Photophysical properties and photodynamic activity in vivo of some tetrapyrroles

Some of the photophysical properties (stationary absorbance and fluorescence, fluorescence decay times and singlet oxygen quantum yields) of pheophorbide a, metal-free, ClAl-, Cu- and Mg-t-butyl-substituted phthalocyanines, metal-free, ClAl- and Cu-t-butyl-substituted naphthalocyanines and of a number of tetraphenylporphyrins (5,10,15,20-tetraphenylporphyrin, 5,10,15,20-tetra(m-hydroxyphenyl)porphyrin, 5,10,15,20-tetra(p-hydroxyphenyl)porphyrin) have been studied in comparison with hematoporphyrin IX in order to select potent photosensitizers for the photodynamic treatment of cancer. The photodynamic activity of these compounds was investigated using Lewis lung carcinoma in mice. As a consequence of the photophysical parameters (relatively short singlet state lifetimes, and high singlet oxygen quantum yields) the photodynamic activities of pheophorbide a, t-butyl-substituted ClAl-phthalocyanine and ClAl-naphthalocyanine were selected for study in greater detail. Under the conditions employed in the present study, pheophorbide a was found to be the most effective sensitizer, as judged from its strong absorption at the excitation wavelength as compared with the hematoporphyrin derivative and greater singlet oxygen quantum yield relative to the phthalocyanines and naphthalocyanines. The photodynamic activity was observed to be strongly dependent on the photophysical parameters of the compounds. The primary mechanism underlying the photodynamic activity of these sensitizers probably consists of energy transfer from the lowest triplet state of the dyes to molecular oxygen, resulting in the formation of singlet oxygen (type II of photosensitization).     

On the possible site of inhibition of photosynthetic electron transport by ultraviolet-B (UV-B) radiation

In Amaranthus chloroplasts that are exposed to ultraviolet-B (UV-B) radiation, the electron flow from water to dichlorophenol indophenol (DCPIP) was inhibited, but the electron flow from reduced DCPIP to methyl viologen remains unaffected. Diphenylcarbazide was ineffective in restoring the activity of DCPIP Hill reaction in UV-B irradiated chloroplasts. Electron flow from water to ferricyanide or dichloro-dimethoxy- p -benzoquinone was inhibited to a degree similar to that of the DCPIP Hill reaction.
The rate of carotenoid photobleaching in the presence of carbonyl cyanide- m -chlorophenylhydrazone, an indicator of the photochemical reaction near the vicinity of reaction centre of photosystem II, was suppressed and paralleled with the inhibition of the DCPIP Hill reaction.
In the UV-B treated chloroplasts, the variable part of the fluorescence transient was diminished. Though the fluorescence yield was lowered by the UV-B radiation, addition of 3-(3,4-dichlorophenyl)-l, l-dimethylurea (DCMU) and/or sodium dithionite increased the emission markedly. With the increase in the dosage of UV-B irradiation, the time required to reach the steady state fluorescence level became longer in the absence of DCMU and shorter in the presence of DCMU. The kinetics of 520 nm absorbance change was markedly unaltered by the UV-B irradiation but its dark decay was prolonged. It is concluded that UV-B irradiation inactivates the photosystem II reaction centre.     

A comparative study of the interaction of 5,10,15,20-tetrakis (N-methylpyridinium-4-yl)porphyrin and its zinc complex with DNA using fluorescence spectroscopy and topoisomerisation.

Binding of 5,10,15,20-tetrakis (N-methylpyridinium-4-yl)porphyrin (H2TMPyP4+) and its zinc complex (ZnTMPyP4+) to DNA is demonstrated by their coelectrophoresis and by absorption and fluorescence spectroscopic methods. Topoisomerisation of pBR322 DNA shows that H2TMPyP4+ unwinds DNA as efficiently as ethidium bromide showing that it intercalates at many sites. ZnTMPyP4+ may cause limited unwinding. Marked changes in the fluorescence spectra of the porphyrins are found in the presence of DNA. The fluorescence intensity of either H2TMPyP4+ or ZnTMPyP4+ is enhanced in the presence of poly (d(A-T)), whereas in the presence of poly (d(G-C] the fluorescence intensity of ZnTMPyP4+ is only slightly affected and that of H2TMPyP4+ markedly reduced. Both the porphyrins photosensitize the cleavage of DNA in aerated solution upon visible light irradiation.     

