Synthesis and luminescent spectral characteristics of porphyrin complexes with platinum group metals

The synthesis of natural and synthetic porphyrin complexes with Pt, Pd, Rh, and Ru is reported. Their electronic absorption spectra, phosphorescence spectra, and lifetimes at room temperature both in the presence and in the absence of oxygen were studied. It has been shown that the variation of the nature of the central metal atom and of the substituents in pyrrole and phenyl rings allows the obtaining of metalloporphyrins with various phosphorescence excitation and phosphorescing emission spectra at room temperature. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.     

Phosphorescent analysis of lipid peroxidation products in isolated human erythrocyte membranes

The room temperature phosphorescence of lipid peroxidation products in the composition of isolated human erythrocyte membranes was registered, and its kinetic parameters were determined. The excitation and emission spectra of phosphorescence of lipid peroxidation products in the composition of erythrocyte membranes at 0 degree C measured. The nature of lipid peroxidation products possessing the phosphorescencing capacity was discussed. Based on the analysis of temperature dependences of the intensity and lifetimes of phosphorescence of lipid peroxidation products in the range -2 divided by 26 degrees C, it is concluded that the deactivation of excited triplet states of lipid chromophores was realized by the dynamic type.     

Phosphorescence of protochlorophyll(ide) and chlorophyll(ide) in etiolated and greening bean leaves

The assignment is presented for the principal phosphorescence bands of protochlorophyll(ide), chlorophyllide and chlorophyll in etiolated and greening bean leaves measured at -196°C using a mechanical phosphoroscope. Protochlorophyll(ide) phosophorescence spectra in etiolated leaves consist of three bands with maxima at 870, 920 and 970 nm. Excitation spectra show that the 870 nm band belongs to the short wavelength protochlorophyll(ide), P627. The latter two bands correspond to the protochlorophyll(ide) forms, P637 and P650. The overall quantum yield for P650 phosphorescence in etiolated leaves is near to that in solutions of monomeric protochlorophyll, indicating a rather high efficiency of the protochlorophyll(ide) triplet state formation in frozen plant material. Short-term (2–20 min) illumination of etiolated leaves at the temperature range from -30 to 20°C leads to the appearance of new phosphorescence bands at about 990–1000 and 940 nm. Judging from excitation and emission spectra, the former band belongs to aggregated chlorophyllide, the latter one, to monomeric chlorophyll or chlorophyllide. This indicates that both monomeric and aggregated pigments are formed at this stage of leaf greening. After preillumination for 1 h at room temperature, chlorophyll phosphorescence predominates. The spectral maximum of this phosphorescence is at 955–960 nm, the lifetime is about 2 ms, and the maximum of the excitation spectrum lies at 668 nm. Further greening leads to a sharp drop of the chlorophyll phosphorescence intensity and to a shift of the phosphorescence maximum to 980 nm, while the phosphorescence lifetime and a maximum of the phosphorescence excitation spectrum remains unaltered. The data suggest that chlorophyll phosphorescence belongs to the short wavelength, newly synthesized chlorophyll, not bound to chloroplast carotenoids. Thus, the phosphorescence measurement can be efficiently used to study newly formed chlorophyll and its precursors in etiolated and greening leaves and to address various problems arising in the analysis of chlorophyll biosynthesis.Abbreviations Pchl protochlorophyll and protochlorophyllide - Chld chlorophyllide - Chl chlorophyll     

Indoles and auxins. I. Spectrophosphorimetry of some natural and synthetic indoles

Phosphorescence characteristics and fluorescence spectra at room temperature and 77°K of 5 indole compounds relevant to 3-indoleacetic acid metabolism have been recorded. Three of these samples were extracted from a biological source and compared to synthetic derivatives.The phosphorescence spectrum of these compounds is very characteristic for the indole chromophore and superior to the fluorescence spectrum for its detection.For the closely related compounds I–V the phosphorescence characteristics are not sufficiently different from each other to allow unambiguous identification.     

