A new fluorescent pH probe for imaging lysosomes in living cells

A new rhodamine B-based pH fluorescent probe has been synthesized and characterized. The probe responds to acidic pH with short response time, high selectivity and sensitivity, and exhibits a more than 20-fold increase in fluorescence intensity within the pH range of 7.5–4.1 with the pK_a value of 5.72, which is valuable to study acidic organelles in living cells. Also, it has been successfully applied to HeLa cells, for its low cytotoxicity, brilliant photostability, good membrane permeability and no ‘alkalizing effect’ on lysosomes. The results demonstrate that this probe is a lysosome-specific probe, which can selectively stain lysosomes and monitor lysosomal pH changes in living cells.     

Methylene blue-based 7-nitro-1,2,3-benzoxadiazole NIR fluorescent probe triggered by H2S

A new Methylene blue–based 7-nitro-1,2,3-benzoxadiazole NIR fluorescent probe 3, 7-bis-dimethylamino-10-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)-10H-phenothiazine (leuco-MB-NBD) was designed and synthesized. Leuco-MB-NBD showed high sensitivity and selectivity for H₂S as a fluorescent probe in C₂H₅OH-PBS (9:1, v/v, pH = 7.4) solution, this fluorescent assay showed a linear range of 0–50.0 μM and a LOD (limit of detection) of 0.43 μM. Moreover, the probe leuco-MB-NBD has lower toxicity at low concentrations to HCT-116 cells and can be used for cell imaging. Additionally, Leuco-MB-NBD is triggered by hydrogen sulfide to generate methylene blue, methylene blue which has potential rescuing effects on the mitochondrial activity can act as an antidote against sulfide intoxication.     

Sensitive detection of strong acidic condition by a novel rhodamine‐based fluorescent pH chemosensor

A novel rhodamine‐based fluorescent pH probe responding to extremely low pH values has been synthesized and characterized. This probe showed an excellent photophysical response to pH on the basis that the colorless spirocyclic structure under basic conditions opened to a colored and highly fluorescent form under extreme acidity. The quantitative relationship between fluorescence intensity and pH value (1.75–2.62) was consistent with the equilibrium equation pH = pKa + log[(I_max – I)/(I – I_min)]. This sensitive pH probe was also characterized with good reversibility and no interaction with interfering metal ions, and was successfully applied to image Escherichia coli under strong acidity. Copyright © 2015 John Wiley & Sons, Ltd.     

Synthesis and application of a “turn on” fluorescent probe for glutathione based on a copper complex of coumarin hydrazide Schiff base derivative

Discrimination and quantification of intracellular biothiols, such as cysteine (Cys), homocysteine (Hcy), glutathione (GSH) under physiological conditions is significant for academic research and disease diagnosis. A new fluorescent probe (complex 1-Cu²⁺) for discriminate detection of GSH was prepared by copper ions coordinate with coumarin carbohydrazide Schiff base derivative 1. In suitable buffer solution (CH₃CN: HEPES = 3:2, v/v) and under appropriate pH condition (pH = 7.2–7.4), the UV–vis spectroscopy experiments showed that compound 1 and copper ion exhibited a 1:1 ratio binding mode and moderate binding ability. Fluorescence quenching of compound 1 was observed when it complexed with Cu²⁺ ions. An obviously fluorescence restoration appeared after addition of GSH to the solution of probe, which also exhibited a highly selectivity relative to cysteine (Cys) and homocysteine (Hcy) in the amino acid competitive experiments. The minimum detection limit was calculated to 0.12 μM by fluorescent method, which was distinctly below the physiological concentration of GSH in live cells. Its biological application to detect the endogenous GSH was further proved by the HepG2 cell fluorescence image test.     

