Pyranine

The pyranine derivative DHPDS (l,3-dihydroxypyrene-6,8-disulfonic acid) retains the high pH sensitivity of pyranine but its acido-basic properties are much less sensitive to ionic strength. This indicator has two pKa values 7.33 + 0.04 and 8.55 + 0.2. Ratiometric measurements in both excitation and emission are possible. 

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Soybean (Glycine max Merr. cv Kent) mechanical agitation in the presence of PGA elicitor pyranine fluorescence [178]... 

Tomato (Lycopersicon esculentum) pressiue (50 mPa) pyranine fluorescence [181]... 

Soybean (Glycine max) direct apphcation of pressure to slide-moimted cells osmotic stress pyranine fluorescence 2, 7 -dichlorofluorescein diacetate [183]... 

Some fluorescent dyes are more stable than others. For example, our experience suggests that Pyranine 1OG is sufficiently stable if samples can be collected within less than 30 min. For wind tunnel measurements of spray drift, success has been obtained with Green S. ... 

Photo-induced proton transfer Aerosol-OT. The efficiency of proton transfer depends on HzO. Pyranine used as a fluorescent probe. Bardez et al., 1984... 

Fig. 1.5. Examples of hydrophobic, hydrophilic and amphiphilic probes. 1 pyrene. 2 8-hydroxypyrene-l,3,6-trisulfonic acid, trisodium salt (pyranine). 3 8-alkoxypyrene-l,3,6-trisulfonic acid, trisodium salt. 4 1-...

Fig. B4.3.2. Fluorescence decay curves for pyranine in various aqueous environments. See text for the meaning of A, B, C and D (reproduced with permission from Gutman et al.b ).

Curve B pyranine trapped in the thin water layer (30 A thickness) of multi-lamellar vesicles made of dipalmitoylphosphatidyl choline (pH 5.5) ... 

Curve C pyranine enclosed in the anion specific pore (18 x 27 A) of the PhoE... 

Curve D pyranine in the heme binding site of apomyoglobin, a site containing 30 water molecules or less. 

Fig. 4.11. pH dependence of emission spectra of pyranine in very acidic media. The residual fluorescence of the acidic form at pH > 3 is due to geminate recombination. 

The effect of various charged species on the pKa of pyranine is shown belowb). 

All these data show that the ionic strength can induce a change in pKa up to 0.5-0.6 unit. Because the ionic strength effect causes spectral changes indistinguishable from those caused by pH, a correction method is desirable. A method based on a double pH indicator system, i.e. pyranine and 4-methylumbellifer-one, was proposed by Opitz and Lubbers 1. 

Fig. 10.4. pH dependence of the excitation and emission spectra of pyranine around neutral pH. 

Various pH sensors have been built with a fluorescent pH indicator (fluorescein, eosin Y, pyranine, 4-methylumbelliferone, SNARF, carboxy-SNAFL) immobilized at the tip of an optical fiber. The response of a pH sensor corresponds to the titration curve of the indicator, which has a sigmoidal shape with an inflection point for pH = pK , but it should be emphasized that the effective pKa value can be strongly influenced by the physical and chemical properties of the matrix in which the indicator is entrapped (or of the surface on which it is immobilized) without forgetting the dependence on temperature and ionic strength. In solution, the dynamic range is restricted to approximately two pH units, whereas it can be significantly extended (up to four units) when the indicator is immobilized in a microhetero-geneous microenvironment (e.g. a sol-gel matrix). 

Use of the pH-sensitive membrane-impermeable flurophore pyranine based on the ratiometric method, which determines directly level of dissociation of pyranine from the ratio between the charged (nnprotonated) pyranine and total pyranine in the intraliposome aqneons phase Addition of impermeable DPX, which acts as a quencher to pyranine fluorescence, into the liposome external medium ensures lack of contribution of extraliposome medium pyranine fluorescence (18,22). This method is considered invasive as the pyranine has to be added in the hydration medium prior to liposome preparation and cannot be used for pH determination of intraliposome aqueous phase... 

Using pyranine (8-hydroxy-1,3,6-pyrene trisulfonate) as intraliposome pH indicator, the liposomes were prepared as above (as in section Preparation of 100 nm SSL Loaded with DOX via Transmembrane AS Gradient ) with the exception that pyranine (0.5 mM) was included in the hydration solution. Removal of untrapped pyranine was achieved by gel filtration on a Sephadex G-50 column, preequilibrated with either NaCl, KCl, sucrose or AS solution (according to need). All these solutions also contained lOmM Hepes buffer at the desired pH (usually pH 7.5). 

We foimd that the ratiometric method is superior because it is not dependent on pyranine concentration and therefore free of error in pipeting (18,22,54). Calibration curves were constructed by preparing liposomes in which the hydration of the lipids to form MLV was done using solutions of high concentration at the desired pH in the range of 3.0 to 10.0. Gel-exclusion chromatography on a Sephadex column, as mentioned above, yielded a series of liposome preparations with a fixed external pH (pH 7.5), but different internal pH values determined by the buffer used for lipid hydration. Neither KI nor DPX, which quench the fluorescence of aqueous solutions of pyranine, has much effect on the fluorescence intensity of pyranine in the void volume after gel-exclusion chromatography, which indicates the complete removal of the pyranine from the extraliposomal medium. 

In the ratiometric method, the fluorescence intensity of the liposomes containing pyranine (F) and in the presence of the quencher DPX was determined at 520 nm upon excitation at two wavelengths 460 nm (of the charged unprotonated pyranine) and 415 nm (of the pH-independent isosbestic wavelength that describe the total pyranine concentration). The ratio of is described as F. The ratiometric measurement is used to determine the intraliposome aqueous phase pH (18,22). Then nigericin (or nonactine) at final concentration of 5pM was added to disrupt the pH and/or ammonium ion gradient that induce complete gradient collapse and the measurement at the above two excitations was repeated, and indeed it demonstrated a shift of the intraliposome aqueous pH to be identical to the extraliposome medium pH (10). 

Recently we found that the presence of ions introduces artifacts in the determination of pH by pyranine (54). This effect is related to the relative position of the ion (both cations and anions) in the Hoffmeir series (54). Compared with other ions, AS was found to have only a minimal effect on this shift, which agrees well with the location of NH4+ in the cation Hoffmeir series and of sulfate in the anion series (54). 

Avnir Y, Barenholz Y. pH determination by pyranine medium related artifacts and their correction. Anal Biochem 2005 347 34-41. 

Pyranine, 8-hydroxypyrene-l,3,6-trisulfonic acid 8-Hydroxypyrene-1,3,6-trisulfonic acid (HPTS, also known as pyranine) can be used as a pH indicator. At low pH, HPTS fluorescence is greater with 405 nm excitation, whereas at neutral pH, fluorescence at 440 nm excitation is greater than at 405 nm excitation. Therefore, a strong fluorescence signal at 405 nm excitation indicates HPTS in acidic compartments (endosomes and lysosomes) a strong signal at 440 nm indicates HPTS in neutral compartments (cytosol) (126 128). 

Straubinger RM, Papahadjopoulos D, Hong KL. Endocytosis and intracellular fate of liposomes using pyranine as a probe. Biochemistry 1990 29(20) 4929-4939. 

Overly CC, Lee KD, Berthiaume E, Hollenbeck PJ. Quantitative measurement of intraorganelle pH in the endosomal-lysosomal pathway in neurons by using ratiometric imaging with pyranine. Proc Natl Acad Sci USA 1995 92(8) 3156-3160. 

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