Development of a new photosensitizer on the basis of ytterbium porphyrazine complex

The tetraphenyltetracyanoporphyrazine complex of ytterbium has been studied as a potential photosensitizer for fluorescence diagnostics and photodynamic therapy (PDT) of cancer. It has been shown that the new compound has an intensive absorption and fluorescence in the "tissue optical window". In particular, the absorption maximum of the complex is at the wavelength of 590 nm, and the fluorescence emission maximum is at 640 nm. A strong fluorescence enhancement with a 50-fold increase in the quantum yield has been revealed in blood serum. The experiments on human cancer cells line have demonstrated that the complex penetrates the cells in vitro and is located around the nuclei. The biodistribution and pharmacokinetics of the complex in animals have been investigated in vivo by a new method of transillumination fluorescence imaging using a peculiar setup. It has been found that the period of maximum uptake of the complex in mouse cervical carcinoma is from 3 to 6 h after i.v. injection, with the half-life in the tumor being 24 h. However, the selectivity of the complex in the tumor is not high enough. The time of clearance from the body is about 48 h. The area of the strongest fluorescence in the abdominal cavity in in vivo images is anatomically recognized as the intestine. This indicates that the new compounds undergo mainly the hepatic clearance mainly. The conventional methods ex vivo (confocal microscopy and point spectroscopic measurements) have detected the largest content of the complex in the intestine, liver, skin and tumor tissue. In general, the optical characteristics of the ytterbium porphyrazine complex as well as the features of its interaction with biological objects make it promising drug candidate for the photodynamic therapy and/or fluorescence diagnostics of cancer. However, a search for other novel formulations possessing a higher tumor selectivity remains an urgent problem.     

Use of fluorescence enhancement technique to study bilirubin–albumin interaction

Bilirubin–albumin solution gave an emission spectrum in the wavelength range 500–600 nm with emission maxima at 528 nm when excited at 487 nm. The magnitude of fluorescence intensity increased on increasing bilirubin/albumin molar ratio. At three different albumin concentrations, namely, 1.0, 2.5 and 10.0 μM, there was an initial linear increase in fluorescence up to a molar ratio 1.0 in all cases beyond which it sloped off or decreased. This fluorescence enhancement was used to calculate the binding parameters of bilirubin–albumin interaction and the value of binding constant was found to be 1.72×10⁷ l/mol similar to the published values obtained with other methods. Different serum albumins, namely, human (HSA), goat (GSA), pig (PSA) and dog serum albumins (DSA) bound bilirubin with almost the same affinity when studied by the technique of fluorescence enhancement. Bilirubin–albumin interaction was also studied at different pH and ionic strengths. There was a decrease in bilirubin–albumin complex formation on either decreasing the pH from 9.0 to 7.0 or increasing the ionic strength from 0.15 to 1.0. These results suggest that the technique of fluorescence enhancement can be used successfully to study the bilirubin–albumin interaction.     

Hydrophobic interaction of isoprenic coumarin ostruthin with nonphosphorylating mitochondrial fragments

New emission maximum at 395 nm appeared in the fluorescence spectrum of ostruthin (6-geranyl-7-hydroxycoumarin) when allowed to interact with Keilin-Hartree preparation. Incubation of Keilin-Hartree preparation with umbelliferone (7-hy-droxycoumarin) did not result in any changes in its fluorescence spectrum. Different changes were observed in the fluorescence spectrum of ostruthin at incubation with bovine serum albumin, which suggest a different nature of interaction. Changes in pH or viscosity influenced the intensity, but the emission maximum of the ostruthin fluorescence spectrum was either not or very little shifted. Increase in concentration of aliphatic alcohols induced similar changes in the fluorescence spectra of both coumarins as Keilin-Hartree preparation did in the case of ostruthin. This indicated the hydrophobic nature of interaction between ostruthin and Keilin-Hartree preparation.Preincubation of Keilin-Hartree preparation with sulfhydryl reagents did not alter the fluorescence response of ostruthin. Removing lipids from Keilin-Hartree preparation resulted in a decrease in the quantum yield of ostruthin fluorescence at 395 nm and in the maximum number of binding sites. On the other hand, mild extraction of neutral lipids with pentane retained the quantum yield unaltered. At least two types of binding sites are present in Keilin-Hartree preparation from which one includes phospholipids, the other probably proteins. The maximum number of binding sites (39–50 nmoles/mg protein) corresponds to the amount of ostruthin needed for uncoupling of rat liver mitochondria.     