Energy transfer by chlorophyllb in detergent micelles

The concentration-dependent depolarization, concentration-dependent quenching, absorption and fluorescence spectra in solutions of chlorophyllb-containing detergent micelles with Triton X-100 were studied in a concentration range ofc = 0.4 μM–0.6mM chlorophyllb andc_d = 0.4–7.0mM Triton X-100. The concentration-dependent depolarization obeys Fo¨rster's theory of depolarization of fluorescence with a transfer distance parameterR₀ = 43 ± 2A?. The concentration-dependent quenching is described by an empirical formula for the relative fluorescence yieldη/η₀=sol1[1 + (cc_1/2)²] given by Kelly and Porter (Kelly A. R. and Porter, G. (1970) Proc. R. Soc. Lond. Ser. A. 315, 149–161). With increasing chlorophyll b concentration the red absorption band at 650 nm is shifted toward a longer wavelength and its width increases by 10 nm, the intensity of the long wave fluorescence band increases about 720 nm. The results analysed in terms of these findings lead to the conclusions that chlorophyllb molecules are (a) locally concentrated in the micelles up to the concentration range of in vivo conditions, (b) partly in an aggregated state capable for fluorescence, (c) the chlorophyllb →chlorophyllb homotransfer may be about 3–26 % of the homotransfer chlorophylla →chlorophyll-a depending on the ratio of their concentrations.     

Energetic factors affecting carbon dioxide fixation in isolated chloroplasts

Light- and HCO₃⁻-saturated (10 millimolar) rates of O₂ evolution (120 to 220 micromoles O₂ per milligram chlorophyll per hour), obtained with intact spinach chloroplasts, are decreased up to 3-fold by changes in assay conditions such as omission of catalase from the medium, the use of high (≥1 millimolar) inorganic phosphate, inclusion of NO₂⁻ as an electron acceptor, or bright illumination at low partial pressures of O₂. These inhibitions may be reversed by addition of uncoupling levels of NH₄Cl or of antimycin concentrations that partially block cyclic electron transfer between cytochrome b₆ and cytochrome f. Measurements of the pH gradient across the thylakoid membrane with the fluorescent probe, 9-aminoacridine, indicate that changes in ΔpH are sufficient to account for both the inhibited and restored rates of electron transport. It follows that the rate of HCO₃⁻-saturated photosynthesis may be restricted by a proton gradient back pressure under these conditions.     

Thermodynamic and EPR characterization of mitochondrial succinate-cytochrome c reductase-phospholipid complexes

Succinate-cytochrome c reductase can be easily solubilized in a phospholipid mixture (1:1, lysolecithin:lecithin) in the absence of detergents. The resulting solution contains two b cytochromes with half-reduction potentials of 95 ± 10 mV (b₅₆₁), and 0 ± 10 mV (b₅₆₆) and cytochrome c₁ (E_{m 7.2} = +280±5 mV). The oxidation-reduction midpoint potentials obtained by optical potentiometric titrations are identical to those determined by the EPR titrations and are 40–60 mV higher than the corresponding midpoint potentials of these cytochromes in intact mitochondria. In contrast to detergent-suspended preparations, no CO-sensitive cytochrome b can be detected in the phospholipid-solubilized preparation or intact mitochondria. The half-reduction potential of cytochrome b₅₆₆ is pH-dependent above pH 7.0 (?60 mV/pH unit) while that of b₅₆₁ is essentially pH-independent from pH 6.7–8.5, in contrast to its pH dependence in intact mitochondria. EPR characterizations show the presence of three oxidized low-spin heme-iron signals with g values of 3.78, 3.41 and 3.37. The identification of these signals with cytochromes b₅₆₆ (b_T), b₅₆₁ (b_K) and c₁ respectively is made on the basis of redox midpoint potentials. No significant amounts of oxidized high-spin heme-iron are detectable. In addition, the preparation contains four distinct types of iron-sulfur centers: S₁ and S₂ (E_{m 7.4} = ?260 mV and 0 mV), and two iron-sulfur proteins which are associated with the cytochrome b-c₁ complex: Rieske's iron-sulfur protein (E_{m 7.4} = +280 mV) and Ohnishi's Center 5 (E_{m 7.4} = +35 mV).     

‘Naked-eye’ quinoline-based ‘reactive’ sensor for recognition of Hg2+ ion in aqueous solution

A new ‘naked-eye’ quinoline-based ‘reactive’ ratiometric fluorescent probe was prepared. The reactive stoichiometry of the probe with Hg²⁺ ion was 2:1. The probe exhibited high selectivity towards Hg²⁺ ion to other metal ions with a 410-fold increase in absorbance intensity ratio (A₄₀₂/A₃₄₀) in aqueous solution over a wide-range pH value (2–12), accompanied by a resonance color change from colorless to pale yellow visible to naked-eye.     