Engineering of near IR fluorescent albumin nanoparticles for in vivo detection of colon cancer

ABSTRACT: BACKGROUND: The use of near-infrared (NIR) fluorescence imaging techniques has gained great interest for early detection of cancer because water and other intrinsic biomolecules display negligible absorption or autofluorescence in this region. Novel fluorescent nanoparticles with potential to improve neoplasm detection sensitivity may prove to be a valuable tool in early detection of colon tumors. METHODS: The present study describes the synthesis and use of NIR fluorescent albumin nanoparticles as a diagnostic tool for detection of colon cancer. These fluorescent nanoparticles were prepared by a precipitation process of human serum albumin (HSA) in aqueous solution in the presence of a carboxylic acid derivative of the NIR dye IR-783 (CANIR). Tumor-targeting ligands such as peanut agglutinin (PNA), anti-carcinoembryonic antigen antibodies (anti-CEA) and tumor associated glycoprotein-72 monoclonal antibodies (anti-TAG-72) were covalently conjugated to the albumin nanoparticles via the surface carboxylate groups by using the carbodiimide activation method. RESULTS AND DISCUSSION: Leakage of the encapsulated dye into PBS containing 4% HSA or human bowel juice was not detected. This study also demonstrates that the encapsulation of the NIR fluorescent dye within the HSA nanoparticles reduces the photobleaching of the dye significantly. Specific colon tumor detection in a mouse model was demonstrated for PNA, anti-CEA and anti-TAG-72 conjugated NIR fluorescent HSA nanoparticles. These bioactive NIR fluorescent albumin nanoparticles also detected invisible tumors that were revealed as pathological only subsequent to histological analysis. CONCLUSIONS: These results may suggest a significant advantage of NIR fluorescence imaging using NIR fluorescent nanoparticles over regular colonoscopy. In future work we plan to broaden this study by encapsulating cancer drugs, such as paclitaxel and doxorubicin, within these biodegradable NIR fluorescent HSA nanoparticles, in order to use them for both detection as well as therapy of colon cancer and others.     

Photobleaching kinetics, photoproduct formation, and dose estimation during ALA induced PpIX PDT of MLL cells under well oxygenated and hypoxic conditions.

Fluorescence photobleaching and photoproduct formation were investigated during delta-aminolevulinic acid (ALA) induced protoporphyrin IX (PpIX) PDT of MLL cells in vitro. Cells were incubated in either 0.1 or 1.0 mM ALA for 4 h and were treated with 532 nm or 635 nm light under well oxygenated or hypoxic conditions. Fluorescence spectra were acquired during treatment. Photobleaching and photoproduct formation were quantified using singular value decomposition fitting of fluorescence spectra to experimentally determined basis spectra for PpIX, photoprotoporphyrin (Ppp), product II (peak at 655 nm), and product III (peak at 618 nm). PpIX photobleaching occurred under both normal and hypoxic conditions. The photobleaching kinetics could not be explained by purely first- or second-order photobleaching kinetics, and were attributed to differences in PpIX binding at the two ALA incubation concentrations. Ppp was the main photoproduct and accumulated in higher levels in the absence of oxygen, likely a result of reduced Ppp photobleaching under hypoxia. Increases in product II fluorescence occurred mainly in the presence of oxygen. To assess potential fluorescence based PDT dose metrics, cell viability was measured at select times during treatment using a colony formation assay. Cell survival correlated well to changes in product II fluorescence, independent of oxygenation, sensitizer concentration, and treatment wavelength, suggesting that this product is primarily a result of singlet oxygen mediated reactions and may potentially be useful to quantify singlet oxygen dose during PDT.     

Reaction of the m-THPC triplet state with the antioxidant Trolox and the anesthetic Propofol: modulation of photosensitization mechanisms relevant to photodynamic therapy?