Tryptophan phosphorescence of the Ca2+-ATPase of sarcoplasmic reticulum

Phosphorescence of protein tryptophan was analyzed in sarcoplasmic reticulum vesicles, and in the purified Ca2+ transport ATPase in deoxygenated aqueous solutions at room temperature. Upon excitation with light of 295 nm wavelength, the emission maxima of fluorescence and phosphorescence were at 330 nm and at 445 nm, respectively. The phosphorescence decay was multiexponential; the lifetime of the long-lived component of phosphorescence was approximately equal to 22 ms. ATP and vandate significantly reduced the phosphorescence in the presence of either Ca2+ or EGTA; ADP was less effective, while AMP was without effect. The quenching by ATP showed saturation consistent with the idea that the ATP-enzyme complex had a lower phosphorescence yield. Upon exhaustion of ATP, the phosphorescence returned to starting level. Significant quenching of phosphorescence with a decrease in phosphorescence lifetime was also caused by NaNO2, methylvinyl ketone and trichloroacetate, without effect on ATPase activity; this quenching did not show saturation and was therefore probably collisional in nature.     

The fine structure of luminescence spectra of azurin.

The spectra of azurin absorption, fluorescence, phosphorescence and fluorescence excitation have been measured in aqueous solutions at ordinary and liquid nitrogen temperatures. The fluorescence spectra of azurin even at ordinary temperatures have a well resolved fine vibrational structure. The frequency analysis reveals practically the same wave number distances between the main structure peaks in fluorescence spectra at room and low temperatures and in phosphorescence spectra. The comparison of the protein absorption and excitation spectra shows that all the energy absorbed by tyrosine residues is transferred onto indole chromophore. These data suggest an unusual tryptophan environment in this protein, which is characterized by the absence of any hydrogen bonding or other polar interaction of tryptophan with its environment. The problem of the possibility of contributions of two electronic transitions (1La in equilibrium A and 1Lb in equilibrium A) in absorption and emission spectra of azurin tryptophan arising from their mirror symmetry is discussed.     

Phosphorescence properties of Trp-84 and Trp-310 in glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus

The phosphorescence spectra of Trp-84 and Trp-310 in glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus in an aqueous glass show distinct 0,0 vibrational bands with peaks at 406.5 and 410.5 nm. With the aid of external heavy-atom perturbation of iodide and the thermal quenching profile, it is concluded that although both chromophores are effectively buried, only one, viz., the 406.5 nm component, is embedded in a sufficiently rigid core of the protein to phosphoresce in fluid solutions at room temperature. From inspection of the crystallographic structure is it evident that only Trp-310 embedded in the beta-sheet of the catalytic domain may satisfy the requirements of a long triplet-state lifetime and slow migration of O2 to its site. This identification confirms previous analysis of the phosphorescence properties of the enzymes from yeast, pig and rabbit muscle.     

Phosphorescent analysis of lipid peroxidation products in liposomes]

It was found that lipid peroxidation products incorporated into liposomes prepared from oxidized preparations of bovine heart phosphatidylcholine and the total lipid fraction of human erythrocyte membranes are able to phosphoresce at room temperature was studied. The temperature dependences of kinetic and spectral parameters of phosphorescence were measured. It is shown that mechanism of phosphorescence quenching of lipid chromophores has a dynamic nature. It is proposed to use endogenic molecules of the lipid peroxidation products capable of phosphorescence as intrinsic phosphorescence probes for studying the slow molecular dynamics of lipids in artificial and biological membranes in a millisecond range.     

Phosphorescence properties and protein structure surrounding tryptophan residues in yeast, pig, and rabbit glyceraldehyde-3-phosphate dehydrogenase

An investigation of the phosphorescence emission properties of tryptophan (Trp) was carried out in glyceraldehyde-3-phosphate dehydrogenase from yeast and from pig and rabbit muscle. Aided by the external heavy-atom effect of iodide, the dependence on excitation wavelength, and thermal quenching profiles, it was established that the 0,0 vibronic band peaked at 406 nm in the pig and rabbit proteins is made up of overlapping contributions from two Trp residues. In contrast to a previous report [Davis, J.M., & Maki, A.H. (1984) Biochemistry 23, 6249-6256], this implies that even in the muscle enzymes all three aromatic side chains are phosphorescent. Further, when the nature of the local environment of each residue is compared to the crystallographic structure of lobster GPDH, it leads to a complete new assignment of the individual phosphorescence spectra. With each protein, a single Trp, identified as Trp-310, was found to display long-lived phosphorescence at room temperature. The decay of this emission gives evidence of conformational homogeneity among the subunits of the tetrameric molecule.     

Reactions of excited triplet states of metal substituted myoglobin with dioxygen and quinone.