Coumarin-suberoylanilide hydroxamic acid as a fluorescent probe for determining binding affinities and off-rates of histone deacetylase inhibitors

Histone deacetylases (HDACs) are intimately involved in epigenetic regulation and, thus, are one of the key therapeutic targets for cancer, and two HDAC inhibitors, namely suberoylanilide hydroxamic acid (SAHA) and romidepsin, have been recently approved for cancer treatment. Because the screening and detailed characterization of HDAC inhibitors has been time-consuming, we synthesized coumarin-SAHA (c-SAHA) as a fluorescent probe for determining the binding affinities (K_d) and the dissociation off-rates (k_off) of the enzyme–inhibitor complexes. The determination of the above parameters relies on the changes in the fluorescence emission intensity (λ_ex = 325 nm, λ_em = 400 nm) of c-SAHA due to its competitive binding against other HDAC inhibitors, and such determination neither requires employment of polarization accessories nor is dependent on the fluorescence energy transfer from the enzyme’s tryptophan residues to the probe. Our highly sensitive and robust analytical protocol presented here is applicable to most of the HDAC isozymes, and it can be easily adopted in a high-throughput mode for screening the HDAC inhibitors as well as for quantitatively determining their K_d and k_off values.     

Characterization of a resolved oxygen-evolving Photosystem II preparation from spinach thylakoids

An oxygen-evolving Photosystem (PS) II preparation was isolated after Triton X-100 treatment of spinach thylakoids in the presence of Mg²⁺. The structural and functional components of this preparation have been identified by SDS-polyacrylamide gel electrophoresis and sensitive spectrophotometric analysis. The main findings were: (1) The concentration of the primary acceptor Q of PS II was 1 per 230 chlorophyll molecules. (2) There are 6 to 7 plastoquinone molecules associated with a ‘quinone-pool’ reducible by Q. (3) The only cytochrome present in significant amounts (cytochrome b-559) occurred at a concentration of 1 per 125 chlorophyll molecules. (4) The only kind of photochemical reaction center complex present was identified by fluorescence induction kinetic analysis as PS II_α. (5) An E_m = ? 10 mV has been measured at pH 7.8 for the primary electron acceptor Q_α of PS II_α. (6) With conventional SDS-polyacrylamide gel electrophoresis, the preparation was resolved into 13 prominent polypeptide bands with relative molecular masses of 63, 55, 51, 48, 37, 33, 28, 27, 25, 22, 15, 13 and 10 kDa. The 28 kDa band was identified as the PS II light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$. In the presence of 2 M urea, however, SDS-polyacrylamide gel electrophoresis showed seven prominent polypeptides with molecular masses of 47, 39, 31, 29, 27, 26 and 13 kDa as well as several minor components. CP I under identical conditions had a molecular mass of 60–63 kDa.     

An easy-to-use FRET protein substrate to detect calpain cleavage in vitro and in vivo

Calpain-1 and -2 are Ca^2 +-activated intracellular cysteine proteases that regulate a wide range of cellular functions through the cleavage of their protein substrates. Unlike degradative proteases, calpains make limited, transformative cleavages, typically in accessible sequences linking discrete subdomains, to irreversibly alter substrate functions. The biological roles of calpain and their interplay with calcium signaling are of significant biomedical interest as biomarkers and potential therapeutic targets in a growing number of diseases including Alzheimer's, cancer and fibrosis. Unfortunately, many of the colorimetric and fluorimetric assays that have been developed to study calpain activity suffer from low sensitivity and/or poor calpain specificity. To address the need for a highly sensitive and calpain-specific substrate suitable for in vitro and in vivo calpain activity analysis, we have developed a protein FRET probe. We inserted the optimized calpain cleavage sequence PLFAAR between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) and modulated its flanking sequences for optimal calpain cleavage. We demonstrate greater sensitivity and calpain-specificity of an optimal 16-residue PLFAAR-based FRET substrate compared to a standard α-spectrin-based probe. The 16-residue PLFAAR protein FRET substrate is not significantly cleaved by trypsin, chymotrypsin, cathepsin-L or caspase-3, and is highly sensitive to both calpain-1 and -2. After transfection of the substrate gene into breast cancer cells the PLFAAR protein FRET product was cut in lysed wild-type cells but not in those with a calpain knock-out phenotype. Blockage of substrate cleavage in the lysates by endogenous and exogenous calpastatin was observed, and was overcome by adding extra calpain.     