Antioxidants may affect the outcome of photodynamic therapy (PDT) through the inactivation of reactive oxygen species. Their direct interaction with photosensitizers excited at the triplet state is also worthy of interest. This process is investigated by laser flash photolysis of m-THPC (meso-tetra(3-hydroxyphenyl)chlorin, Foscan) hydroalcoholic solutions added with Trolox (TrOH), a standard antioxidant or Propofol (PfOH, Diprivan(?)), a common anesthetic agent also characterized for its antioxidant properties. Transient UV-visible absorption spectra, kinetics at selected wavelengths and final spectra after extensive laser irradiation show that both compounds react with the m-THPC triplet state, (3)m-THPC, to ultimately restore the photosensitizer in its ground state. For PfOH, this process mainly appears as a single step obeying pseudo-first order kinetics. The bimolecular rate constant for the quenching of (3)m-THPC by PfOH is around 2 × 10(6) M(-1) s(-1), a value increased to some extent by the water content of the solution. A bimolecular reaction between (3)m-THPC and TrOH is observed with a rate constant of similar magnitude and dependence upon water. However, the reaction leads, at least partly, to intermediate species assigned to the TrO˙ radical and the m-THPC anion radical. Within a few ms, these species back react to yield m-THPC in its ground state. A general mechanism involving an intermediate activated complex with some charge transfer character is proposed. Depending on the redox potentials for the oxidation of the antioxidant, this complex evolves predominantly either toward the formation of radicals (TrOH) or back to the photosensitizer ground state (PfOH). Notably, the kinetics data suggest that Propofol may quench (3)m-THPC at concentrations relevant of clinical situation in PDT involving anesthesia.     

Correlation of death modes of photosensitized cells with intracellular ATP concentration

The impact of intensity of glycolysis and oxidative phosphorylation on death of photosensitized murine hepatoma MH22 cells in vitro has been investigated. Cells photosensitized with meso-tetra(4-sulfonatophenyl)-porphine localized to lysosomes died mostly by necrosis, and the mode of cell death did not depend on the energy metabolism. Photosensitization with 5-aminolevulinic acid-stimulated endogenous porphyrins localized mainly in mitochondria or 5,10,15,20-tetrakis(m-hydroxyphenyl)-chlorine localized to cell membranes, including mitochondria, led to cell death mostly by apoptosis. In this case, the mode of cell death depended on the medium: under conditions unfavorable to glycolysis the ratio apoptosis/necrosis decreased significantly.     

A central role for intermolecular dityrosine cross-linking of fibrinogen in high molecular weight advanced oxidation protein product (AOPP) formation

Background

Advanced oxidation protein products (AOPPs) are dityrosine cross-linked and carbonyl-containing protein products formed by the reaction of plasma proteins with chlorinated oxidants, such as hypochlorous acid (HOCl). Most studies consider human serum albumin (HSA) as the main protein responsible for AOPP formation, although the molecular composition of AOPPs has not yet been elucidated. Here, we investigated the relative contribution of HSA and fibrinogen to generation of AOPPs.

Methods

AOPP formation was explored by SDS-PAGE, under both reducing and non-reducing conditions, as well as by analytical gel filtration HPLC coupled to fluorescence detection to determine dityrosine and pentosidine formation.

Results

Following exposure to different concentrations of HOCl, HSA resulted to be carbonylated but did not form dityrosine cross-linked high molecular weight aggregates. Differently, incubation of fibrinogen or HSA/fibrinogen mixtures with HOCl at concentrations higher than 150 μM induced the formation of pentosidine and high molecular weight (HMW)-AOPPs (> 200 kDa), resulting from intermolecular dityrosine cross-linking. Dityrosine fluorescence increased in parallel with increasing HMW-AOPP formation and increasing fibrinogen concentration in HSA/fibrinogen mixtures exposed to HOCl. This conclusion is corroborated by experiments where dityrosine fluorescence was measured in HOCl-treated human plasma samples containing physiological or supra-physiological fibrinogen concentrations or selectively depleted of fibrinogen, which highlighted that fibrinogen is responsible for the highest fluorescence from dityrosine.