The triplet state absorption and phosphorescence of Zn and Pd derivatives of myoglobin were compared. Both metal derivatives exhibit long triplet state lifetimes at room temperature, but whereas the Pd derivative showed exponential decay and an isosbestic point in the transient absorption spectra, the decay of the Zn derivative was nonsingle exponential and the transient absorption spectra showed evidence of more than one excited state species. No difference was seen in triplet quenching by oxygen for either derivative, indicating that differences in the polypeptide chain between the two derivatives are not large enough to affect oxygen penetrability. Quenching was also observed by anthraquinone sulfonate. In this case, the possibility of long-range transfer by an exchange mechanism is considered.     

Phosphorimetric method to determine Zn-porphyrins in microorganisms and biological liquids

A phosphorimetric technique of direct quantitative determination of Zn-porphyrins in microorganisms and biological liquids is described. The technique is based on the registration of visible light-induced afterglow and phosphorescence of Zn-porphyrins at room temperature under anaerobic conditions. The sensitivity of the technique is not less than 10(-8) M. Certain types of Zn-porphyrins can be identified with respect to the spectra of excitation and radiation of afterglow. The technique has been applied to study the afterglow of microorganisms (E. coli, St. aureus, B. subtilis, B. pioceaneum, M. tuberculosis) and of milk, serum, and urine.     

Tryptophan phosphorescence of G-actin and F-actin

The tryptophan phosphorescence spectrum, intensity and decay kinetics of G-actin and F-actin were measured over a temperature range of 140-293 K. The fine structure in the phosphorescence spectra at low temperature, with O,O vibrational bands centered at 405 nm and 415.5 nm for both species, reveals a marked heterogeneity of the chromophore environment. The thermal quenching profile distinguishes these sites in terms of their flexibility, and shows that probably only one of the four tryptophan residues is still phosphorescent at ambient temperature due to its location in a relatively rigid buried core. Although some differences are demonstrated between G-actin and F-actin at low temperature, the identity of the triplet lifetime at ambient temperature strongly supports the notion that the conformation of the macromolecule is largely unaffected by polymerization. Preliminary phosphorescence anisotropy measurements demonstrate both the occurrence of singlet-singlet energy transfer among tryptophan residues and a strong immobilization of actin in the polymerized state.     

The luminescence properties of concanavalin A.

1. The luminescence properties of native concanavalin A, both at room temperature and at 77 degrees K, are similar to those of other proteins containing tyrosine and tryptophan. 2. Binding of methyl alpha-D-glucopyranoside to concanavalin A causes a slight reduction of its fluorescence at room temperature. 3. Removal of Mn2+ and Ca2+ ions from concanavalin A causes a small increase in its fluoresence. The fluorescence: phosphorescence ratio and phosphorescence lifetime of apo-concanavalin A are similar to those of tryptophan. 4. Denaturation of concanavalin A by urea and by guanidine hydrochloride apparently takes place in two stages. Apo-concanavalin A is more easily denatured than the native molecule, but concavalin A combined with methyl alpha-D-glucopyranoside is more resistant to denaturation. 5. The luminescence properties of concanavalin A are pH-dependent. 6. The results have been interpreted in terms of the known structure and properties of concanavalin A.     

Photochemistry of 2-acyloxycarbazoles. A potential tool in the synthesis of carbazole alkaloids.

The photochemistry of two 2-acyloxycarbazoles, 2-acetyl- and 2-benzoyloxycarbazole, in different solvents has been studied. Irradiation of the 2-acyloxycarbazoles in organic media at 254 or 313 nm yields the [1,3]-migrated photoproducts, 1-acyl-2-hydroxycarbazole, 3-acyl-2-hydroxycarbazole and 2-hydroxycarbazole. The effects of the solvent, the atmosphere and the intensity of the light source on the photochemistry of 2-acyloxycarbazole have been studied. Laser flash photolysis as well as photosensitization experiments were performed in order to determine the photoreactive excited state. Electronic spectra (absorption, fluorescence and phosphorescence emission spectra) of the 2-acyloxycarbazoles have been recorded in homogeneous media at 298 K and in solid matrices at 77 K. The dynamic properties of the lowest singlet excited state in terms of fluorescence lifetime and fluorescence quantum yield have been measured in different organic solvents at room temperature. The photo-Fries rearrangement as a mild and clean one-pot reaction for the preparation of an advanced intermediate precursor in the total synthesis of carbazole alkaloids is described.     

