocyanine succinimidyl esters.

Ten carboxymethylindocyanine dyes which form the basis of a new series of fluorescent probes have been synthesized and converted into succinimidyl active esters for fluorescent labeling of proteins or other amino-containing substances. Fluorescence emission maxima for members of the series range from 575 to 780 nm. Hydrophilic, water-soluble reagents have been obtained which yield labeled antibodies with little tendency to form precipitates. The fluorescence intensities achieved are higher than those produced by labeling with the cyanine isothiocyanates described previously (Mujumdar et al.: Cytometry 10:11-19, 1989). The utility of these reagents has been demonstrated in antibody labeling for two-color immunofluorescent imaging of internal structures in a mammalian cell and for two-color flow-cytometry experiments. The use of values of chromophore-equivalent weight (W/Ceq), calculated from quantitative absorption data on dye samples, is proposed as an aid in formulating labeling procedures.

Calcium signals recorded from two new purpurate indicators inside frog cut twitch fibers.

Two new Ca indicators, purpurate-3,3'diacetic acid (PDAA) and 1,1'-dimethylpurpurate-3,3'diacetic acid (DMPDAA), were synthesized and used to measure Ca transients in frog cut muscle fibers. These indicators are analogues of the purpurate components of murexide and tetramethylmurexide, in which two acetate groups have been incorporated into each molecule to render it membrane impermeant. The apparent dissociation constant for Ca is 0.95 mM for PDAA and 0.78 mM for DMPDAA. One of the indicators was introduced into a cut fiber, which was mounted in a double Vaseline-gap chamber, by diffusion from the end-pool solutions. The time course of indicator concentration, monitored optically in the middle of the fiber in the central-pool region, suggests that 19% of the PDAA or 27% of the DMPDAA became bound or sequestered inside the fiber. In resting fibers, the absorbance spectrum of either indicator was well fitted by the indicator's [Ca] = 0 mM cuvette absorbance spectrum, which is consistent with the idea that PDAA and DMPDAA do not enter the sarcoplasmic reticulum as tetramethylmurexide appears to be able to do (Maylie, J., M. Irving, N.L. Sizto, G. Boyarsky, and W. K. Chandler, 1987. Journal of General Physiology. 89:145-176). After an action potential, the absorbance of either indicator underwent a rapid and transient change that returned to the prestimulus baseline within 100-200 ms. The amplitude of this change had a wavelength dependence that matched the indicator's Ca-difference spectrum. The average amplitude of peak free [Ca] was 21 microM (PDAA or DMPDAA) if all the indicator inside a fiber was able to react with Ca as in cuvette calibrations, and was 26 (PDAA) or 28 microM (DMPDAA) if only freely diffusible indicator could so react. These results suggest that PDAA and DMPDAA are the first Ca indicators that provide a reliable estimate of both the amplitude and time course of (the spatial average of) free [Ca] in a twitch muscle fiber after an action potential.

Skin sensitization with the new reagents NOPYE (4-nitro-1-cyclohexyl-3-ethoxy-2-oxo-3-pyrroline) and NOPY-L-phenylalanine.

The aim of this study was to investigate the compound 4-nitro-1-cyclohexyl-3-ethoxy-2-oxo-3-pyrroline (NOPYE) and some related compounds for skin sensitization in guinea pigs, as the first step in a search for more effective skin sensitizers for immunotherapy of cutaneous tumors. In guinea pigs, NOPYE and NOPYE-L-alanine produce far milder delayed hypersensitivity reactions than DNCB. Both NOPYE and DNCB fail to act as adjuvants for skin sensitization to tuberculin purified protein derivative (PPD) and ovalbumin (OV). This suggests an explanation for the lack of effectiveness of DNCB in immunotherapy of metastases: DNCB may be relatively ineffective as an adjuvant for production of specific antitumor immunity. Such adjuvant activity may be essential if the action of the immunotherapeutic reagent is not to be confined to its site of application but is to be effective at the site of distant metastases.

4-Nitro-1-cyclohexyl-3-ethoxy-2-oxo-3-pyrroline. A reversible, amino-specific, protein reagent.

4-Nitro-1-cyclohexyl-3-ethoxy-2-oxo-3-pyrroline reacts with both amino and sulfhydryl groups. The instability of the product with sulfhydryl groups makes the reagent a useful amino-group specific protein reagent. The advantages of this compound include (1) rapid reaction with protein (less than 15 min at pH 9), (2) EASE OF REVERSAL UNDER MILDLY ALKALINE CONDITIONS (PH larger than or equal to 8) with formation of a water-soluble by-product (lambdamax = 363 nm), and (3) ease of quantitation utilizing the high extinction coefficients of the amino derivative (lambdamax = 383 and 397 nm, epsilon397 = 20 200 M-1 . cm-1) and the reversal by-product (lambdamax = 363 nm, epsilon = 16 300 M-1 . cm-1). With these characteristics and the stability of the amino derivative under physiological conditions (t1/2 for reversal = 167 h at pH 7.0 and room temperature), nitrocyclohexylethoxyoxopyrroline can be a useful reagent in a wide variety of protein sequencing and structure studies.