Conclusions

A central role for intermolecular dityrosine cross-linking of fibrinogen in HMW-AOPP formation is shown.

General significance

These results highlight that oxidized fibrinogen, instead of HSA, is the key protein for intermolecular dityrosine formation in human plasma.     

Spectroscopic investigation on the interaction of J-aggregate with human serum albumin

The interactions of three cyanine dyes, which exhibit different meso substituent in polymethine chain, with human serum albumin (HSA) have been investigated by the means of absorption, fluorescence and circular dichroism (CD) spectra. In phosphate buffer solution (PBS), the mentioned dyes exist not as isolated monomers but rather in the formation of J-aggregation. In the presence of HSA, the absorption and fluorescence emission spectra indicated that the J-aggregation was decomposed to monomer because of the strong affinity between dye molecules and HSA. Besides the association of cyanine dyes with HSA, binding to HSA gave rise to the J-aggregation CD signals. The meso substituent in the polymethine plays an important role in the interaction of HSA and the J-aggregation. Spectral studies showed that the dye bound with HSA in a 1:1 formation. The apparent constant (K(a)) value was roughly identified by analysis of the corresponding fluorescence data at various HSA concentrations. The higher affinity of the molecule with meso phenyl towards HSA with respect to molecules with meso ethyl or methyl can be attributed to the arrangement of molecules in J-aggregation and the hydrophobic force between the molecules and HSA.     

A comparative study of water soluble 5,10,15,20-tetrakis(2,6-dichloro-3-sulfophenyl)porphyrin and its metal complexes as efficient sensitizers for photodegradation of phenols.

5,10,15,20-Tetrakis(2,6-dichloro-3-chlorosulfophenyl)porphyrin and its tin and zinc complexes were synthesized with high yields and fully characterized. The corresponding water-soluble 5,10,15,20-tetrakis(2,6-dichloro-3-sulfophenyl)porphyrins were obtained by hydrolysis with water. An extensive photophysical study of the new water soluble porphyrinic compounds was carried out including absorption and fluorescence spectra, fluorescence quantum yields, triplet absorption spectra, triplet lifetimes, triplet and singlet oxygen quantum yields. These sensitizers were successfully used in the photodegradation of 4-chlorophenol and 2,6-dimethylphenol. A comparison is made of their efficiencies, and some mechanistic considerations are highlighted.     

Surface-enhanced resonance Raman scattering of porphyrins on gold nanoparticles attached to silanized glass plates

Surface-enhanced resonance Raman scattering (SERRS) spectra of cationic 5,10,15,20-tetrakis(1-methyl-4-pyridyl) porphyrin (TMPyP) and anionic 5,10,15,20-tetrakis(4-sulfonatophenyl) porphyrin (TSPP) were measured from gold surfaces prepared by attaching citrate-reduced colloidal nanoparticles to glass slides silanized by 3-aminopropyltrimethoxysilane. SERRS spectra of both porphyrins obtained in a large concentration range (1 x 10(-4) to 1 x 10(-7)M) of primary solution do not show any sign of porphyrin metalation or perturbation of its native structure. Optimal adsorption time (15-20 min) and covering concentration limit (lower than 1 x 10(-5)M) of porphyrins have been estimated from the concentration and soaking time dependences of SERRS spectra.     

Time-resolved luminescence energy transfer immunobinding study using a ruthenium-ligand complex as a donor label

A novel immunosystem is described that exploits the effect of luminescence energy transfer from a luminescently labeled antigen to a fluorescent antibody. A luminescent ruthenium-ligand complex (D-455) with absorption/emission maxima at 456/639 nm, respectively, was employed as the donor label, and a squaraine-type cyanine label (636/655 nm), as the fluorescent acceptor label. Specifically, the system human serum albumin (HSA)/anti-HSA was studied. HSA was labeled with the donor dye D-455, and anti-HSA was labeled with the acceptor dye A-631. On formation of the antigen-antibody complex, energy transfer occurs. The radiationless energy transfer affects both the decay time of D-455 and the intensities of the emissions of both D-455 and A-631. The decay time of around 500 ns of D-455 allows frequency-domain measurements in the low kilohertz range and therefore can be based on the use of conventional optoelectronics. This also suggests gated measurements to be performed. The major difference from existing HSA immunosystems is the use of a slow decaying ruthenium-ligand complex as the donor and of a long-wave emitting cyanine acceptor dye having a high quantum yield and a decay kinetics that is governed by the rate of energy transfer from the slow decaying donor.     