Characterization of lens alpha-crystallin tryptophan microenvironments by room temperature phosphorescence spectroscopy

Room temperature phosphorescence techniques were used to study the structural and dynamic features of the tryptophan residues in bovine alpha-crystallin. Upon excitation at 290 nm, the characteristic signature of tryptophan phosphorescence was observed with an emission maximum at 442 +/- 2 nm. The phosphorescence intensity decay was biphasic with lifetimes of 5.4 ms (71%) and 42 ms (29%). Phosphorescence quenching measurements strongly suggest that each component corresponds to one class of tryptophans with the more buried residues having the longer emission lifetime. Three small-molecule quenchers were surveyed, and in order of increasing quenching efficiency: iodide less than nitrite less than acrylamide. A heavy-atom effect was observed in iodide solutions, and an upper limit of 5% was placed on the quantum yield of triplet formation in iodide-free solutions, while the phosphorescence quantum yield was estimated to be approximately 3.2 x 10(-4). The temperature dependence of the phosphorescence lifetime was measured between 5 and 40 degrees C. Arrhenius plots exhibited discontinuities at 26 and 29 degrees C for the short- and long-lived components, respectively, corresponding to abrupt transitions in segmental flexibility. Denaturation studies revealed conformational transitions between 1 and 2 M guanidine hydrochloride, and 4 and 6 M urea. Long-lived phosphorescence lifetimes of 3 and 7 ms were measured in 6 M guanidine hydrochloride and 8 M urea, respectively, suggesting that some structural features are preserved even at very high concentrations of denaturant. Our studies demonstrate the sensitivity of room temperature phosphorescence spectroscopy to the structure of alpha-crystallin, and the applicability of this technique for monitoring conformational changes in lens crystallin proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

A spectroscopic analysis of vitamin B12 derivatives.

1. Several vitamin B12 derivatives including descobalt B12 show unusual inversion of their CD signs upon rapid cooling to liquid N2 temperature, although the room temperature CD signs are conserved by a slower cooling to the same temperature. As a possible explanation for this puzzling observation, a micro-environmental birefringence around the chromophore imbedded in an organic glass is proposed. 2. Absorption, fluorescence, phosphorescence, and polarization spectra of descobalt B12 can be correlated with those of porphyrin free bases, as these two molecular systems share many similarites in their electronic structure. 3. Molecular orbital calculations of polarization directions further support the analoby between the spectroscopic characteristics of corrins and porphyrins, and are generally in good agreement with the fluorescence polarization data.     

Simplex optimization of the variables affecting the micelle-stabilized room temperature phosphorescence of 6-methoxy-2-naphthylacetic acid and its kinetic determination in human urine

This article reports the kinetic determination of 6-methoxy-2-naphthylacetic acid (6-MNA), the major metabolite of nabumetone, from micelle-stabilized room temperature phosphorescence (MS-RTP) measurements made by using the stopped-flow mixing technique. This methodology allows one to determine analytes in complex matrices without the need for a tedious separation process. It also shortens analysis times substantially. The proposed method uses simplex methodology to optimize the chemical and instrumental variables affecting the phosphorescence. It was applied to the determination of 6-MNA in human urine. The maximum phosphorescence signal is obtained within only 10 s after the sample is prepared. The maximum slope of the kinetic curve, which corresponds to the maximum rate of the phosphorescence development, is measured at lambda(ex)=273 nm and lambda(em)=516 nm. Least-squares regression was used to fit experimental data, and the detection limit, repeatability, and standard deviation for replicate samples were determined.     

Tryptophan interactions with glycerol/water and trehalose/sucrose cryosolvents: infrared and fluorescence spectroscopy and ab initio calculations