Interaction of α-lactalbumin with dimyristoyl phosphatidylcholine vesicles. I. A microcalorimetric and fluorescence study

α-Lactalbumin and dimyristoyl phosphatidylcholine were used as a prototype to study the influence of a protein conformational change, induced by the pH, on the interaction between that protein and a phospholipid.The enthalpy changes associated with the interaction of α-lactalbumin with dimyristoyl phosphatidylcholine vesicles were measured as a function of the molar ratio of phospholipid to protein, pH and temperature. Gel-filtration, electron-microscopic and fluorescence data for the same experimental conditions were also obtained. At pH 4 and 5, the enthalphy changes (ΔH) are not only larger than at physiological pH, but also show a maximum at about 23°C in the ΔH vs. temperature graph. At pH 6 and 7, on the contrary, ΔH increases with decreasing temperature without a maximum in the curve. Gel-chromatographic and electron-microscopic data show that at pH 6 and 7, the morphological characteristics of the vesicles are unchanged upon addition of α-lactalbumin, while at pH 4 and 5 at 23°C an extra peak appears in the gel-filtration graphs between the pure vesicles and α-lactalbumin. The new fraction contains lipid-protein complexes. Electron micrographs show that bar-shaped entities are formed. A red shift at 23°C and a blue shift at 37°C, both to 336 nm, are observed for λ_max of the fluorescence emission spectra at pH 4 when α-lactalbumin is brought into contact with the phospholipid. At the same time, a strong increase in the fluorescence intensity is observed. The chromatographic and fluorescence data indicate that a lipid-protein complex with a molar ratio of approx. 80 is formed. At pH 7 and different temperatures, the emission maximum remains at the wavelength of pure α-lactalbumin, the change in the fluorescence intensity, however, indicates that interaction with the lipid occurs.The results can be explained on the basis of an electrostatic interaction at pH 6 and 7, and a hydrophobic interaction at pH 4 and 5.     

Structural analysis of seminal and serum human transferrin by second derivative spectrometry and fluorescence measurements

Denaturation of human seminal transferrin (HSmT) compared with human serum transferrin (HSrT) was followed to check structural differences between these two proteins. Second derivative UV spectroscopy indicated that treatment with 6 M guanidine hydrochloride (Gnd·HCl) induced greater structural changes in HSrT than in HSmT and, in particular; (i) the exposure value of tyrosinyl residues was almost 2.5-fold higher in native HSmT than in native HSrT; and (ii) a much more pronounced movement of tryptophanyl residues toward a higher polar environment could be noticed in HSrT after incubation with denaturating agent. Fluorescence measurements showed that: (i) a shift of the maximum emission wavelength of HSmT occurred (maximum emission was centered at 333 nm instead of 323 nm as for HSrT; excitation = 280 nm); (ii) the intrinsic tryptophan fluorescence intensity of HSmT increased after 36 hr in the range of 1.5–4.0 M of denaturant, whereas an opposite behavior was found for HSrT in the range 0.0–2.0 M; and (iii) the wavelength maximum of fluorescence emission changed in a biphasic manner for HSrT and, conversely, under the same experimental conditions, HSmT gave a linear and parallel increase of fluorescence emission after 1 and 36 hr. We can conclude that this different behavior of HSmT with respect to HSrT might be due mainly to the fact that both the number and the exposure of tyrosinyl and tryptophanyl residues are different. Lately, these effects are discussed in relationship with the fact that HSmT contains less than half disulphide bridges than HSrT.     