A simple boronic acid-based fluorescent probe for selective detection of hydrogen peroxide in solutions and living cells

An approach of high sensitivity and selectivity for hydrogen peroxide (H₂O₂) detection is highly demanded due to its important roles in regulating diverse biological process. In this work, we introduced an easily synthesized fluorescent “turn off” probe, BNBD. It is designed based on the core structure of 4-chloro-7-nitrobenzofurazan as a fluorophore and incorporated with a specific H₂O₂-reactive group, aryl boronate, for sensitive and selective detection of H₂O₂. We demonstrated its selectivity by incubating the probe with other types of ROS, and measured the limit of detection of BNBD as 1.8 nM. BNBD is also conducive to H₂O₂ detection at physiological conditions. We thus applied it to detect both exogenous and endogenous changes of H₂O₂ in living cells by confocal microscopy, supporting its future applications to selectively monitor H₂O₂ levels and identify H₂O₂-related physiological or pathological responses from live cells or tissues in the near future.     

Quinacrine is not a vital fluorescent probe for vesicular ATP storage

Quinacrine, a fluorescent amphipathic amine, has been used as a vital fluorescent probe to visualize vesicular storage of ATP in the field of purinergic signaling. However, the mechanism(s) by which quinacrine represents vesicular ATP storage remains to be clarified. The present study investigated the validity of the use of quinacrine as a vial fluorescent probe for ATP-storing organelles. Vesicular nucleotide transporter (VNUT), an essential component for vesicular storage and ATP release, is present in very low density lipoprotein (VLDL)-containing secretory vesicles in hepatocytes. VNUT gene knockout (Vnut^−/−) or clodronate treatment, a VNUT inhibitor, disappeared vesicular ATP release (Tatsushima et al., Biochim Biophys Acta Molecular Basis of Disease 2021, e166013). Upon incubation of mice’s primary hepatocytes, quinacrine accumulates in a granular pattern into the cytoplasm, sensitive to 0.1-μM bafilomycin A₁, a vacuolar ATPase (V-ATPase) inhibitor. Neither Vnut^−/− nor treatment of clodronate affected quinacrine granular accumulation. In vitro, quinacrine is accumulated into liposomes upon imposing inside acidic transmembranous pH gradient (∆pH) irrespective of the presence or absence of ATP. Neither ATP binding on VNUT nor VNUT-mediated uptake of ATP was affected by quinacrine. Consistently, VNUT-mediated uptake of quinacrine was negligible or under the detection limit. From these results, it is concluded that vesicular quinacrine accumulation is not due to a consequence of its interaction with ATP but due to ∆pH-driven concentration across the membranes as an amphipathic amine. Thus, quinacrine is not a vital fluorescent probe for vesicular ATP storage.     

Positive surface charge inhibition of phospholipase A2 in mixed monolayer systems

Inhibition of pancreatic phospholipase A₂ by surface-active local anesthetics was recently reported by this laboratory to be due to enzyme-anesthetic interaction in the subphase and surface effects. In order to study surface effects in the absence of subphase effects, a long-chain tetracaine analog which was completely insoluble in the subphase, dimethylaminoethyl p-decoxybenzoate, was synthesized. To determine if inhibition was due to the positive surface charge of the analog or some other effect related to structure, the analog's inhibitory effects were compared with those of octadecylamine. Analog-didecanoyl lecithin (PC) monolayers showed nonideal mixing as evidenced by a condensing effect, while octadecylamine-didecanoyl PC monolayers showed ideal mixing. The apparent pK′_a of octadecylamine-dioctanoyl PC micelles (1:4) was 9.9, while that of the analog-dioctanoyl PC micelles (1:4) was 7.6. At pH values where both amines were fully protonated, inhibition of both porcine pancreatic and Crotalus adamanteus phospholipase A₂ on the mixed films was maximal and similar (94–97%). Inhibition decreased with increasing pH and decreasing surface charge on both mixed films and at pH values where both amines were 50% protonated, inhibition was half-maximal. At pH 8.5, where the analog was unprotonated, no inhibition was observed. Thus, inhibition of phospholipase A₂ appears to be due to a positive surface charge alone rather than any effects related to anesthetic structure or spacing in the monolayer.     