In order to correlate how the solvent affects emission properties of tryptophan, the fluorescence and phosphorescence emission spectra of tryptophan and indole model compounds were compared for solid sugar glass (trehalose/sucrose) matrix and glycerol/water solution and under the same conditions, these matrices were examined by infrared spectroscopy. Temperature was varied from 290 to 12 K. In sugar glass, the fluorescence and phosphorescence emission spectra are constant over this temperature range and the fluorescence remains red shifted; these results are consistent with the static interaction of OH groups with tryptophan in the sugar glass. In sugar glass containing water, the water retains mobility over the entire temperature range as indicated by the HOH infrared bending frequency. The fluorescence of tryptophan in glycerol/water shifts to the blue as temperature decreases and the frequency change of the absorption of the HOH bend mode is larger than in the sugar glass. These results suggest rearrangement of glycerol and water molecules over the entire temperature change. Shifts in the fluorescence emission maximum of indole and tryptophan were relatively larger than shifts for the phosphorescence emission-as expected for the relatively smaller excited triplet state dipole for tryptophan. The fluorescence emission of tryptophan in glycerol/water at low temperature has maxima at 312, 313, and 316 nm at pH 1.4, 7.0, and 10.6, respectively. The spectral shifts are interpreted to be an indication of a charge, or Stark phenomena, effect on the excited state molecule, as supported by ab initio calculations. To check whether the amino acid remains charged over the temperature range, the infrared spectrum of alanine was monitored over the entire range of temperature. The ratio of infrared absorption characteristic of carboxylate/carbonyl was constant in glycerol/water and sugar glass, which indicates that the charge was retained. Tryptophan buried in proteins, namely calcium parvalbumin from cod and aldolase from rabbit, showed temperature profiles of the fluorescence spectra that were largely independent of the solvent (glycerol/water or sugar glass) and temperature whereas the fluorescence and phosphorescence yields were dependent. The results demonstrate how the rich information found in tryptophan luminescence can provide information on the dipolar nature and dynamics of the matrix.     

Characterization of tryptophan environments in glutamate dehydrogenases from temperature-dependent phosphorescence

Tryptophan room temperature phosphorescence in solution was detected in glutamic dehydrogenase from bovine liver and Escherichia coli with lifetimes of 1.2 and 0.65 s, respectively. Although these enzymes possess three and five tryptophanyl residues per polypeptide chain, respectively, the temperature dependence of the phosphorescence quantum yield estimates that the room temperature emission is due, in either case, to a single residue. Long triplet-state lifetimes and very small rates of O2 quenching indicate that these tryptophanyl side chains are embedded in a highly inflexible internal region of the macromolecule. Aided by sequence homology with dehydrogenases of known structure and theoretical predictions of secondary structure [Wootton, J.C. (1974) Nature (London) 252, 542-546; Brett, M., Chambers, G.K., Holder, A. A., Fincham, J.R.S., & Wootton, J.C. (1976) J. Mol. Biol. 106, 1-22], the phosphorescing tryptophans have been tentatively placed in the catalytic coenzyme binding domain of each enzyme. The particular sensitivity of the triplet-state lifetime in probing local changes in conformation provides a strong indication that within the time window of phosphorescence measurements the six subunits in the hexameric enzymes are equivalent. Furthermore, while in the bovine enzyme this parameter is markedly affected by the interaction with ligands which have a functional role, the constancy of the phosphorescence lifetime at various degrees of polymerization suggests that the association process is not accompanied by important conformational changes in the macromolecule.     

Time-resolved room temperature protein phosphorescence: nonexponential decay from single emitting tryptophans.

The single room temperature phosphorescent (RTP) residue of horse liver alcohol dehydrogenase (LADH). Trp-314, and of alkaline phosphatase (AP), Trp-109, show nonexponential phosphorescence decays when the data are collected to a high degree of precision. Using the maximum entropy method (MEM) for the analysis of these decays, it is shown that AP phosphorescence decay is dominated by a single Gaussian distribution, whereas for LADH the data reveal two amplitude packets. The lifetime-normalized width of the MEM distribution for both proteins is larger than that obtained for model monoexponential chromophores (e.g., terbium in water and pyrene in cyclohexane). Experiments show that the nonexponential decay is fundamental; i.e., an intrinsic property of the pure protein. Because phosphorescence reports on the state of the emitting chromophore, such nonexponential behavior could be caused by the presence of excited state reactions. However, it is also well known that the phosphorescence lifetime of a tryptophan residue is strongly dependent on the local flexibility around the indole moiety. Hence, the nonexponential phosphorescence decay may also be caused by the presence of at least two states of different local rigidity (in the vicinity of the phosphorescing tryptophan) corresponding to different ground state conformers. The observation that in the chemically homogeneous LADH sample the phosphorescence decay kinetics depends on the excitation wavelength further supports this latter interpretation. This dependence is caused by the wavelength-selective excitation of Trp-314 in a subensemble of LADH molecules with differing hydrophobic and rigid environments. With this interpretation, the data show that interconversion of these states occurs on a time scale long compared with the phosphorescence decay (0.1-1.0 s). Further experiments reveal that with increasing temperature the distributed phosphorescence decay rates for both AP and LADH broaden, thus indicating that either 1) the number of conformational states populated at higher temperature increases or 2) the temperature differentially affects individual conformer states. The nature of the observed heterogeneous triplet state kinetics and their relationship to aspects of protein dynamics are discussed.