荧光法研究血清白蛋白与药物的结合作用

本文应用荧光光谱法,观测了药物分子头孢菌素Ⅳ、异烟肼、维生素B_6和氟哌酸对白蛋白荧光的猝灭。由Lineweaver-Burk双倒数作图法,确定了药物与白蛋白作用的离解常数。并通过Forster偶极-偶极无辐射能量转移机理确定了药物分子氟哌酸在人血清白蛋白中与色氨酸残基之间的距离R为2.55nm,由这一距离确定了药物分子可能进入的区域和位置。     

Fluorescent labeling of proteins with amine-specific 1,3,2-(2H)-dioxaborine polymethine dye

A novel water-soluble amine-reactive dioxaborine trimethine dye was synthesized in a good yield and characterized. The potential of the dye as a specific reagent for protein labeling was demonstrated with bovine serum albumin and lysozyme. Its interaction with proteins was studied by fluorescence spectroscopy and gel electrophoresis. The covalent binding of this almost nonfluorescent dye to proteins results in a 75- to 78-fold increase of its emission intensity accompanied by a red shift of the fluorescence emission maximum by 27 to 45 nm, with fluorescence wavelengths of labeled biomolecules being more than 600 nm. The dye does not require activation for the labeling reaction and can be used in a variety of bioassay applications.     

Design and testing of a new photosensitizer based on an ytterbium porphyrazine complex

The ytterbium tetraphenyltetracyanoporphyrazine complex (YbPz) has been studied as a potential photosensitizer for fluorescence diagnostics and photodynamic therapy of cancer. A water-miscible formulation of YbPz has been prepared using PEG 6000. The new compound shows intense absorption and fluorescence in the “optical window” of biological tissues (absorption maximum ∼590 nm, emission maximum ∼625 nm). Fluorescence enhancement (a 50-fold increase in YbPz quantum yield) is observed in blood serum. In cultured human cancer cells, YbPz quickly accumulates around the nuclei, presumably in lysosomes. The distribution and pharmacokinetics of YbPz in mice have been investigated by a novel method of in vivo transillumination fluorescence imaging. The highest level of YbPz in grafted mouse cervical carcinoma is maintained from 3 to 6 h after i.v. injection, its half-life in the tumor being ∼24 h. However, the selectivity of YbPz for the tumor vs. normal tissue is not high enough. Most of the drug is cleared from the body (mainly via liver and intestine) in two days. Conventional ex vivo methods (confocal microscopy and point spectroscopic measurements) have revealed the largest content of YbPz in the intestine, liver, tumor, and skin. In general, the optical characteristics of YbPz as well as the features of its interaction with biological objects make it a promising candidate drug for photodynamic therapy and/or fluorescence diagnostics of cancer, though searching for formulations with higher tumor selectivity remains a topical task.     

Localization of polyphosphates in Saccharomyces fragilis, as revealed by 4′,6-diamidino-2-phenylindole fluorescence

The fluorescent dye 4′,6-diamidino-2-phenylindole has its emission maximum at 456 nm. Fluorescence intensity at this wavelength is significantly increased by various negatively-charged polyelectrolytes. Among several polyelectrolytes tested, polyphosphates appeared to be unique in the sense that they shifted the emission maximum from 456 to 526 nm. Addition of Saccharomyces fragilis cells to a diamidinophenylindole solution caused an immediate shift of the emission maximum to 526 nm, followed by a gradual increase of fluorescence at 456 nm. The 526 nm, but not the 456 nm fluorescence was instantly quenched by non-penetrating cations, like UO²⁺₂. These results suggest a momentary interaction of diamidinophenylindole with polyphosphate, localized outside the plasma membrane, followed by a slow penetration of the dye into the cells, yielding increased fluorescence at 456 nm by interaction of the dye with e.g., nucleic acids. This was confirmed by fluorescence microscopy. After addition of diamidinophenylindole the yeast cells exhibited an immediate green-yellow fluorescence of the membrane, that was suppressed by UO²⁺₂. After longer incubation times the cytoplasm and nucleus developed a blue fluorescence.     