Action of DCFH and BODIPY as a Probe for Radical Oxidation in Hydrophilic and Lipophilic Domain

Fluorogenic probes such as 2',7'-dichlorofluorescin (DCFH) have been extensively used to detect oxidative events and to measure antioxidant capacity. At the same time, however, the inherent drawbacks of these probes such as non-specificity towards oxidizing species have been pointed out. The present study was carried out to analyze the action and dynamics of 4, 4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (BODIPY) and DCFH as a fluorescent probe in the free radical-mediated lipid peroxidation in homogeneous solution, aqueous suspensions of liposomal membranes and LDL and plasma. The rate constant for the reaction of BODIPY with peroxyl radicals was estimated as 6.0×10₃ M_-1s_-1, which makes BODIPY kinetically an inefficient probe especially in the presence of potent radical-scavenging antioxidants such as tocopherols, but a convenient probe for lipid peroxidation. On the other hand, the reactivity of DCFH toward peroxyl radicals was as high as Trolox, a water-soluble analogue of α-tocopherol. Thus, DCFH is kinetically more favored probe than BODIPY and could scavenge the radicals within lipophilic domain as well as in aqueous phase. The partition coefficients for BODIPY and DCFH were obtained as 4.57 and 2.62, respectively. These results suggest that BODIPY may be used as an efficient probe for the free radical-mediated oxidation taking place in the lipophilic domain, especially after depletion of α-tocopherol, while it may not be an efficient probe for detection of aqueous radicals.     

Biodegradable and biocompatible multi-arm star amphiphilic block copolymer as a carrier for hydrophobic drug delivery

Multi-arm star amphiphilic block copolymers (SABCs) with approximately 32 arms were synthesized and characterized for drug delivery applications. A hyperbranched polyester, boltorn^® H40 (H40), was used as the macroinitiator for the ring-opening polymerization of ?-caprolactone (?-CL). The resulting multi-arm H40-poly(?-caprolactone) (H40-PCL-OH) was further reacted with carboxyl terminated methoxy poly(ethylene glycol) (MPEG-COOH) to form H40-PCL-b-MPEG copolymers. The resulting SABCs were characterized by ¹H NMR spectroscopy and gel permeation chromatography (GPC). The critical aggregation concentration (CAC) of H40-PCL-b-MPEG was 3.8 mg/L as determined by fluorescence spectrophotometry. Below the CAC, stable unimolecular micelles were formed with an average diameter of 18 nm as measured by TEM. Above the CAC, unimolecular micelles exhibited agglomeration with an average diameter of 98 nm. The hydrodynamic diameter of these agglomerates was found to be 122 nm, as measured by dynamic light scattering (DLS). The drug loading efficacy of the H40-PCL-b-MPEG micelles was 26 wt%. Drug release study showed an initial burst followed by a sustained release of the entrapped hydrophobic model drug, 5-fluorouracil, over a period of 9–140 h. These results indicate that the H40-PCL-b-MPEG micelles have great potential as hydrophobic drug delivery carriers.     

Ratiometric fluorescence imaging of nuclear pH in living cells using Hoechst-tagged fluorescein

Small-molecule fluorescent sensors that allow specific measurement of nuclear pH in living cells will be valuable for biological research. Here we report that Hoechst-tagged fluorescein (hoeFL), which we previously developed as a green fluorescent DNA-staining probe, can be used for this purpose. Upon excitation at 405 nm, the hoeFL–DNA complex displayed two fluorescence bands around 460 nm and 520 nm corresponding to the Hoechst and fluorescein fluorescence, respectively. When pH was changed from 8.3 to 5.5, the fluorescence intensity ratio (F₅₂₀/F₄₆₀) significantly decreased, which allowed reliable pH measurement. Moreover, because hoeFL binds specifically to the genomic DNA in cells, it was applicable to visualize the intranuclear pH of nigericin-treated and intact living human cells by ratiometric fluorescence imaging.     