Acetoacetate promotes the formation of fluorescent advanced glycation end products (AGEs)

Acetoacetate (AA) is an important ketone body, which produces reactive oxygen species (ROS). Advanced glycation end products (AGEs) are defined as final products of glycation process whose production is influenced by the levels of ROS. The accumulation of AGEs in the body contributes to pathogenesis of many diseases including complications of diabetes, and Alzheimer’s and Parkinson’s disease. Here, we evaluated the impact of AA on production of AGEs upon incubation of human serum albumin (HSA) with glucose. The effect of AA on the AGEs formation of HSA was studied under physiological conditions after incubation with glucose for 35 days. The physical techniques including circular dichroism (CD) and fluorescence spectroscopy were used to assess the impact of AA on formation and structural changes of glycated HSA (GHSA). Our results indicated that the secondary and tertiary structural changes of GHSA were increased in the presence of AA. The fluorescence intensity measurements of AGEs also showed an increase in AGEs formation. Acetoacetate has an activator effect in formation of AGEs through ROS production. The presence of AA may result in enhanced glycation in the presence of glucose and severity of complications associated with accumulation of AGEs.     

Papaverine increases human serum albumin glycation

Glycation is a non-enzymatic reaction that is initiated by the primary addition of sugars to amino groups of proteins. In the early phase of glycation, the synthesis of intermediates leads to formation of Amadori compounds. In the last phase, advanced glycation end products (AGE) are irreversibly formed following a complex cascade of reactions. It has recently been shown that glycation also affects diabetes-related complications and Alzheimer’s disease. In this study, human serum albumin at a concentration of 10 mg/ml was incubated in PBS with 40 mM of glucose and in different concentrations of papaverine (25, 100, 250, 500 μM) for 42 days at 37 °C. HSA with no additives as well as with glucose 40 mM were incubated as a control and as a glycated sample, respectively. Following the incubation, the samples were prepared for circular dichroism, fluorescence and absorbance techniques. The results showed that in presence of papaverine and glucose, the glycation of HSA increased notably compared with the glycated sample. In conclusion, in this work, we showed that papaverine affects HSA and increases its glycation level.     

Photodynamic inactivation of Escherichia coli immobilized on agar surfaces by a tricationic porphyrin

The photodynamic activity of 5,10,15-tris[4-(3-N,N,N-trimethylammoniumpropoxy)phenyl]-20-(4-trifluoromethylphenyl)porphyrin iodide (A3B3+) has been studied in vitro on a typical Gram-negative bacterium Escherichia coli immobilized on agar surfaces. The results obtained for the tricationic A3B3+ porphyrin were compared with those of 5,10,15,20-tetra(4-N,N,N-trimethylammoniumphenyl)porphyrin p-tosylate (TTAP4+), which is a standard active sensitizer established to eradicate E. coli in cellular suspension. The photobleaching of these porphyrins in solution was evaluated by decay in absorbance and in fluorescence. In both cases, a higher photostability was found for A3B3+ than for TTAP4+. Photodynamic inactivation capacities of these sensitizers were analyzed in E. coli cells immobilized on agar surfaces. Small colonies were treated with different amount of sensitizer (0-14 nmol) and irradiated with visible light for 3h. The light source used was either a projector or midday sun. The A3B3+ porphyrin produced a growth delay of E. coli colonies on agar surfaces. Similar result was obtained irradiating only one isolated colony through an optical fiber. Under these conditions, A3B3+ porphyrin shows a high activity to inactivate localized bacterial cells. The higher photodynamic activity of A3B3+ was confirmed by mechanical spreading of the colonies before treatment. This procedure produces complete inactivation of E. coli cells on the agar surface. Therefore, tricationic A3B3+ porphyrin is an interesting sensitizer with potential applications in photodynamic inactivation of bacteria growing as localized foci of infection.     