Isolation of nuclei from epidermis cells of larvae of the blowfly Calliphora vicina R.-D

Rapid microspectrofluorometry has been used to evaluate 1-pyrene-butyric acid as an oxygen probe in single living EL2 ascites tissue culture cells. Despite instrumental conditions preventing detection of the pyrene butyric acid maxima at 380 and 400 nm, the probe having penetrated the cell can be easily identified (maximum around 440 nm in unconnected spectra) from the fluorescence emission spectrum, as compared with NAD(P)H emission in controls (maximum around 460 nm). Fluorescence changes during gradually increasing anaerobiosis under nitrogen flow, are compatible with a linear relationship between the reciprocal of the fluorescence intensity and the intracellular oxygen concentration (increase in 430, 434, 442/461 nm ratios at anaerobiosis). The cells having absorbed the probe continue to catabolize glycolytic substrate, but some inhibition is noticeable (e.g. from the amplitude of the NAD(P)H fluorescence increase spectrum due to intracellular addition of glucose-6-P). In principle rapid microspectrofluorometry allows a multiprobe (e.g. 1-pyrene-butyric acid for oxygen, vs NAD(P)H for metabolism) exploration of the living cell.     

Fluorescence changes from single striated muscle fibres injected with labelled troponin C (TnCDANZ)

A fluorescently labelled derivative of the calcium binding subunit of troponin, TnC, has been injected into isolated striated muscle fibres from the barnacle Balanus nubilus. The Ca2+ affinity of isolated TnC is close to that of intact troponin when located in the thin filament. Excitation of the TnCDANZ within the muscle cell (325nm) revealed a marked fluorescence at 510 nm and was similar to that observed in vitro, which was absent at 400 or 600 nm after subtraction of the fibre autofluorescence. High Ca2+ salines increased the fluorescence at 510 nm by roughly 2 times. Single voltage clamp pulses produced a rapid rise in fluorescence at 510 nm after allowing for any non-specific changes at 400 nm, and this signal preceded force development by approx. 55 ms at 22 degrees C. It reached a maximum at the same time as force and subsequently decayed more slowly. The fluorescence signal increased in magnitude with increase in stimulus intensity. These results suggest that Ca2+ attaches rapidly to the contractile filament, but is lost relatively slowly and imply a slow decay of the activation process.     

Electron spin resonance (esr) investigations on blood of patients with leukemia.

ESR investigations of lyophilized blood of patients with acute lymphatic leukemia exhibit an increase in spin concentration and an additional peak not present in control samples. This peak disappears almost completely concomittantly with a reduction in spin concentration after treating the patients with prednisolone, vincristine, and doxorubicin or after addition of 5mM of CuCl2 to blood samples. The results show that the ESR spectra of lyophilized blood samples can be utilized to follow the effect of therapy. Moreover, they suggest that the leukemic blood possesses a high concentration of antioxidants.     

Characterization of the fluorescence of the antitumor agent, mitoxantrone

Studies have been conducted on the absorption, fluorescence excitation and fluorescence emission of mitoxantrone, an important antineoplastic agent. Mitoxantrone has been found to fluoresce with excitation maxima at 610 and 660 nm and emission maximum at 685 nm. Further characterizations of the fluorescence were undertaken to study its usefulness in biological studies. Mitoxantrone fluorescence intensity is altered by pH and the emission spectrum is red-shifted by DNA. Furthermore, the fluorescence polarization is enhanced by DNA, confirming the binding of the antitumor agent to DNA. The fluorescence spectra are slightly modified by changes in ionic strength and the addition of albumin. Data establishing the usefulness of fluorescence to measure serum concentrations in the range of 0.0 to 100 nM are presented. Such determinations can distinguish serum mitoxantrone from its non-fluorescent primary metabolite.     

Influence of succinylation of bovine serum albumin on its conformation and bilirubin binding

In order to investigate the role of lysine residues in the interaction of bilirubin with bovine serum albumin, five succinylated preparations of albumin, namely: 23%, 39%, 49%, 55% and 87%, were prepared, and their conformational and bilirubin-binding properties were studied by the techniques of gel filtration, ultraviolet and visible spectroscopy, and fluorescence quenching. Gel filtration experiments performed at pH 7.0 and ionic strengths 0.15 and 1.0 suggested that the albumin molecule undergoes gradual disorganization with increase in succinylation. The Stokes radius and frictional ratio at ionic strength 0.15 increased from 3.7 nm and 1.36, respectively, for the native protein to 6.3 nm and 2.26 for maximally (87%) succinylated albumin. Interestingly, increase in ionic strength to 1.0 caused significant refolding in succinylated preparations as evidenced by a decrease in Stokes radius and frictional ratio (5.3 nm and 1.90 for 87% succinylated albumin). Progressive succinylation produced a steady decline in the intensity of bilirubin-induced fluorescence quenching, and in the visible spectral changes of the bilirubin-albumin complex at 480 nm. Both of these changes had a good correlation with increase in Stokes radius. Increase in ionic strength to 1.0 produced a significant reversal in these properties. From these results we conclude that probably none of the surface lysine residues is involved in bilirubin-albumin interaction, and that if lysine residues are involved in this interaction they must be buried in the protein interior.     