Biochemical characterization of a highly active O2-evolving Photosystem II preparation from maize

An O₂-evolving Photosystem (PS) II preparation was isolated from maize by a Triton X-100 procedure (Kuwabara, T. and Murata, N. (1982) Plant Cell Physiol. 23, 533–539). A highly active O₂-evolving preparation was obtained which evolved O₂ at 76% the rate of fresh chloroplasts (H₂O → 2,6-dichloro-p-benzoquinone) and was very sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea. There was no detectable PS I activity in the preparation (2,3,5,6-tetramethyl-p-phenylenediamine → methyl viologen). When analyzed by lithium dodecyl sulfate (LDS) polyacrylamide gel electrophoresis the O₂-evolving preparation was shown to be highly depleted in CP I, CF₁, and devoid of cytochromes f and b-563 (the absence of which was confirmed by difference spectroscopy). The preparation was enriched in the PS II reaction center polypeptides I and II, the 34 kDa polypeptide (Metz, J., Wong, J. and Bishop, N.I. (1980) FEBS Lett. 114, 61–66), the Coomassie blue-stainable 32 kDa polypeptide (Kuwabara, T. and Murata, N. (1979) Biochim. Biophys. Acta 581, 228–236), LHCP-associated polypeptides and cytochrome b-559. Polypeptides of unknown function at 40.5, 25, 24, 22, 16.6 and 14 kDa were also present in the O₂-evolving preparation. Triton X-114 phase partitioning (Bricker, T.M. and Sherman, L.A. (1982) FEBS Lett. 149, 197–202) indicated that the majority of these polypeptides were intrinsic. Only the polypeptides at 32, 25, 24 and 14 kDa were extrinsic. When examined by the octylglucoside procedure of Camm and Green (Camm, E.L. and Green, B.R. (1980) Plant Physiol. 66, 428–432) the PS II O₂-evolving preparation was shown to contain the chlorophyll-proteins CP 27, CP 29, CP II1, D, and CP a-1 and CP a-2. Chlorophyll-proteins associated with PS I were highly depleted. The visible absorption spectra indicated an enrichment of chlorophyll b and carotenoids in the preparation. The 77 K fluorescence emission spectrum (excitation wavelength = 435 nm) exhibits a strong F-686 with little F-695 shoulder and a broad, low-intensity F-735 emission.     

Isothermal titration calorimetry of membrane protein interactions: FNR and the cytochrome b6f complex

Ferredoxin-NADP⁺ reductase (FNR) was previously inferred to bind to the cytochrome b₆f complex in the electron transport chain of oxygenic photosynthesis. In the present study, this inference has been examined through analysis of the thermodynamics of the interaction between FNR and the b₆f complex. Isothermal titration calorimetry (ITC) was used to characterize the physical interaction of FNR with b₆f complex derived from two plant sources (Spinacia oleracea and Zea maize). ITC did not detect a significant interaction of FNR with the b₆f complex in detergent solution nor with the complex reconstituted in liposomes. A previous inference of a small amplitude but defined FNR-b₆f interaction is explained by FNR interaction with micelles of the undecyl β-D maltoside (UDM) detergent micelles used to purify b₆f. Circular dichroism, employed to analyze the effect of detergent on the FNR structure, did not reveal significant changes in secondary or tertiary structures of FNR domains in the presence of UDM detergent. However, thermodynamic analysis implied a significant decrease in an interaction between the N-terminal FAD-binding and C-terminal NADP⁺-binding domains of FNR caused by detergent. The enthalpy, ΔH^o, and the entropy, ΔS^o, associated with FNR unfolding decreased four-fold in the presence of 1 mM UDM at pH 6.5. In addition to the conclusion regarding the absence of a binding interaction of significant amplitude between FNR and the b₆f complex, these studies provide a precedent for consideration of significant background protein-detergent interactions in ITC analyses involving integral membrane proteins.