Role of salt bridge(s) in the binding and photoconversion of bilirubin bound to high affinity site on human serum albumin

The role of salt bridge(s) (between epsilon-NH(2) groups of lysine residues of human serum albumin (HSA) and carboxyl groups of bilirubin) in the binding and photoconversion of bilirubin bound to high affinity site on HSA was investigated by covalent modification of approximately 20% internal (buried) lysine residues of HSA with acetic anhydride, succinic anhydride and O-methylisourea and white light irradiation of their complexes with bilirubin. The different HSA derivatives, namely, acetylated HSA (aHSA), succinylated HSA (sHSA) and guanidinated HSA (gHSA), thus obtained, were found to be homogeneous with respect to charge and size and characterized in detail in terms of mean residue ellipticity, Stokes radius, tryptophan fluorescence, bilirubin binding and the photochemistry of their complexes with bilirubin. All the three derivatives retained helical contents and molecular size (Stokes radius) similar to HSA except for sHSA which showed a slight increase in the Stokes radius from 3.56 to 3.64 nm. Further, fluorescence properties of aHSA and sHSA were also found to be different from HSA and gHSA. Based on difference spectral change, fluorescence quenching and fluorescence enhancement results of bilirubin bound to HSA and its derivatives, nearly 46 and 48% reduction in bilirubin binding was observed in the case of aHSA and sHSA, respectively. Both aHSA and sHSA showed a decrease of 8- and 10-fold, respectively, in association constant compared to native HSA. Although the bisignate circular dichroism (CD) spectra of an equimolar (1:1) bilirubin-HSA complex was retained by all three HSA derivatives, the intensity of both positive and negative CD Cotton effects decreased significantly in both aHSA and sHSA. gHSA which retained net charge identical to native HSA, showed little decrease in bilirubin binding and the intensity of bisignate CD Cotton effects. The photochemical reaction of bilirubin bound to aHSA and sHSA produced opposite results to those observed with HSA and gHSA. A brief (2 min) irradiation of an equimolar complex of bilirubin with both aHSA and sHSA accompanied a rapid shift (14-15 nm) in the absorption spectrum of the bound pigment towards the blue region and almost complete elimination of negative CD Cotton effects while only moderately affecting the magnitude of positive CD Cotton effects. On the other hand, similar treatment of the complexes of bilirubin with HSA and gHSA did not show any change in the absorption spectrum, only a slight decrease in the intensity of both positive and negative CD Cotton effects was observed. The fluorescence intensity of bilirubin bound to HSA and gHSA was increased upon irradiation with white light and after 30 min it was nearly twice the value observed at 0 min irradiation. Interestingly, no change in the fluorescence intensity of bilirubin bound either to aHSA or sHSA was observed upon irradiation, even on increasing the duration of irradiation to 1 h. Taken together, the results on fluorescence quenching, fluorescence enhancement, CD spectral changes and visible absorption spectroscopy suggest that salt bridge(s) of the type (-COO(-).(+)H(3)N-) in which the epsilon-NH(2) group(s) contributed by lysine residues, are not only involved in the enantioselective binding of bilirubin but also in the stereospecific photoisomerization of bilirubin bound to a high affinity site on HSA.     

Entrapment and in vitro release of delta-sleep inducing peptide from polymer hydrogels based on modified polyvinyl alcohol