High-performance liquid chromatographic assay for the measurement of the novel microtubule inhibitor 1069C85 in biological tissues and fluids

1069C85 is a novel tubulin binder developed to circumvent the resistance associated with the Vinca alkaloids. Cytotoxic activity has been demonstrated in vitro against a variety of tumour cell lines, including a variant of the P388 leukaemia with acquired resistance to vincristine. A phase I clinical trial is planned and an assay suitable for preclinical and clinical pharmacokinetics has been developed. A high-performance liquid chromatographic (HPLC) assay is described which allows measurement of 1069C85 in plasma, urine, and tissue samples. The method uses reversed-phase chromatography with isocratic elution and detection by fluorescence at 406 nm following excitation at 340 nm. The assay is specific, sensitive (limit of sensitivity 0.25 ng/ml) and reproducible (coefficient of variation <5%). The method has been used to study the pharmacokinetics of 1069C85 in Balb C mice following a single oral dose of 1 mg/kg. The maximum plasma concentration was reached 15 min after administration and subsequent elimination was slow with a half life of 6.5 ± 2.2 h. The drug remained detectable in plasma, at 1 ± 0.5 ng/ml, 24 h after this dose. This assay will be used to determine the pharmacokinetic profile of 1069C85 in mice and in a forthcoming phase I clinical trial.     

Binding of saccharides to ricin E isolated from small castor beans

The binding of saccharides to ricin E isolated from small castor beans was studied by equilibrium dialysis and spectroscopy. Equilibrium dialysis data indicate that ricin E has two galactose-binding sites, a high affinity site (HA-site) and a low affinity site (LA-site). The binding of specific saccharides to ricin E induces a shift of the fluorescence spectrum to shorter wavelength by 3 nm and UV-difference spectra with a maximum at 290 nm and a negative intensity around 300 nm. The interaction of ricin E with its specific saccharides was analyzed in terms of the variation of the intensity at 320 nm in the fluorescence spectrum and the magnitude of the negative intensity at 300 nm in the UV-difference spectra as functions of saccharide concentration. The results indicate that these spectroscopic changes are representative of the binding of saccharides to the LA-site, which contains a tryptophan residue. By comparing the association constants of saccharides for ricin E with those for ricin D, isolated from the large castor beans, it was found that the HA of ricin E binds saccharides with an affinity of less than one-half that of ricin D, while the saccharide-binding abilities of the LA-site of the two ricins were about the same.     

Modulation in the photosensitivity of albumin-bound bilirubin

Exposure of BR–albumin complexes to visible light at pH 8.0 led to a change in the fluorescence intensity at 525 nm, which was found to be different for different serum albumins. Whereas a complex of BR with human serum albumin (HSA) showed a marked increase in fluorescence upon photoirradiation, BR–sheep serum albumin (SSA) complex failed to produce a marked increase. On the other hand, a complex of pig serum albumin (PSA) with BR produced a remarkable decrease in fluorescence upon photoirradiation. Equilibration of these complexes with 20 mM chloroform for 1 h resulted in alteration in the photoinduced fluorescence. These photoinduced fluorescence modulations were found to be concentration dependent. Photoirradiation of BR–HSA complex led to a significant decrease in the positive CDCEs of the bisignate CD spectra in a time dependent manner that can be reconciled, to a significant extent, in the presence of chloroform. Taken together, all these results suggest that chiroptical properties/stability of albumin-bound BR varies with albumin species, protein concentration and the presence of chloroform.     