The aim of this study was to entrap delta-sleep inducing peptide (DSIP) in cross-linked poly(vinyl alcohol)-based hydrogels of different structures and to determine kinetics of the peptide release from these hydrogels using an in vitro model. Isotropic and macroporous hydrogels based on poly(vinyl alcohol) acrylic derivative (Acr-PVA) and also macroporous epoxy groups containing hydrogels synthesized by copolymerization of this macromer and glycidyl methacrylate, have been used in this study. Isotropic hydrogels were prepared at positive temperatures while macroporous ones were obtained by formation in cryo-conditions. The peptide was entrapped into macroporous PVA hydrogels by adding the peptide solution onto preformed matrices, while peptide immobilization on PVA-GMA hydrogels, containing free epoxy groups, was carried out by sorption of peptide from its aqueous solution. In the case of DSIP entrapment into isotropic PVA gel the peptide solution was added into the polymer mixture at hydrogel formation. The kinetics of peptide release from hydrogels was studied by incubating matrices in PBS solution (pH 7.4), in physiological solution (0.9% NaCl) and in water. DSIP concentration in supernatants was determined by reverse-phase HPLC. Incubation of macroporous PVA gels in PBS, 0.9% NaCl, and water for 30 min caused release of 74, 70, and 64% DSIP, respectively, and this processes completed within 3 h. From hydrogel containing epoxy groups the release of neither peptide nor its degradation products was observed even after incubation for 48 h. For freshly prepared isotropic hydrogel the release kinetics was as follows: 27 and 78% DSIP were released within first 30 min and 33 h, relatively. For the lyophilized hydrogel samples the peptide release was 63% after incubation for 30 min, while drying of samples at room temperature for 3 days caused significant peptide loss because of its structure damage.     

Analysis of collagen cyanogen bromide peptides using electrophoresis in continuous concave gradient polyacrylamide gels.

A rapid and simple spectrophotometric method is described for the estimation of microgram quantities of glycosaminoglycans following the formation of soluble complexes with alcian blue dye. The method is based on the different absorption spectra of the dye and dye-glycosaminoglycan complexes. No heating, centrifugation, lengthy equilibration, or sophisticated instrumentation which hamper other methods are required. Samples are mixed with freshly prepared dye solution and absorbance readings at 480 nm are compared to an appropriate standard curve. Albumin and individual monosaccharides do not interfere with the assay but high concentrations of chloride ion do. The method is suitable for the estimation of total glycosaminoglycan levels in biological fluids such as urine and blood.     

A kinetic study of the reconstitution of azurin from Cu(II) and the apoprotein.

The kinetics of reconstitution of Pseudomonas aeruginosa azurin from its apoprotein and copper(II) salts have been studied using absorbance at 625 nm and fluorescence emission at 308 nm as monitors of the process. At low Cu(II) concentrations the rates of both absorbance and fluorescence changes are linearly dependent on Cu(II) concentration. At higher Cu(II) concentrations the rate of absorbance change is independent of Cu(II) concentration. The rates of both absorbance and fluorescence changes as a function of pH suggest that the titration of a single ionizable group is important for the Cu(II)-dependent reaction. Overall analysis of the kinetics suggests that the fluorescence change and the absorbance change are associated with at least two steps in the overall pathway of the formation of the metal-protein complex, and that the copper(II) and tryptophan environments in this protein, though perhaps spatially close, may be distinct.     

Photodegradation mechanism and reaction kinetics of recombinant human interferon-alpha2a.

The photodegradation mechanism of recombinant human interferon-alpha2a (IFNalpha2a) has been investigated using absorption, fluorescence, and circular dichroism (CD) spectroscopies, and fluorescence photobleaching kinetics measurements under various conditions. After photobleaching, the absorption profile of aromatic amino acid residues in IFNalpha2a was almost absent, and an absorption profile showing a monotonic increase toward short wavelengths was observed. According to the CD spectrum analysis, partial unfolding of IFNalpha2a was accompanied by a complete loss of fluorescence. This unfolding was attributed to tryptophan-mediated photoinduced disulfide bond cleavage. Photooxygenation and photoionization of tryptophan (Trp) residues followed by subsequent radical reactions were the main photodegradation pathways of IFNalpha2a. Photobleaching kinetics was faster in acidic solution (pH 2.5) than in neutral solution (pH 7.4). The variation of photobleaching kinetics seemed to be caused by the structural differences in IFNalpha2a according to the solution pH. The relationship between the protein conformation and photobleaching rate could be explained based on the competition between excited state energy transfer and the photoionization process in Trp residues.