Frequency-domain fluorescence studies of an extracellular metalloproteinase of Staphylococcus aureus

A frequency-domain fluorescence study of calcium-binding metalloproteinase from Staphylococcus aureus has shown that this two-tryptophan-containing protein exhibits a double-exponential fluorescence decay. At 10 degrees C in 0.05 M Tris-HCl buffer (pH 9.0) containing 10 mM CaCl2, fluorescence lifetimes of 1.2 and 5.1 ns are observed. Steady-state and frequency-domain solute-quenching studies are consistent with the assignment of the two lifetimes to the two tryptophan residues. The tryptophan residue characterized by a shorter lifetime has a maximum of fluorescence emission at about 317 nm and the second one exhibits a maximum of its emission at 350 nm. These two residues contribute almost equally to the protein's fluorescence. These results, as well as fluorescence-quenching studies using KI and acrylamide as a quencher, indicate that in calcium-loaded metalloproteinase, the tryptophan residue characterized by the shorter lifetime is extensively buried within the protein. The second residue is exposed on the surface of the protein. The tryptophan residues of metalloproteinase have acrylamide dynamic-quenching rate constants, kq values, of 2.3 and 0.26 X 10(9) M-1 X s-1 for the exposed and buried residue, respectively. A study of the temperature dependence of the fluorescence lifetime for the two tryptophan components gives activation energies, Ea values, for thermal quenching of 1.8 and 2.2 kcal/mol for the buried and the exposed residue, respectively. Dissociation of Ca2+ from the protein causes a change in the protein's structure, as can be judged from dramatic changes which occur in the fluorescence properties of the buried tryptophan residue. These changes include an approx. 13 nm red-shift in the maximum of the fluorescence emission and an increase in the acrylamide-quenching rate constant, and they indicate that the removal of Ca2+ results in an increase in the exposure and the polarity of the microenvironment of this 'blue' residue.     

High-affinity binding of NADPH to camel lens zeta-crystallin

Fluorescence spectrum of camel lens zeta-crystallin, a major protein in the lens of camelids and histicomorph rodents, showed maximum emission at 315 nm. This emission maximum is blue shifted compared to most proteins, including alpha-crystallin, and appeared to be due to tryptophan in highly hydrophobic environment. Interaction of NADPH with zeta-crystallin quenched the protein fluorescence and enhanced the fluorescence of bound NADPH. Analysis of fluorescence quenching suggested high-affinity interaction between NADPH and zeta-crystallin with an apparent Km<0.45 microM. This value is at least an order of magnitude lower than that suggested by activity measurements. Analysis of NADPH fluorescence showed a biphasic curve representing fluorescence of free- and bound-NADPH. The intersection between free- and bound-NADPH closely paralleled the enzyme concentration, suggesting one mole of NADPH was bound per subunit of the enzyme. Phenanthrenequinone (PQ), the substrate of zeta-crystallin, also was able to quench the fluorescence of zeta-crystallin, albeit weaker than NADPH. Quantitative analysis suggested that zeta-crystallin had low affinity for PQ in the absence of NADPH, and PQ binding induced significant conformational changes in zeta-crystallin.     

Interaction of virstatin with human serum albumin: spectroscopic analysis and molecular modeling

Virstatin is a small molecule that inhibits Vibrio cholerae virulence regulation, the causative agent for cholera. Here we report the interaction of virstatin with human serum albumin (HSA) using various biophysical methods. The drug binding was monitored using different isomeric forms of HSA (N form ～pH 7.2, B form ～pH 9.0 and F form ～pH 3.5) by absorption and fluorescence spectroscopy. There is a considerable quenching of the intrinsic fluorescence of HSA on binding the drug. The distance (r) between donor (Trp214 in HSA) and acceptor (virstatin), obtained from Forster-type fluorescence resonance energy transfer (FRET), was found to be 3.05 nm. The ITC data revealed that the binding was an enthalpy-driven process and the binding constants K(a) for N and B isomers were found to be 6.09×10(5 )M(-1) and 4.47×10(5) M(-1), respectively. The conformational changes of HSA due to the interaction with the drug were investigated from circular dichroism (CD) and Fourier Transform Infrared (FTIR) spectroscopy. For 1:1 molar ratio of the protein and the drug the far-UV CD spectra showed an increase in α- helicity for all the conformers of HSA, and the protein is stabilized against urea and thermal unfolding. Molecular docking studies revealed possible residues involved in the protein-drug interaction and indicated that virstatin binds to Site I (subdomain IIA), also known as the warfarin binding site.